Mechanistic insights into inflammatory cytokines in adenomyosis‑induced infertility (Review)

Adenomyosis (AM) is a gynecological disorder characterized by the infiltration of endometrial tissue into the myometrium, clinically manifesting as dysmenorrhea, menorrhagia and infertility ( 1 ). Epidemiological data indicate an incidence of ~21.9% ( 2 ), with 5-25% of cases occurring in women under the age of 39. Although advances in imaging techniques such as transvaginal ultrasound and magnetic resonance imaging (MRI) have significantly improved the diagnostic rate of AM in women of reproductive age ( 3 ), the increasing trend of delayed childbearing has made the disease a major contributing factor to secondary infertility, with chronic inflammatory response and immune dysregulation emerging as the core drivers linking the pathological progression of AM to reproductive impairment. Moreover, due to its insidious nature, early-stage and mild cases are often underdiagnosed, suggesting that the actual prevalence may be underestimated. Numerous studies have confirmed that women with infertility frequently present with sonographic features of AM and exhibit significant immune dysfunction ( 4 , 5 ). AM has increasingly become a critical factor impacting both female fertility and the outcomes of assisted reproductive technology (ART) ( 6 , 7 ). The abnormal infiltration of tissue-specific immune cell subsets, such as macrophages, natural killer (NK) cells and mast cells, combined with an imbalance between pro-inflammatory cytokines [such as interleukin (IL)-6, IL1B and C-X-C motif chemokine ligand 8 (CXCL8)] and anti-inflammatory cytokines (such as IL-4 and IL-10), contributes to pathological angiogenesis, impaired endometrial decidualization and reduced endometrial receptivity (ER). These changes ultimately lead to embryo implantation failure ( 8 ), thereby resulting in adverse pregnancy outcomes. Elevated levels of inflammatory markers in both serum and peritoneal fluid confirm that chronic inflammation and immune dysregulation are not isolated local phenomena but systemic disruptions of the immune-inflammatory network in AM ( 9 ). Therefore, elucidating the unique immunoregulatory network centered on chronic inflammation and immune dysregulation in AM, identifying diagnostic inflammatory cytokines and developing targeted intervention strategies are essential to improving reproductive outcomes in affected patients.

NSAIDs exert therapeutic effects in AM primarily by inhibiting COX activity and subsequently reducing prostaglandin synthesis ( 201 ). Studies have demonstrated that NSAIDs can decrease the depth of endometrial infiltration into the myometrium, inhibit the migration and invasion of ESCs and induce apoptosis ( 202 , 203 ). In addition, NSAIDs suppress key pathological processes such as fibrosis, angiogenesis and EMT ( 204 ). Therefore, NSAIDs are considered promising pharmacological candidates for the clinical management of AM. Celecoxib, a highly selective COX-2 inhibitor, significantly reduces inflammatory responses and the depth of lesion infiltration in EMs by suppressing the expression of pro-inflammatory cytokines such as IL1B, IL-6 and TNF-α, as well as the transcription factor NF-κB ( 205 ). Studies have suggested that in ART, localized inflammatory responses may impair embryo implantation by inducing abnormal uterine contractility and reducing ER via prostaglandin-mediated mechanisms ( 10 , 206 ). Theoretically, NSAIDs may improve ART outcomes by mitigating the detrimental effects of prostaglandins; however, this hypothesis requires further experimental validation ( 207 ). GnRH is a hypothalamic peptide hormone that acts on the anterior pituitary via the hypophyseal portal system to regulate the synthesis and pulsatile secretion of follicle-stimulating hormone and luteinizing hormone. A study by Zippl et al ( 89 ) reported no significant differences in endometrial immune characteristics between women with AM and those with recurrent pregnancy loss or RIF. The study also found that GnRH-a suppress the production of inflammatory cytokines such as TNF-α and IL1B by modulating the mononuclear phagocyte system, thereby inhibiting inflammation and angiogenesis. Similarly, Khan et al ( 208 ) demonstrated that GnRH-a treatment downregulates the secretion of cytokines (such as IL1B, MCP-1 and VEGF) from adenomyotic endometrial cells, attenuates the immune response of von Willebrand factor and improves the inflammatory microenvironment, ultimately enhancing ER. Furthermore, GnRH-a therapy has been shown to upregulate the expression of key receptivity markers in the endometrium, including HOXA10, HOXA11, integrin β3 and LIF, thereby promoting embryo implantation ( 209 ). Several case reports have confirmed successful pregnancies in previously infertile women with AM following GnRH-a treatment ( 210 , 211 ). Notably, the combination of GnRH-a with surgical intervention appears to yield superior outcomes. In a large prospective study, 55 out of 165 women with AM conceived after surgery alone or surgery followed by GnRH-a therapy, achieving a clinical pregnancy rate of 77.5%, with 69.0% delivering successfully by the end of a 2-year follow-up period ( 212 ). GnRH-As exert their effects by competitively blocking GnRH receptors in the pituitary gland, thereby directly inhibiting the secretion of gonadotropins. Compared with GnRH-a, GnRH-As offer the advantage of avoiding the initial 'flare-up effect' (a transient surge in gonadotropins at the onset of treatment) and allow for a more rapid recovery of ovarian function after drug withdrawal, making them particularly suitable for patients desiring fertility preservation ( 213 ). Currently available GnRH-As include elagolix, relugolix and linzagolix. A clinical study has shown that the administration of GnRH-A prior to ART procedures or surgical intervention may improve embryo transfer outcomes and increase surgical success rates ( 209 ). Lessey et al ( 214 ) demonstrated that treatment with elagolix for 2 months led to the upregulation of several anti-inflammatory mediators, including IL-10, IL-13, CCL18 and TWIST2. Simultaneously, miRNA-mediated downregulation of inflammatory components such as NOTCH1, NF-κB, T cells and NK cells was observed, suggesting that elagolix significantly modulates the systemic inflammatory milieu. Moreover, elagolix has been associated with higher pregnancy and live birth rates compared with oral contraceptive pills, although further research is warranted to confirm these findings ( 215 ). DNG is a progestin with high selectivity for PRs that exerts mild ovulation-inhibiting effects; it can directly suppress the proliferation of AM stromal cells and induce apoptosis. As the primary target of DNG, the activation of the PR also regulates inflammation in the endometrium ( 216 ). A prospective study confirmed that dienogest and danazol can alleviate the production of TNF-α-induced IL-6 and CXCL8 in endometriotic stromal cells by inactivating NF-κB ( 217 ). Oral administration of dienogest also increases the number of infiltrating NK cells in ectopic endometrial glands, benefiting pregnancies that occur after cessation of treatment in terms of embryo implantation and fetal protection ( 218 ). Immunomodulators represent a promising therapeutic strategy for preserving fertility as they target inflammatory responses, modulate the immune microenvironment and inhibit the growth of ectopic lesions. Most notably, these agents can improve tubal patency, enhance oocyte quality and restore the pelvic microenvironment without interfering with estrogen secretion or ovulatory cycles, making them an optimal choice for patients seeking to retain reproductive potential ( 219 ). A study has demonstrated that tocilizumab, an IL-6 receptor antagonist, can effectively inhibit ectopic lesion cell proliferation and migration induced by endometrial cell-derived exosomes ( 220 ). Tocilizumab induces G1/S cell cycle arrest and significantly suppresses activation of the IL-6/JAK2/STAT3 signaling axis, thereby exerting potent anti-inflammatory and antiproliferative effects ( 25 ). AMY109, a long-acting circulating antibody targeting CXCL8, has shown promising antitumor activity by reducing fibrosis and immunosuppression in cancer models ( 221 ). In a non-human primate model of EMs, AMY109 significantly reduced the volume of nodular lesions and improved fibrosis and adhesions; its mechanism is associated with the inhibition of neutrophil recruitment and MCP-1 production, and notably, it does not impair ovarian function ( 222 ). The classic antidiabetic drug metformin has demonstrated therapeutic potential in AM ( 223 ); it exerts beneficial effects by modulating oxidative stress, inflammation and apoptosis. Specifically, metformin downregulates pro-inflammatory cytokines (including IL-6 and CXCL8), angiogenic factors (including VEGF and MMP-9), and simultaneously upregulates LIF and HOXA10, thereby enhancing ER and supporting implantation ( 224 ). A clinical study has confirmed that the combination of high-intensity focused ultrasound and metformin significantly reduces uterine volume and lowers recurrence rates in AM, largely due to the suppression of inflammatory mediators ( 225 ). Anakinra, an IL-1 receptor antagonist that competitively binds to IL-1R1, has been shown in a clinical study to significantly reduce visual analogue scale (VAS) pain scores and improve quality of life in patients with EMs, without adversely affecting ovulatory function or menstrual regularity during treatment ( 226 ). Lipoxin A4, an anti-inflammatory and pro-resolving lipid mediator, significantly reduces the size of endometriotic lesions by downregulating pro-inflammatory cytokines (including IL1B and IL-6) and inhibiting the expression of COX-2 and PGE2 in both lesions and peritoneal fluid cells ( 227 ). Additionally, it suppresses the expression of angiogenic factors (such as VEGF, HIF-1α and MMP-9), thereby inhibiting neovascularization, and regulates TGF-β isoform expression, reducing adhesion and invasive properties of endometriotic cells ( 228 ). These effects collectively contribute to lesion regression and potential improvements in fertility. Disulfiram, a derivative of thiamine, has recently been shown to exert anti-endometriotic effects. An animal study has demonstrated that disulfiram significantly reduces serum TNF-α and IL1B levels and inhibits angiogenesis within ectopic lesions by downregulating VEGF-A and MMP-2 expression via suppression of NF-κB nuclear translocation ( 229 ). Moreover, disulfiram alleviates postoperative inflammatory adhesions associated with EMs, thereby helping to prevent infertility caused by adhesion formation ( 230 ). Animal experiments have also shown that melatonin exerts therapeutic effects in AM by blocking the NF-κB signaling pathway. Melatonin downregulates ROS and pro-inflammatory cytokines such as TNF-α and IL1B, while upregulating VEGF, thereby improving the endometrial microenvironment, reducing apoptosis of endometrial cells and restoring ER. These effects ultimately improve pregnancy outcomes in AM mouse models ( 231 ). Furthermore, melatonin alleviates uterine oxidative stress and improves uteroplacental hemodynamics, creating a favorable environment for embryo implantation and development ( 232 ). Etanercept, a TNF inhibitor, effectively blocks TNF-α/β-mediated NF-κB activation ( 233 ). Experimental studies have shown that administration of etanercept in baboon models of EMs significantly reduces both the number and volume of endometriotic lesions ( 234 ). In mouse models, treatment with etanercept results in reduced lesion size along with downregulation of VEGF, IL-6 and TNF-α levels ( 235 ). A retrospective study involving 68 infertile women with EMs reported that a single intramuscular injection of 50 mg etanercept on day 2 of the menstrual cycle in the treatment group (n=19) led to improved clinical pregnancy and embryo implantation rates ( 236 ). Pentoxifylline, a non-selective phosphodiesterase inhibitor, has been shown to suppress the secretion of pro-inflammatory cytokines such as TNF-α and IL1B, while also downregulating key angiogenic factors ( 237 ). A randomized controlled trial confirmed that postoperative treatment with pentoxifylline significantly reduced VAS pain scores and improved long-term outcomes in patients with EMs ( 238 ). Another randomized controlled trial demonstrated that in patients with mild or asymptomatic EMs, the pregnancy rate in the pentoxifylline-treated group was twice as high as that in the control group ( 239 ). Intralipid, an intravenous lipid emulsion derived from soybean polyunsaturated fatty acids and egg yolk phospholipids, has gained significant attention in reproductive medicine due to its immunomodulatory properties. Basic research indicates that intralipid exerts broad anti-inflammatory and immune homeostasis effects by specifically suppressing NK cell activity ( 240 ). In patients with RIF complicated by NK cell hyperactivity, intralipid treatment has been associated with live birth rates ranging from 33 to 42%, while clinical observations in patients with recurrent pregnancy loss report even higher live birth rates of 75 to 91%, markedly improving adverse pregnancy outcomes ( 241 ). Consequently, lipid-based intralipid administration could be considered as a novel delivery route for patients with AM ( 242 ). Recent advances in nanoparticle-based precision diagnostics and therapeutics have shown breakthrough potential. A digital droplet enzyme-linked immunosorbent assay system constructed with SiO 2 nanoparticles achieved femtomolar sensitivity for detecting inflammatory markers such as OPN, IL-6 and IL-10 in menstrual blood, unveiling for the first time the molecular-level immune-inflammatory signatures in patients with EMs and providing reliable non-invasive biomarkers ( 243 ). Furthermore, 10 nm magnetic iron oxide nanoparticles enable in situ visualization and localization of ectopic lesions via MRI, markedly enhancing diagnostic accuracy ( 244 ). Outer membrane vesicle-modified poly (lactic-co-glycolic acid) nanoparticles have been developed to deliver IL-4 plasmids specifically to macrophages, successfully reversing M2 polarization and markedly inhibiting lesion progression without compromising ovarian reserve ( 245 ). In addition, cerium dioxide nanoparticles have been shown to reduce oxidative stress and inhibit angiogenesis, thereby alleviating induced endometrial lesions in mouse models while preserving both the quantity and quality of oocytes ( 246 ). miRNAs, small ncRNAs that suppress target gene translation by binding mRNAs, regulate angiogenesis and inflammatory responses during EMs pathogenesis. Elevated levels of angiogenic and inflammatory factors such as VEGF-A, IL-6 and CXCL8 in ectopic lesions are closely linked to aberrant activation of miRNA regulatory networks, including miR-21 and miR-135b ( 247 ). Notably, miR-199a targets and suppresses IKKβ in ESCs, inhibiting NF-κB activation and CXCL8 production, thereby attenuating ESC adhesion, migration and invasiveness ( 248 ), miR-25-3p, delivered via EMs-derived extracellular vesicles (EMs-EVs), targets the PTEN/AKT pathway, inducing collagen deposition and inhibiting decidualization, ultimately decreasing ER ( 220 ). The U.S. FDA has approved RNA interference therapeutics such as patisiran, which employs lipid nanoparticles to silence pathogenic RNAs in lesions ( 249 ). Additionally, exosomes have been explored as natural carriers for therapeutic ncRNAs (such as miR-141-3p or let-7b), enabling targeted delivery to ectopic lesions and enhancing local uptake ( 250 ). Patients with AM exhibit aberrant expression of 21 fertility-related miRNAs in EMs-EVs secreted by the endometrium, potentially underlying infertility mechanisms ( 251 ). Small-molecule drugs targeting EMs-associated miRNAs (such as inhibitors of miR-135 or miR-196) have entered phase II clinical trials, highlighting ncRNA-based therapies as promising novel strategies for fertility preservation in patients with EMs. The AI-powered drug target discovery platform PandaOmics ( https://pandaomics.cn/access ) has recently made breakthrough progress in EMs research. Through intelligent algorithmic screening, the platform identified two novel immune-regulatory therapeutic targets, guanylate binding protein 2 Gene (GBP2) and hemopoietic cell kinase (HCK). Key validation experiments demonstrated that gene silencing of GBP2 or HCK significantly inhibited EMs cell proliferation and induced apoptosis, confirming their potential as therapeutic targets ( 252 ). Therapeutically, outer membrane vesicle-functionalized poly(lactic-co-glycolic acid) nanoparticles precisely targeting macrophages modulate the immune microenvironment, markedly suppressing lesion progression without impairing ovarian reserve, offering a novel non-hormonal treatment option for reproductive-aged patients ( 245 ). Comprehensive TCM therapies, including oral herbal formulations, acupuncture and topical applications, have been shown to reduce inflammatory cytokine levels and demonstrate unique advantages in the diagnosis and treatment of AM. For instance, Shaoyao-Gancao decoction and its active ingredient paeoniflorin significantly decrease PGE2 and PGF2α levels, alleviating AM-associated dysmenorrhea ( 253 ). Resveratrol, a polyphenolic compound, has both anti-inflammatory and antioxidant properties. Animal studies have demonstrated that it can effectively inhibit myometrial infiltration and alleviate pain-related characteristics without disrupting hormonal homeostasis ( 254 ). Clinical trials have demonstrated that a daily dose of 30 mg resveratrol combined with oral contraceptives significantly reduces the expression of COX-2 and aromatase expression, potentially via suppression of prostaglandin biosynthesis pathways, thereby diminishing the invasive potential of ectopic endometrial tissue ( 255 ). Oleuropein, a novel natural product, selectively inhibits ERβ activity without affecting ERα. As ERβ can activate the NLRP3 inflammasome, potentiate IL-1β-mediated proliferation and modulate signaling cascades such as TNFα/NF-κB, ROS and IL-6/JAK/STAT3, selective inhibition by oleuropein may impede disease progression ( 256 ). Experiments have further indicated that oleuropein restores defective endometrial decidualization and attenuates excessive inflammatory responses, ultimately improving pregnancy rates in EMs models ( 257 ). Quercetin, a natural flavonoid polyphenol, exhibits antioxidant, anti-inflammatory, antiproliferative and antitumor activities; it dose-dependently suppresses the migration and invasion of ectopic ESCs by downregulating invasion-related proteins including Fascin, Ezrin and MMP-2/MMP-9 ( 110 ), aligning with the natural bioactive compound-based interventions featured in the multi-modal therapeutic framework of Fig. 6 . Furthermore, sodium tanshinone IIA sulfonate inhibits platelet aggregation and suppresses the TGF-β1/Smad3 and NF-κB signaling pathways, thereby attenuating fibrosis ( 258 ). Oral emodin dose-dependently inhibits IL1B-induced NF-κB expression and Wnt/β-catenin signaling activation to treat AM ( 109 ). Berberine significantly reduces LPS-induced endometrial invasion and downregulates IL-6 and CXCL8 expression, promoting apoptosis of proliferative ESCs ( 259 ). Collectively, a growing body of evidence supports these findings. Guided by TCM principles of syndrome differentiation and individualized therapy, patients can achieve symptomatic relief and improved fertility, with robust clinical efficacy. These TCM-derived bioactive components are included in Table SII , which comprehensively summarizes the full spectrum of medications for AM treatment. These agents target inflammatory cytokines while preserving female reproductive capacity.

AM is commonly regarded as a chronic inflammatory immune disease. We consider that chronic inflammation acts as the upstream inducer of immunological abnormalities in AM, with the subsequent imbalance between proinflammatory and anti-inflammatory signals, serving as a downstream pathological consequence; together, this cascade constitutes the critical core factors affecting fertility in affected women. Notably, this pathogenic process originates from persistent TIAR at the EMJ: Repeated menstrual microtrauma at the EMJ triggers sustained release of proinflammatory cytokines, which in turn initiates chronic inflammation. This chronic inflammatory milieu further induces EMT in eutopic endometrial cells, enhancing their invasiveness to infiltrate the myometrium, while simultaneously disrupting local immune homeostasis to cause immune dysregulation, establishing a reciprocal amplification loop between inflammation and immune dysfunction that underpins AM pathogenesis. Inflammatory cytokines, key immunological markers, abnormally accumulate in endometrial lesions, serum and peritoneal fluid and play a central role in AM-related infertility. Driven by chronic inflammation and synergized by immune dysregulation, their dysregulated expression exerts multi-stage reproductive impairment. On the one hand, they sustain an abundant blood supply to ectopic lesions by promoting neovascularization and the inflammatory changes at the EMJ (exacerbated by EMT-mediated tissue remodeling) lead to reduced ER and impaired decidualization. On the other hand, immune dysregulation disturbs the maternal-fetal immune tolerance required for embryo implantation. Together, these effects ultimately hinder embryo implantation, causing infertility, IVF failure and recurrent pregnancy loss. In addition, disturbances in inflammatory cytokine levels are associated with adverse immune environments during childbirth and postpartum, contributing to risks such as preterm birth, postpartum hemorrhage and premature rupture of membranes. Other symptoms of AM, including menorrhagia and dysmenorrhea, are also rooted in this chronic inflammation-immune dysregulation cascade, as proinflammatory factors sensitize pain pathways and disrupt endometrial hemostasis. At present, the clinical diagnosis of AM relies primarily on non-invasive methods, such as transvaginal ultrasound or MRI, with CA-125 as the only routinely measured biomarker. However, CA-125 has limited diagnostic potential, with insufficient sensitivity for detecting early and mild AM, and lacks the capacity for timely diagnosis and prevention of chronic pain and infertility associated with AM ( 260 ). Therefore, the development of novel serum biomarkers is essential for patients with AM who have long-term fertility plans. Research has demonstrated that inflammatory cytokines possess diagnostic sensitivity and specificity ( 261 ), with IL-6, CXCL8 and IL1B showing the most notable potential for clinical translation. Serum IL-6 is a reliable marker for the early diagnosis of infertility related to EMs. CXCL8 may serve as a specific predictor for evaluating embryo and oocyte quality in patients with AM undergoing IVF and can independently assess pretransfer embryonic developmental potential ( 238 ). Elevated IL1B levels may also act as a highly sensitive and specific potential indicator for early AM diagnosis ( 262 ). Additionally, the neutrophil-to-lymphocyte ratio in peripheral blood has been recognized as a valuable marker for infertility in women with EMs, while increased levels of anti-inflammatory cytokines, such as IL10, IL4 and IL-1RA, have been linked to the disease prognosis ( 263 ). Endometrial autoantibodies also offer a novel, non-invasive diagnostic approach ( 264 ). However, to enable effective diagnosis or treatment of AM based on inflammatory cytokines, high-sensitivity cytokine arrays and multivariable statistical analyses (accounting for inter-individual phenotypic variability) are necessary, highlighting the need for optimized diagnostic tools. When multiple biomarkers are evaluated, multivariable statistical analyses are needed, with careful consideration of the phenotypic variability of EMs across different individuals. Thus, further advancements in diagnostic tools and techniques are crucial. The present review also discussed recent advances in pharmacological therapies targeting inflammatory cytokines such as IL1B, IL-6, CXCL8, IL-10, NF-κB and COX-2. As treatment strategies for AM often depend on the specific patient's symptoms, a number of medications effective for dysmenorrhea and menorrhagia may compromise fertility, with some significantly compromising reproductive function ( 242 ). Therefore, prior to treatment, individualized assessment of the inflammatory and immune status of the patient is essential for determining the most appropriate therapeutic approach, which may support the development of more effective, safer management options for AM-associated infertility. Future research should focus on three specific priorities to advance the field: First, large-scale prospective studies should be conducted to validate the diagnostic cut-off values of IL-6 and IL1B for early AM and AM-related infertility and establish combined biomarker panels to improve diagnostic accuracy; second, clinical trials should be launched to evaluate the efficacy of IL-6-targeted therapies (such as tocilizumab) for improving IVF implantation rates in patients with AM and explore the synergistic effects of combining NF-κB inhibitors with IL1B antagonists; third, the crosstalk mechanism between chronic inflammation-induced EMT and immune dysregulation at the EMJ should be clarified, to identify new intervention nodes that simultaneously target lesion invasion and immune tolerance for fertility preservation. In conclusion, chronic inflammation-driven immune dysregulation is the core pathogenic link underlying AM-related reproductive impairment and IL-6, CXCL8 and IL-1β represent the most promising targets for diagnostic optimization and therapeutic innovation. Further research into these directions will provide critical support for improving reproductive outcomes in patients with AM.

The present review summarizes the role of inflammatory cytokines in the pathogenesis of AM-related infertility, particularly highlighting that the pathological progression and fertility impairment of AM are largely dependent on the regulation of inflammatory cytokines. These cytokines directly or indirectly affect the fertility of patients with AM by modulating multiple signaling pathways to regulate critical physiological processes, including cell proliferation and invasion, EMT, TIAR, angiogenesis and ER. Given their irreplaceable and core role in disease progression, these inflammatory cytokines hold great promise as important biomarkers for the early diagnosis and targeted treatment of AM-related infertility. However, current research on their clinical application value is still in the exploratory stage. Further studies are needed to conduct targeted investigations to further explore their potential in optimizing diagnosis methods and therapeutic strategies.