FOXA2 loss results in an increase of endometriosis development and LIF reveals a therapeutic effect for endometriosis

We previously identified FOXA2 as a critical transcription factor essential for the development of endometrial glands, normal uterine function, and fertility ( 9 , 15 ). Clinical evidence shows that FOXA2 expression is significantly lower in the eutopic endometrium of endometriosis patients compared to disease-free controls ( 17 ). One month after endometriosis induction, we confirmed the development of endometriotic lesions, yet fertility—including implantation and decidualization—remained unaffected ( 23 ). By three months post-induction, however, mice with endometriosis exhibited implantation failure and defective decidual responses compared to control and sham groups ( 23 ). These findings indicate that endometriosis leads to implantation failure and impaired decidualization over time. Our mouse model closely recapitulates the implantation and decidualization defects observed in humans ( 21 , 28 , 29 ), making it a valuable tool for investigating the impact of endometriotic lesions on endometrial gene expression and implantation. To examine the expression profile of FOXA2 and its target protein during endometriosis progression, we performed IHC for FOXA2 and CXCL15 at one and three months after endometriosis induction.. Our IHC results revealed a significant decrease ( p < 0.05) in FOXA2 expression in the eutopic endometrium of endometriosis-induced mice compared to sham controls at three-months post-surgery ( Figure 1 ). These findings, along with prior human data ( 17 ), suggest that endometriosis results in downregulation of FOXA2 in eutopic endometrium. Notably, although the uteri of adult FOXA2-deleted mice ( Ltf iCre/+ Foxa2 f/f ) appeared morphologically normal and contained glands, several gland-specific genes, including chemokine C-X-C motif ligand 15 (Cxcl15), were not expressed ( 10 ). Consistent with the reduction in FOXA2, CXCL15 expression was also significantly decreased in the eutopic uteri of endometriosis-induced mice at both 1- and 3-months post-surgery ( p < 0.05 and p < 0.01, respectively) compared to sham controls ( Figure 2 ). To further examine the effect of FOXA2 loss in the development of endometriosis, we performed autologous endometriosis surgery on both Foxa2 d/d Rosa26 mTmG/+ and control mice, followed by examining ectopic lesion development for one month. Foxa2 d/d Rosa26 mTmG/+ mice developed significantly more ectopic lesions ( p < 0.01) and had a higher cumulative lesion weight ( p < 0.01) compared to controls ( Figure 3 ). These results provide strong evidence that FOXA2 plays a protective role in suppressing lesion formation and limiting the progression of endometriosis, emphasizing its importance in maintaining normal uterine function. The role of PGR is critical in the pathophysiology of endometriosis, as disruptions in its activity result in altered steroid signaling, leading to P4 resistance, impaired decidualization, and the persistence of ectopic lesions ( 21 , 30 ). P4 resistance in endometriosis patients might be due to decreased responsiveness of PGR, its chaperone immunophilins, and deregulated P4-response genes in the eutopic endometrium ( 20 ). Notably, FOXA2 expression is regulated by P4 in the mouse uterus ( 22 ). Therefore, we examined PGR expression in both eutopic uteri and ectopic lesions of Foxa2 d/d Rosa26 mTmG/+ and control mice with endometriosis. Our findings demonstrate that epithelial PGR expression is significantly reduced in both tissues of Foxa2 d/d Rosa26 mTmG/+ mice compared to controls ( p < 0.001) ( Figure 4 ), suggesting a potential link between FOXA2 deficiency and impaired P4 signaling in endometriosis. Given the essential role of PGR in steroid signaling and decidualization ( 21 ), these results indicate that FOXA2 loss may contribute to P4 resistance, exacerbating endometriosis pathophysiology. To confirm the histological identity of the ectopic lesions, we performed immunohistochemical staining for vimentin and E-cadherin, which revealed well-defined endometrial glands and stromal layers, resembling the eutopic uterus ( Figure S1 ). This supports the characterization of these structures as bona fide endometriotic lesions ( 31 ). To determine whether the altered PGR expression was exclusively due to FOXA2 loss during endometriosis, we next evaluated PGR expression in the eutopic uteri of control and Foxa2 d/d Rosa26 mTmG/+ mice without endometriosis at GD 3.5. Importantly, the expression of PGR was not significant different between control and Foxa2 d/d Rosa26 mTmG/+ mice ( Figure S2 ). This finding indicates that Foxa2 d/d Rosa26 mTmG/+ mice do not alter PGR expression in the uterus during early pregnancy. Together, our findings highlights the role of FOXA2 in modulating endometriosis severity and its potential impact on PGR-mediated progesterone responsiveness. Foxa2 d/d Rosa26 mTmG/+ mice exhibited impaired fertility attributed to failed implantation and decreased expression of LIF on GD 3.5 ( 9 ). Of particular interest, LIF treatment successfully rescued embryo implantation in these mice ( 10 ). To determine the impact of LIF on ectopic lesion establishment and growth, Foxa2 d/d Rosa26 mTmG/+ mice, following autologous endometriosis surgery, received either vehicle or 0.5 mg/kg recombinant LIF for one month ( Figure 5A ). Evaluation of ectopic lesions revealed a significant reduction in both lesion formation and associated radiant efficiency in the LIF-treated group compared to the vehicle-treated mice ( p < 0.001) ( Figure 5B ). These findings suggest that the increased lesion development in Foxa2 d/d Rosa26 mTmG/+ mice is due to the absence of LIF, underscoring its therapeutic potential in addressing endometriosis in FOXA2-deficient mice.

All animal procedures were approved by the University of Missouri’s Animal Care and Use Committee. Mice were housed with no more than five per cage and bred in a dedicated facility with controlled temperature, humidity, and a 12-hour light/dark cycle to maintain optimal conditions. Food and water were provided ad libitum. Pgr cre/+ Foxa2 f/f Rosa26 mTmG/+ ( Foxa2 d/d Rosa26 mTmG/+ ) mice ( 9 ) were used to examine the effect of FOXA2 lose on endometriosis. The detailed procedure for inducing endometriosis has been thoroughly described in our previous work ( 23 ). In brief, endometriosis was surgically induced in Pgr cre/+ Rosa26 Luc/mTmG mice by transplanting either autologous or syngeneic endometrial tissue into the peritoneal cavity. To investigate the role of FOXA2 in the development and progression of endometriosis, syngeneic endometriosis surgery was performed in 8-week-old Pgr cre/+ Rosa26 Luc/mTmG mice. A sham group receiving PBS without endometrial tissue served as the control. Samples were collected at one- and three-months post-surgery to analyze FOXA2 expression in the eutopic uteri of endometriosis and sham groups (n=5 per group per time point). Additionally, CXCL15 expression in the eutopic uteri was evaluated at both time points and compared between sham and endometriosis mice to assess its relationship with endometriosis progression (n=5 per group). To further examine the impact of FOXA2 loss on endometriosis development, autologous endometriosis was performed in 8-week-old Foxa2 d/d Rosa26 mTmG/+ and control ( Pgr cre/+ Rosa26 Luc/mTmG ) mice, allowing lesions to develop for one month ( n = 5 for the control group, n = 4 for the Foxa2 d/d Rosa26 mTmG/+ group). In each case, endometriotic lesions were identified, counted, and removed using a Nikon fluorescent dissection microscope ( 24 ). Uterine samples from pregnant mice at gestational day 3.5 (GD 3.5) were collected by mating control and Foxa2 d/d Rosa26 mTmG/+ females (n = 5 per group) with C57BL/6 males. The presence of a vaginal plug was recorded as GD 0.5, and tissue collection was performed at GD 3.5. Pregnancy was confirmed by embryo flushing on the morning of GD 3.5, ensuring the presence of blastocysts in the uterus ( 25 ). For all cases, uterine tissues were either flash-frozen immediately after dissection or fixed in 4% paraformaldehyde, then paraffin-embedded for further analysis. To evaluate the effects of LIF treatment on endometriosis, 8-week-old Foxa2 d/d Rosa26 mTmG/+ mice underwent autologous endometriosis surgery and were assigned to two groups: one treated with vehicle and the other with recombinant mouse LIF (0.5 mg/kg) via intraperitoneal injection, administered three times per week for four weeks (n=5 per group). At the end of the treatment period, ectopic lesions were identified and collected using fluorescence-guided dissection with a Nikon fluorescent dissection microscope. Afterward, further imaging of ectopic lesions was conducted using the in vivo imaging system (IVIS, PerkinElmer, Waltham, MA) to measure and compare the relative GFP signal intensity, ensuring precise assessment of lesion progression. Lesion sizes and outcomes were then compared between the vehicle- and LIF-treated groups. Immunohistochemical analysis was performed following previously established protocols ( 23 , 26 ). Briefly, uterine sections from paraffin-embedded tissue were cut at a thickness of 5 μm, mounted on silane-coated slides, then deparaffinized and rehydrated using graded series of alcohols. Afterward, the sections were blocked using 10% normal goat serum in PBS (pH 7.5) and incubated overnight at 4°C with primary antibodies diluted in the same blocking solution. The antibodies used were anti-FOXA2 (1:20,000 dilution, WRAB-1200, Seven Hills BioReagent), anti-CXCL15 (1:2000 dilution, ab197016, Abcam), and anti-PGR (1:500 dilution, 8757S, Cell Signaling). The following day, sections were incubated with species-specific HRP-conjugated secondary antibodies for 1 hour at room temperature: anti-mouse (1:500 dilution, BA-9200, Vector Laboratories) and anti-rabbit (1:500 dilution, BA-1000, Vector Laboratories). Immunoreactivity was detected using the Vectastain Elite DAB kit (Vector Laboratories), producing a brown staining pattern for the positive cells. Visualization was carried out using the PhenoImager HT system (Akoya Biosciences Inc.), with whole-slide viewing facilitated by Phenochart 2.1.0 software (Akoya Biosciences Inc.). To assess staining intensities, the H-score system was applied. The H-score was calculated using the equation: H-score = ∑ Pi, where the intensity of staining was assigned a value of 0 (no staining), 1 (weak), 2 (moderate), or 3 (strong), multiplied by the percentage of cells at each intensity ( 27 ). The H score ranged from 0 to 300. To evaluate the statistical significance of parametric data, we applied appropriate tests based on the number of groups being compared. For comparisons between two groups, Student’s t -test was used, while one-way ANOVA followed by Tukey’s post hoc multiple comparison test was employed for comparisons involving three or more groups. All statistical analyses were conducted using GraphPad Prism version 10 (GraphPad Software, San Diego, CA). A p -value of less than 0.05 ( P < 0.05) was considered statistically significant, indicating a significant difference between the groups.

In this study, we revealed that endometriosis results in attenuation of FOXA2 in eutopic endometrium and determined the effect of FOXA2 loss in endometriosis. At the molecular level, the loss of FOXA2 disrupts endometrial steroid hormone signaling and impairs the function of key glandular proteins. These dysfunctions likely contribute to more aggressive ectopic lesion formation, exacerbating the severity of endometriosis. Notably, LIF treatment significantly reduced ectopic lesion formation, thereby offering a promising therapeutic approach for endometriosis. FOXA2 plays a crucial role in the development and function of endometrial glands ( 9 , 10 ). Mice with uterine-specific ablation of Foxa2 ( Pgr cre/+ Foxa2 f/f Rosa26 mTmG/+ ) fail to develop endometrial glands, retaining only luminal epithelium ( 9 ). Additionally, FOXA2-deficient mouse models are infertile due to defects in embryo attachment and lack of LIF expression on GD 4 ( 10 , 32 ). In our study, we observed a marked reduction in FOXA2 expression in the eutopic uteri of endometriosis mice compared to sham controls. This downregulation coincided with a significant decrease in CXCL15, a chemokine and secreted protein reported to play a crucial role in gland function ( 15 ). CXCL15 is known to regulate immune responses and maintain epithelial integrity ( 33 ). The loss of FOXA2, along with the reduction in CXCL15, underscores the importance of glandular proteins in preserving uterine health and preventing the pathological changes associated with endometriosis. Our findings suggest that FOXA2 downregulation may drive the progression of endometriosis, as impaired glandular function likely supports the survival and growth of ectopic lesions. While this study primarily examines FOXA2 and CXCL15 changes in the eutopic endometrium, a temporal evaluation of ectopic lesions is essential to determine whether these alterations are specific to the eutopic environment or indicative of broader disease-associated changes in ectopic lesions. A key finding in this study is that FOXA2 deficiency leads to increased ectopic lesion formation in endometriosis. In our autologous endometriosis model, Foxa2 d/d Rosa26 mTmG/+ mice developed significantly more ectopic lesions and had a higher cumulative lesion weight compared to control mice. These results partially align with prior research demonstrating that FOXA2 plays a protective role in uterine function ( 10 ), including the suppression of endometrial carcinogenesis ( 34 ). The increased ectopic lesion formation in FOXA2-deficient mice suggests that FOXA2 may regulate inflammatory and immune responses that drive ectopic tissue growth ( 35 – 37 ). Indeed, FOXA2 mutations are present in 10% of endometrial cancers ( 38 ), suggesting that the loss of FOXA2 may lead to a more favorable environment for the establishment and survival of ectopic endometrial cells ( 22 ). One mechanism for survival and progression be the promotion of epithelial-to-mesenchymal transition (EMT), a highly conserved cellular process that allows polarized and generally immotile epithelial cells to convert to motile mesenchymal cells in response to injury ( 39 ). EMT has been implicated in the progression of experimental ectopic lesions development in baboons ( 40 ). FOXA2 expression has been found to inhibit EMT in studies of human breast cancer and endometrial cancer ( 34 , 41 ), and it is possible that loss of expression may contribute to EMT in ectopic lesions, leading to increased invasiveness and survival. These findings highlight the critical role of FOXA2 in maintaining uterine health and that targeting FOXA2 pathways could be a therapeutic strategy to limit lesion formation in endometriosis patients. P4 resistance is a hallmark of endometriosis, and disruptions in PGR signaling are thought to contribute to the persistence of ectopic lesions ( 42 – 44 ). We found that PGR expression was significantly reduced in both eutopic uteri and ectopic lesions of Foxa2 d/d Rosa26 mTmG/+ mice with endometriosis. Interestingly, there was no difference in PGR expression between control and Foxa2 d/d Rosa26 mTmG/+ mice without endometriosis, suggesting that FOXA2 deficiency specifically impairs PGR signaling in the context of endometriosis. As FOXA2 is not expressed in the luminal epithelium ( 9 , 10 ), its effect on luminal epithelial PGR expression is likely indirect, underscoring the importance of gland-specific PGR analysis for more precise insights. However, due to the uterine gland development defects observed in Foxa2 d/d Rosa26 mTmG/+ mice ( 10 ), assessment of glandular PGR expression in FOXA2-deleted mice was not feasible in this study. Altogether the reduction in PGR expression in FOXA2-deficient mice may contribute to P4 resistance, which is commonly observed in endometriosis patients ( 21 , 44 , 45 ). P4 resistance impairs the decidualization process ( 46 ), which is critical for normal uterine function and the prevention of ectopic lesion growth ( 47 ). This finding highlights the importance of FOXA2 in regulating PGR expression and suggests that FOXA2 loss may exacerbate the hormonal imbalances seen in endometriosis. Given the essential role of FOXA2 in regulating uterine glands and maintaining normal uterine function, we explored the therapeutic potential of LIF, a key implantation factor secreted by the glands. In our study, recombinant LIF administration significantly reduced ectopic lesion formation in Foxa2 d/d Rosa26 mTmG/+ mice. This suggests that the increased lesion formation in FOXA2-deficient mice may be partially due to decreased LIF secretion. Of note, Zutautas et al . ( 48 ) demonstrated in a murine model of endometriosis that LIF treatment over the course of two weeks alters the local peritoneal immune response, leading to the prevention of ectopic tissue growth. These findings provide strong evidence that LIF supplementation could be a viable therapeutic approach for mitigating the effects of FOXA2 deficiency in endometriosis. By restoring LIF levels, it may be possible to counteract the glandular dysfunction and P4 resistance associated with FOXA2 loss. In contrast, LIF administration has been shown to promote tumor growth and vascularization in cancer ( 48 ), future studies should investigate the long-term efficacy of LIF treatment in reducing lesion formation and improving fertility outcomes in endometriosis patients with FOXA2 deficiencies. In conclusion, our study demonstrates that FOXA2 plays a critical role in suppressing ectopic lesion formation and maintaining normal uterine function. FOXA2 downregulation in endometriosis leads to disrupted glandular protein expression, reduced PGR signaling, and increased ectopic lesion formation. The therapeutic potential of LIF in mitigating these effects highlights the importance of glandular secretions in the pathogenesis of endometriosis and offers a promising avenue for endometriosis therapy.

Endometriosis is a prevalent gynecological condition, affecting approximately 10% of women of reproductive age. It is characterized by the presence of ectopic endometrial-like tissue outside the uterine cavity ( 1 , 2 ). This ectopic tissue responds to the cyclic hormonal changes of the menstrual cycle ( 3 ), leading to repeated bleeding, inflammation, fibrosis, and adhesion formation ( 4 ). Clinically, it manifests as severe pelvic pain, infertility, and an increased risk of ovarian malignancies, significantly impacting reproductive health ( 1 , 5 , 6 ). Despite its prevalence and the substantial impact on quality of life, the exact molecular mechanisms driving endometriosis remain incompletely understood ( 7 ), posing significant challenges to the development of targeted therapeutic interventions. The pathogenesis of endometriosis is multifactorial, involving complex hormonal, immunological, and genetic interactions ( 8 ). Among these, transcriptional regulators have been identified as crucial drivers of both normal uterine physiology and the pathological processes observed in endometriosis ( 9 – 11 ). Forkhead box A2 (FOXA2), a pioneer transcription factor in the Forkhead family ( 9 ), plays a critical role in the development and function of the uterine glandular epithelium (GE) ( 10 , 12 ). FOXA2 is predominantly expressed in the uterine GE ( 9 , 13 , 14 ) and is essential for successful pregnancy establishment ( 9 , 10 , 15 , 16 ). Disruptions in FOXA2 function in murine models result in impaired glandular function, reduced uterine receptivity, and infertility despite normal uterine morphology ( 9 , 10 , 12 ). The partial rescue of implantation defects via exogenous leukemia inhibitory factor (LIF) administration, a cytokine regulated by FOXA2, further highlights FOXA2’s role in regulating uterine glandular products ( 9 , 10 ). Given its central role in uterine function, FOXA2 dysregulation may contribute to the pathogenesis of endometriosis, though its biological role in the disease remains unclear. In the context of endometriosis, FOXA2 is implicated in the disease’s etiology ( 8 ). Clinical evidence indicates a significant reduction in FOXA2 expression in the eutopic endometrium of women with endometriosis compared to healthy controls ( 17 , 18 ). These findings suggest that FOXA2 downregulation may contribute to endometriosis pathophysiology, but direct in vivo evidence is still limited. Steroid hormone signaling, through estrogen (E2) and progesterone (P4), is integral to regulating uterine function, including maintaining uterine receptivity, facilitating implantation, and driving stromal decidualization ( 19 , 20 ). In endometriosis, disruptions in these pathways are well-documented, with P4 resistance, likely through the reduced expression of progesterone receptors (PGRs), emerging as a key driver of the disease’s pathology ( 21 ). Since PGR signaling is essential for stromal decidualization and pregnancy maintenance, its dysregulation leads to inadequate hormonal responses in affected tissues, exacerbating the condition ( 22 ). Our previous studies revealed that FOXA2 levels in the uterus are significantly reduced by E2 treatment, with this effect amplified when combined with P4 ( 9 ). This suggests that FOXA2 is directly involved in steroid hormone-driven uterine regulation, and its downregulation may contribute to the hormonal dysfunction observed in endometriosis. In addition to its role in hormone signaling, FOXA2 has been shown to regulate GE expression of LIF, which is essential for blastocyst implantation and stromal decidualization ( 10 , 12 ). The FOXA2-LIF axis has thus been explored as a potential therapeutic target in the management of endometriosis-related infertility ( 10 , 12 ). In FOXA2-deficient mice, recombinant LIF administration has been shown to partially restore implantation capacity ( 9 , 10 ), suggesting that therapies aimed at restoring LIF levels could mitigate the effects of FOXA2 downregulation and improve reproductive outcomes in patients with endometriosis. In this study, we demonstrate that endometriosis is significantly reduced the expression of FOXA2 in eutopic endometrium. Using uterine specific Foxa2 knockout mice, we reveal that FOXA2 loss increased development of ectopic lesions, which coincided with downregulated PGR expression in both eutopic endometrium and ectopic lesions. Importantly, we evaluated the therapeutic potential of LIF treatment, which successfully reduced lesion development in this FOXA2-deficient endometriosis mouse model. These findings offer novel insights into the molecular mechanisms of endometriosis and suggest new avenues for therapeutic intervention.