Adenomyosis (ADS) is a common gynecological disease, and the main pathological feature is ectopic growth of endometrial glands and stroma into the myometrium [1]. The ectopic growth results in a cascade of clinical problems, including dysmenorrhea, dyspareunia, abnormal uterine bleeding, and infertility [2, 3], which seriously impair the quality of life of reproductive-age women [1]. The estimated prevalence of adenomyosis in women of reproductive age ranges from 20% to 34.6% on the basis of radiographic assessment to 10% to 88% on the basis of pathological diagnosis in resected uterine specimens, so this diagnostic heterogeneity underscores the underrecognition of this disease [4].

It has been demonstrated by recent studies that endometrial basal cell abnormal proliferation and invasion have intimate connections with epithelial-mesenchymal transition (EMT) [5]. EMT is a salient biological program: through reprogramming of some molecules, epithelial cells are able to transform into extremely migratory and invasive mesenchymal cells. Despite the inevitability of the process in embryonic development and tissue restoration, its pathological activation is widely implicated in inflammatory reactions and metastasis of tumors [6, 7]. In adenomyosis, EMT may lead to the phenotypic transformation of endometrial epithelial cells, which lose their original features step by step, acquire invasive stromal cell characteristics, and further breach the basal barrier and invade the myometrium to form the typical ADS lesions [8].

Because the dynamic equilibrium of mitochondria is in charge of cell homeostasis and even the body’s homeostasis, as the core center of cellular energy metabolism, the dynamic equilibrium of mitochondria is thus very crucial. In the regulating network, Mitofusin 2 (Mfn2) is also the core executor of mitochondrial fusion and an important safeguard of mitochondrial network structure [9]. By interacting with Mitofusin 1 (Mfn1) and other proteins to form functional complexes, Mfn2 initiates processes of membrane fusion between the mitochondria [10], thus ensuring mitochondrial function and morphology integrity. The regulation has a profound effect on basic processes such as energy metabolism, signaling by calcium, and cell life and death processes [11, 12, 13]. It should be noted that the abnormal expression or dysfunction of Mfn2 has been demonstrated to be closely related to a series of severe diseases, ranging from neurodegenerative diseases, metabolic disorders, to cardiovascular diseases, and even throughout the whole process of tumor occurrence, development, and metastasis [14, 15, 16, 17].

With an increasing depth of research on cell biology and molecular mechanisms, the regulatory network between Mfn2 and EMT has been progressively exposed. Present data suggest that Mfn2 has a tumor suppressor function by dynamically regulating the balance of mitochondrial fusion/fission in thyroid cancer progression and directly regulates the EMT process [18]. In ovarian cancer xenograft models, Mfn2 may be induced by genetic or drug approaches to cause mitochondrial fusion, which strongly inhibits the growth, migration, invasion and EMT phenotype of cancer cells by reducing the level of reactive oxygen species (ROS) [19]—the mechanism is mitochondrial fusion-mediated F-actin remodeling, decreasing the formation of lamella pseudopodia, a key connection in EMT. These findings collectively describe a multi-organ role of Mfn2 as an EMT mediator.

Surprisingly, despite accumulating evidence, the functional role of Mfn2 in ADS remains largely unknown. Based on confirmed Mfn2-EMT regulation in oncology models, and EMT’s established role in adenomyosis pathogenesis, we hypothesize that Mfn2 inhibits adenomyosis progression by suppressing EMT in endometrial cells. This study is the first to test this mechanism in benign gynecological disease. By combining the three-dimensional verification system of in vitro cell model, clinical sample analysis, and animal experiment, the principal role of Mfn2 in the occurrence and development of diseases will be studied deeply, which will provide a new understanding of ADS pathological mechanism and create the possibility of translational medicine for targeted intervention.

We found that Mfn2 protein levels were significantly lower in the endometrium of adenomyosis patients compared to healthy women. This hints that Mfn2 might be involved in the disease. Looking closer at a process called EMT—which is important in diseases like this—we saw changes in key markers: the “epithelial” marker E-cadherin (both mRNA and protein) was down (p 0.05), while the “mesenchymal” marker N-cadherin, Vimentin was up (p 0.05). This confirms EMT is happening in adenomyosis. Since Mfn2 was low at the same time, we suspected its decrease might be linked to triggering EMT (Fig. 1).

Fig. 1.

Immunohistochemical, Western Blot, and qPCR results of clinical samples. (A,B) Immunohistochemistry was used to compare the expression of Mfn2 in normal endometrium and adenomyosis endometrium. Scale bar: 100 µm ($\times$200). (C,D) Western Blot was used to detect the protein expression of E-cadherin, N-cadherin, Vimentin, and Mfn2 in the endometrium of the two groups. (E–H) qPCR was used to detect the expression of E-cadherin, N-cadherin, Vimentin, and Mfn2 mRNA in the two groups. Values represent mean $\pm$ SD, Adenomyosis group n = 15, Control group n = 8 (t-test). *p 0.05, **p 0.01, ***p 0.001, ****p 0.0001. qPCR, quantitative polymerase chain reaction; Mfn2, Mitofusin 2; SD, standard deviation.

To test this link, we used a lab model of EMT (treating Ishikawa cells with TGF-$\beta$1). Just like in patients, Mfn2 levels dropped significantly in these treated cells (p 0.05). The EMT markers also changed as expected: E-cadherin down (p 0.05), N-cadherin and Vimentin up (p 0.05). Importantly, these cells became much better at moving and invading (p 0.05)—classic signs of EMT. Then, we boosted Mfn2 levels in this model. This successfully reversed the EMT changes: E-cadherin increased (p 0.05), N-cadherin and Vimentin decreased (p 0.05). Crucially, it also significantly reduced the enhanced cell migration and invasion caused by TGF-$\beta$1 (p 0.05). This strongly suggests Mfn2 acts like a brake, slowing down EMT and the invasive cell behavior seen in adenomyosis (Figs. 2,3).

Fig. 2.

Ishikawa cells were treated with TGF-$\beta$1 to induce EMT model and the expression levels of each factor were detected by qPCR and Western Blot after plasmid transfection. The Mfn2 negative control plasmid was represented by Mfn2-NC. The Mfn2 overexpression plasmid was represented by Mfn2-OE. (A–D) qPCR was used to detect the mRNA expressions of E-cadherin, N-cadherin, Vimentin and Mfn2 in the control group, TGF-$\beta$1 model group, Mfn2-NC group and Mfn2-OE group. Values represent mean $\pm$ SD, n = 3 (ANOVA). (E,F) Western Blot was used to detect the protein expression of E-cadherin, N-cadherin, Vimentin and Mfn2 in the control group vs the TGF-$\beta$1 model group and the Mfn2 negative control group (Mfn2-NC) vs the Mfn2 overexpression group (Mfn2-OE). Values represent mean $\pm$ SD, n = 3 (ANOVA). *p 0.05, **p 0.01, ***p 0.001. TGF-$\beta$1, transforming growth factor-beta 1; EMT, epithelial-mesenchymal transition; ANOVA, analysis of variance.

Fig. 3.

Cell scratch assay and Transwell invasion assay. Functional verification: (A,C) cell scratch assay. Scale bar: 200 µm ($\times$100). (B,D) Transwell invasion assay showed that EMT enhanced migration and invasion, while Mfn2 overexpression inhibited this effect. Scale bar: 200 µm ($\times$100). Values represent mean $\pm$ SD, n = 3 (ANOVA). *p 0.05, **p 0.01, ***p 0.001.

To see if Mfn2 protects against adenomyosis in a living system, we used a mouse model of the disease. Mice with adenomyosis showed the expected problems: disrupted boundary between the endometrium and muscle layer, glands invading the muscle, and increased fibrosis (p 0.05). Giving these mice a control virus (LV-NC) didn’t help. However, using a virus to increase Mfn2 levels (LV-Mfn2) in the uterus significantly improved things: the tissue boundary was less disrupted, gland invasion was reduced, and fibrosis was lessened (p 0.05). We confirmed Mfn2 levels were indeed higher in the treated mice (p 0.05). Just like in the cells and patients, the diseased mice showed EMT activation (low E-cadherin (p 0.05), high N-cadherin, Vimentin) (p 0.05). Boosting Mfn2 reversed these EMT marker changes too (Figs. 4,5).

Fig. 4.

Related pathological changes in mouse uterus. (A) H&E staining of the uterus of mice in each group: Control, adenomyosis model (ADS), Mfn2 negative control (LV-NC), lentiviral Mfn2 overexpression (LV-Mfn2). H&E staining showed the endometrial-myometrial junction. Scale bar: 200 µm ($\times$60), 25 µm ($\times$300). (B,C) Masson staining of the uterus in each group. Masson staining revealed the deposition of collagen fibers in the uterine tissue (in blue). The percentage of collagen area was quantitatively analyzed using the ImageJ software. Scale bar: 200 µm ($\times$60). Values represent mean $\pm$ SD, n = 9 (ANOVA). ****p 0.0001. H&E, hematoxylin and eosin.

Fig. 5.

Method validation of relevant markers in mouse uterus by qPCR and WB. (A–D) qPCR: the expression of E-cadherin, N-cadherin, Vimentin and Mfn2 mRNA in the uterus of the control vs model group (ADS) and Mfn2 negative control (LV-NC) vs Mfn2 overexpression group (LV-Mfn2). Values represent mean $\pm$ SD, n = 9 (t-test). (E,F) Western Blot was used to detect the protein expression of E-cadherin, N-cadherin, Vimentin and Mfn2 in the uterus of each group. Values represent mean $\pm$ SD, n = 9 (ANOVA). *p 0.05, **p 0.01, ***p 0.001, ****p 0.0001.

Together, our results show Mfn2 levels are consistently low in adenomyosis across different systems. Increasing Mfn2 counteracts the EMT process, reduces the invasive potential of endometrial cells, and significantly improves disease features in mice. This strongly indicates that Mfn2 plays a protective role in adenomyosis by inhibiting EMT, making it a promising potential target for treatment.

This study provides the first evidence that Mfn2 critically inhibits adenomyosis progression by suppressing EMT. Through integrated clinical, cellular, and animal analyses, we consistently demonstrated significant Mfn2 downregulation in adenomyosis endometrium, which strongly correlated with aberrant EMT activation ($\downarrow$ E-cadherin/$\uparrow$ N-cadherin, $\uparrow$ Vimentin). Mechanistically, Mfn2 overexpression in TGF-$\beta$1-stimulated Ishikawa cells reversed EMT-driven cell invasion, while in vivo lentiviral delivery of Mfn2 restored uterine architecture and reduced fibrosis in tamoxifen-induced adenomyosis mice. Critically, unlike tumors, adenomyosis has no malignant transformation; thus, the anti-EMT effect of Mfn2 is primarily directed against tissue invasion and fibrosis rather than metastasis. This aligns with its documented role in suppressing pathological fibrosis in other organs, establishing Mfn2 as a novel therapeutic target for this benign gynecological disorder.

Taken together with our findings, we propose a comprehensive hypothesis that Mfn2 may suppress ROS burst and reverse pathological metabolic reprogramming by inhibiting mitochondrial homeostasis (fusion/fission), ultimately inhibiting EMT. The basic mechanism may include the following interrelated levels:

1. Regulation of mitochondrial morphology and function: as a key regulatory protein of mitochondrial outer membrane fusion, down-regulation of Mfn2 expression directly leads to excessive mitochondrial fission (fragmentation) [13, 23, 24]. The morphological defect impairs mitochondrial electron transport chain (ETC) function, leading to the overproduction of ROS. Overproduced ROS act as key signaling molecules, activating nuclear factor kappa B (NF-$\kappa$B) and hypoxia-inducible factor-1 alpha (HIF-1$\alpha$) signaling pathways [6]. The activation of these pathways also induces the phosphorylation of the TGF-$\beta$/Smad pathway, which eventually initiates the EMT process, which is manifested as the suppression of the expression of E-cadherin and the up-regulation of Vimentin and other mesenchymal markers.

2. Inhibition of metabolic reprogramming: Mitochondrial fragmentation not only leads to ROS accumulation (as described above), but also forces cells to forego efficient oxidative phosphorylation (OXPHOS) in favor of inefficient glycolysis (i.e., the Warburg effect) [25], a metabolic shift that is itself a hallmark feature of EMT. Overexpressed Mfn2 might restore the efficiency of OXPHOS by restoring the normal structure and function of mitochondrial cristae. At the same time, it is able to inhibit the activity of key glycolytic enzymes such as lactate dehydrogenase A (LDHA) [23, 26], effectively opposing the aberrant metabolic adaptation that EMT depends on.

3. Regulation of mitochondrial-endoplasmic reticulum coupling: In addition to regulating mitochondrial fusion, Mfn2 is also involved in the formation and maintenance of mitochondrial-endoplasmic reticulum contact sites (MERCs), which are important for calcium (Ca²⁺) signaling [27]. Calcium homeostasis disruption activates calcium-dependent proteases such as Calpain, which cleave E-cadherin and destabilize cell polarity, thereby inducing the EMT process. Therefore, Mfn2 could suppress the calpain-mediated EMT pathway through the stabilization of MERCs’ integrity and calcium signaling.

The major finding of the present study—that Mfn2 overexpression substantially suppressed TGF-$\beta$1-induced EMT and cell invasion—provides important experimental validation of this hypothesis. Of course, the precise molecular network of Mfn2 regulating EMT is yet to be completely elucidated. Follow-up studies can track the effect of Mfn2 on ROS levels by knocking down Mfn2 or, more directly, quantify the OXPHOS/glycolytic flux alternation with the assistance of metabolomics technology to more comprehensively map the role of Mfn2.

In summary, this study demonstrates for the first time that Mfn2 plays a vital protective role in adenomyosis by inhibiting the process of EMT, which introduces new ideas for clinical diagnosis and treatment of adenomyosis. Studies have found that the low expression mode of Mfn2 in the endometrium of patients shows that Mfn2 can be used as an effective auxiliary diagnostic biomarker, and if combined with serum EMT markers (such as Vimentin), it is expected to significantly improve the accuracy of disease staging and invasiveness. In the therapeutic field, therapies targeting Mfn2 have shown broad prospects. Small-molecule compounds (e.g., natural product derivatives) with selective activation of Mfn2 can be developed through high-throughput screening and combined with in utero sustained release systems to achieve lesion-precise intervention. Lentiviral or adeno-associated virus (AAV) vector-mediated gene therapy has been shown to improve pathological phenotypes in animal models. Maximizing delivery efficiency or creating tissue-specific promoters may further improve efficacy and reduce toxicity in the future. Of particular interest is whether the combination of Mfn2 activators with existing antifibrotic treatments, e.g., gonadotrophin-releasing hormone (GnRH) agonists, can enhance efficacy through synergistic mechanisms. However, it is still necessary to systematically tackle key problems to facilitate clinical translation: to clarify the correlation of Mfn2 expression with clinical stage and symptom severity, to stringently evaluate the in vivo safety and off-target risk of Mfn2 agonists, and to fully explore the mechanisms of interaction between Mfn2 regulating mitochondrial function and Wnt/$\beta$-catenin and other key EMT pathways. Overcoming these challenges will accelerate the process of Mfn2 from bench to bedside and provide ever more accurate and efficient therapeutic approaches for patients with adenomyosis.

This study confirms that Mfn2 is a key repressor of adenomyosis progression. We consistently demonstrated that Mfn2 is significantly downregulated in human adenomyosis endometrium, in a cell model of EMT, and in a mouse adenomyosis model. Functionally, restoring Mfn2 expression effectively reversed TGF-$\beta$1-induced EMT, suppressed endometrial cell migration and invasion in vitro, and, in a mouse model of adenomyosis, ameliorated key pathological features in vivo, including tissue invasion, fibrosis, and dysregulation of EMT markers. These results strongly suggest that Mfn2 plays a protective role in adenomyosis mainly by inhibiting the EMT process. Thus, Mfn2 holds promise as a novel diagnostic biomarker and therapeutic target for future interventions to mitigate this prevalent gynecologic disorder.

The data supporting the results of this study are included in this paper and its online Supplementary Material.

XW: Responsible for the conceptualization and design of this study, and experimented. NNX: Responsible for writing the first draft of the manuscript and improving the study design. XQ: assisted in the H&E staining of histological samples, immunohistochemical staining, and the scoring of their results. QH and CD: participated in the execution of cell culture, animal model establishment, and tissue sampling. HY: supervised the whole research process, guided the implementation of the study, and analyzed and interpreted the data. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

The Ethics Committee of Yichang Central People’s Hospital approved this human subject/tissue sample study (Approval No. [2024-252-01]). All subjects gave written informed consent in compliance with the Declaration of Helsinki. Anonymity and confidentiality of the patients were strictly maintained during the study. All animal procedures were conducted according to the ARRIVE Guidelines for the Care and Use of Laboratory Animals and were endorsed by the Experimental Animal Ethics Committee of China Three Gorges University (Protocol No. [2024070I]). All animal experiments complied with ethical guidelines and the principles of Replacement, Reduction, and Refinement (3Rs). Efforts were made to minimize animal suffering, including the use of anesthesia during surgical procedures and the application of humane endpoints for euthanasia.

This study was strongly supported by the Central Laboratory of Wujia Branch of Yichang Central People’s Hospital in terms of research sites and equipment, which is greatly appreciated. At the same time, we sincerely thank the Laboratory Animal Center of China Three Gorges University for providing professional assistance in the feeding and management of laboratory animals and the technical operation of gavage.

This study was supported by the Natural Science Foundation of Hubei Province (Joint Fund Project: 2024AFD189).

The authors declare no conflict of interest.

Supplementary material associated with this article can be found, in the online version, at https://doi.org/10.31083/CEOG46464.