Effects of E2/ERβ on follicular granulosa cells and on AMH/Smad signalling in endometriosis

Patients with endometriosis have greater risk of infertility, which is associated with compromised ovarian function. Dysfunction in follicular granulosa cells and hyperactivation of oestrogen receptor beta (ERβ) are evident in endometriosis. It is also known that anti-Müllerian hormone (AMH)/Smad signalling pathway regulates ovarian activity. In this study, we aimed to explore effects of oestradiol (E2)/ERβ on follicular granulosa cells and on AMH/Smad signalling in endometriosis. The human ovarian granulosa cells were obtained from patients with tubal factor infertility and ovarian endometriosis, respectively, who underwent IVF/ICSI-ET using GnRH antagonist protocol in our hospital. A human ovarian granulosa tumour cell line (KGN) was cultured and subject to treatments with oestradiol and/or PHTPP (a selective ERβ antagonist). Cell viability, apoptosis, migration, and invasion were assessed. The mRNA and protein expressions were also investigated. It was found that AMH/Smad signalling pathway was suppressed in human ovarian granulosa cells in patients with ovarian endometriosis. Then, in KGN cells, E2 treatment (10−10 mol/L for 72 h) did not alter cell viability or apoptosis, but E2 decreased migration and invasion via ERβ. Further, E2 inhibited AMH/Smad signalling pathway via ERβ in KGN cells. Conversely, selective inhibition of ERβ could reverse these effects. In conclusion, activation of E2/ERβ compromised the function of follicular granulosa cells in endometriosis, which may be mediated by AMH/Smad signalling pathway.

Endometriosis is defined as the presence of endometrial-like tissue outside of the uterine cavity. It affects about 190 million females worldwide (Horne & Missmer, Citation2022). Dysmenorrhoea and chronic pelvic pain are common symptoms caused by endometriosis. More importantly, patients with endometriosis have greater risk of infertility. However, the mechanism of endometriosis-associated infertility is complex, involving endometriosis subtype, inflammation, adhesions, disrupted ovarian function, and compromised endometrial receptivity (Bonavina & Taylor, Citation2022). To improve fertility, surgery seems to be futile in patients with endometriosis, while assisted reproductive technologies (ART) is effective (Ata & Somigliana, Citation2024). During ART, the oocyte yield and fertilisation rate is decreased in patients with endometriosis (Benlioglu et al., Citation2025; Horton et al., Citation2019). In non-donor oocyte in vitro fertilisation-embryo transfer (IVF-ET), a lower oocyte yield and lower fertilisation rate were observed in patients with endometriosis compared with those with tubal factor infertility, though their implantation and clinical pregnancy rates were similar (Singh et al., Citation2014). In donor oocyte cycles, pregnancy, implantation, miscarriage, and live birth rates were not affected by endometriosis, compared with patients without endometriosis (Díaz et al., Citation2000). Moreover, in patients with endometriosis, oocyte donation could improve the live birth rate after recurrent failures during autologous IVF cycles (Maignien et al., Citation2023). These studies indicate that infertility is attributed to suboptimal oocyte quality resulting from compromised ovarian function in endometriosis. The mechanism of detrimental effect of endometriosis on oocyte quality needs further investigation. Dysregulation of steroidogenesis compromises oocyte quality during endometriosis. Lower oestradiol (E2) concentrations at the preovulatory stage, at the luteinizing hormone surge, and even on the day of human chorionic gonadotropin (hCG) trigger during ART were observed in endometriosis (Sanchez et al., Citation2017). The level of oestrogen in the follicular fluid is also lower in patients with endometriosis (Fan et al., Citation2023). These are caused by dysfunction in follicular granulosa cells. However, the oestrogen receptor beta (ERβ), one of the receptors of E2, mainly expressed in ovarian granulosa cells, is upregulated and enhanced in activity during endometriosis (Tang et al., Citation2019). The hyperactivation of ERβ could promote cell proliferation and tissue invasion of ectopic lesions in endometriosis (Tang et al., Citation2019). However, the effects of E2/ERβ on follicular granulosa cells in endometriosis are elusive and further research is necessary. The anti-Müllerian hormone (AMH)/Smad signalling pathway regulates ovarian activity (Spector et al., Citation2024). AMH is produced by the granulosa cells and is crucial for folliculogenesis (Sinha et al., Citation2022). The Smad signalling pathway is involved in the regulation of oogenesis as well as folliculogenesis (Rossi et al., Citation2016). Activation of Smad signalling pathway can promote follicle development (Kim et al., Citation2020; Li et al., Citation2022). It was reported that E2 inhibited AMH via ERβ in granulosa cells (Grynberg et al., Citation2012). Moreover, E2/ERβ could regulate Smad signalling during cardiac fibrosis (Pedram et al., Citation2010). The effects of E2/ERβ on AMH/Smad signalling in follicular granulosa cells remain unknown in endometriosis. In this study, we aimed to find whether AMH/Smad signalling pathway was altered in human ovarian granulosa cells in patients with ovarian endometriosis, as well as explore the effects of E2/ERβ on a human ovarian granulosa tumour cell line (KGN), and demonstrate that E2 regulates AMH/Smad signalling pathway via ERβ in KGN cells. Thus, this study could confirm whether activation of E2/Erβ could compromise the function of ovarian granulosa cells via AMH/Smad signalling in patients with endometriosis, which may contribute to disrupted ovarian function, resulting in infertility.

Patients The human ovarian granulosa cells were obtained from patients with tubal factor infertility and ovarian endometriosis, respectively, who underwent IVF/ICSI-ET using GnRH antagonist protocol in our hospital. A total of 18 patients were recruited in each group. Inclusion criteria for the ovarian endometriosis group were patients were diagnosed with ovarian endometriosis via laparoscopic surgery. The control patients were infertile solely due to tubal factors, and inclusion criteria were as follows: (i) regular menstrual cycles; (ii) normal ovarian reserve; (iii) no history of steroid hormone therapy within the past 6 months; and (iv) the presence of ≥3 follicles with a diameter ≥18 mm on the day of hCG injection. The exclusion criteria were as follows: (i) polycystic ovary syndrome (PCOS); (ii) history of ovarian surgery; (iii) history of pelvic tuberculosis; and (iv) concurrent endocrine disorders (e.g. thyroid dysfunction, hyperprolactinaemia, adrenal disorders). hCG was administered 36 h before oocyte retrieval. The follicular fluid from follicles ≥16 mm in the same patient was pooled as one independent sample during transvaginal oocyte retrieval. Centrifugation was performed at room temperature for 5 min (700 × g) within 2 h. The upper follicular fluid was aspirated and gently pipetted to resuspend the settled cells and 3 mL of 50% lymphocyte separation solution was added before centrifugation at room temperature for 10 min (700 × g). The second layer of granulosa cells was carefully aspirated and 5 mL of PBS added and mixed before centrifugation at room temperature for 5 min (500 × g). The supernatant was discarded, and the settled cells resuspended in the remaining 300 μL of PBS to which 10 μL of hyaluronidase (final concentration: 70 μg/mL) was added. This was incubated in a 37 °C incubator for 2 min to digest the mucus between granulosa cells and then 300 μL of 20% foetal bovine serum added and mixed to terminate the digestion, then centrifuged to discard the supernatant. The granulosa cells were then suspended in 5 mL of PBS, centrifuged at room temperature for 5 min (300 × g), and the white pellet of granulosa cells collected. The granulosa cells from a single patient were treated as one independent sample. After resuspending the granulosa cells in PBS, the cell concentration was adjusted to 1 × 106/mL using a haemocytometer, and the cell suspension aliquoted into tubes at 1 mL per tube. This study protocol was reviewed and approved by Medical Ethics Committee of West China Second University Hospital, Sichuan University, approval number 2022-289. All patients signed consent form before the procedure and the privacy rights of human subjects have been observed. All procedures were performed in compliance with relevant laws and institutional guidelines. Cell culture and treatments The KGN cell line is an immortalised tumour cell line from a patient with ovarian granulosa tumour. KGN cells were chosen because they maintain the physiological characteristics of ovarian granulosa cells (Li et al., Citation2019, Citation2024). A KGN cell line was bought from Procell (Pricella Biotechnology Co., Ltd., Wuhan, China). The KGN cells were routinely cultured in steroid-deprived medium consisting of phenol red-free DMEM/F-12 (HyClone, USA, Cat. No. SH30023.01) supplemented with 10% charcoal-stripped foetal bovine serum (FBS; Lonsera, Uruguay) and 1% penicillin–streptomycin (BIOSHARP, China). Cells were passaged using 0.25% trypsin (Beyotime, China) when they reached approximately 80% confluence. Oestradiol (E2; E8140, Solarbio, Beijing, China), and PHTPP (HY-103456, MedChemExpress LLC, Shanghai, China), alone or in combination, were used to treat the cells for the indicated conditions before further analysis. PHTPP was prepared as a 10 mM stock in 100% DMSO and stored at −20 °C, protected from light. Immediately before use, the stock was diluted 1:1000 in fresh complete medium to a final concentration of 10 μM (0.1% DMSO v/v) and applied to the cells. The treatments were performed in steroid-deprived medium. 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay The MTT assay was used to assess the cell viability and performed according to the manufacturer’s instruction (Sigma-Aldrich, Shanghai, China). The final concentration of MTT used was 0.5 mg/mL. In brief, KGN cells were seeded into a 96-well plate. After culture and indicated treatments, 10 μL MTT was added following by incubation for 4 h. Then, after removal of the medium, 150 μL DMSO was added, followed by shaking for 10 min at room temperature. The optical density of the cells was measured by a Scientific MultiskanMK3 microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) at 492 nm. Apoptosis assay by flow cytometry using Annexin V-FITC/propidium iodide (PI) After the indicated treatments, KGN cells and the medium from all wells were collected into a plastic tube. The tubes were centrifuged at 300 × g for 5 min at room temperature. Afterwards, the supernatant was discarded. Then, after washing with cold PBS, centrifugation, and removing the supernatant, 300 µL of 1 × binding buffer was added, followed by adding 5 µL Annexin V-FITC (401006; BestBio, Suzhou, China). Finally, 10 µL PI (C1052, Beyotime, Shanghai, China) was added 10 min before flow cytometric analysis (FACSVerse, BD Biosciences, MA, USA). Wound-healing assay A total of 3 × 105 cells were added to a 3.5 cm dish and scratched by a 200 µL pipette tip after reaching 90% confluence. The cells were then softly washed with PBS to remove any debris. Scratched cells were captured immediately by a microscope (IX71, Olympus, Tokyo, Japan). Afterwards, cells were treated and photographed after 72 h. Transwell migration assay Transwell chambers (Costar, Corning Incorporated, NY, USA) with 8-μm-pore-size polycarbonate membranes were applied. A total of 10,000 cells with indicated treatments were added to the upper serum-free chambers. The corresponding lower chambers served as a chemo-attractant containing 10% FBS medium. After incubation at 37 °C for 72 h, cells that migrated onto the lower side of the insert membrane were fixed with formaldehyde for 30 min and then stained with crystal violet for 10 min. After removing polycarbonate membranes, the cells that migrated to the lower compartment of the chamber were captured and counted in five randomly selected views under a microscope. Transwell invasion assay Transwell chambers (Costar, Corning Incorporated, NY, USA) with 8-μm-pore-size polycarbonate membranes and Matrigel (BD Biosciences, MA, USA) were used. Briefly, a total of 100 µL Matrigel was added to the upper compartment of each chamber at 4 °C, and the plate incubated at 37 °C for 2 h. A total of 10,000 cells with indicated treatments were added to the upper serum-free chambers. The corresponding lower chambers served as a chemo-attractant containing 10% FBS medium. After incubation at 37 °C for 72 h, cells that invaded to the lower side of the insert membrane were fixed with formaldehyde for 30 min and then stained with crystal violet for 10 min. After removing polycarbonate membranes, the cells that invaded to the lower compartment of the chamber were captured and counted in five randomly selected views under a microscope. Real-time quantitative polymerase chain reaction (rt-qPCR) RNA was extracted by TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Reverse transcription was performed by a cDNA kit (#K1622, Thermo Fisher Scientific, Waltham, MA, USA). Rt-qPCR was performed by using a SYBR Green qPCR Kit (Thermo Fisher Scientific, Waltham, MA, USA). All reactions were run in triplicate by ABI-7500 real-time PCR detection system (Thermo Fisher Scientific, Waltham, MA, USA). The expressions of mRNA were normalised to GAPDH or β-actin by using 2 − ΔΔCt method. Results were shown as fold changes to the control group. Primers were designed with Primer Premier 5.0 software (Sangon Biotech, Shanghai. China) and are listed in Supplementary Table 1. Western blot (WB) The whole proteins from cells were extracted by using RIPA buffer with PMSF on ice. A total of 30 μg proteins from each cell sample were resolved by 10% SDS-PAGE and transferred to PVDF membrane (Millipore, Billerica, MA, USA). Then, the membrane was blocked with 5% nonfat dry milk dissolved in TBST at 37 °C for 2 h and was subsequently incubated with corresponding primary antibodies overnight at 4 °C. Blots were washed in TBST afterward and incubated with appropriate secondary antibodies for 2 h with agitation at room temperature. Bands were visualised by using a western blot detection kit (ECL-0011, Dingguo Changsheng, Beijing, China) with ChemiScope 5300 Pro (Clinx Science, Shanghai, China). Clinx ChemiScope software (Clinx Science, Shanghai, China) was used to measure intensity of the bands. Protein expression levels were normalised to β-actin or GAPDH. The results were shown as fold changes to the control group. Antibody lists could be found in Supplementary Table 2-3. Statistics At least three replicates were used in all experiments. All data were presented as the mean ± standard deviation (SD). The data were analyzed by SPSS 19.0 software (SPSS, Chicago, IL, USA). For quantitative data, the Student’s t-test was used for comparison between 2 groups, while one-way ANOVA followed by SNK multiple comparison tests was used for multi-group comparisons. A p-value < 0.05 was considered statistically significant.

AMH/Smad signalling pathway was down regulated in ovarian granulosa cells in patients with ovarian endometriosis The human ovarian granulosa cells were obtained from follicular fluid from patients with tubal factor infertility (control group) and ovarian endometriosis (EMS group), respectively. By WB, protein expressions of AMH, AMHR2, SMAD1, SMAD5, ALK2, ALK3, ALK6, and BMP15 were significantly reduced, in ovarian granulosa cells in the EMS group, compared with the control group (p 0.05; ). Oestradiol (E2) inhibited migration and invasion of a human ovarian granulosa tumour cell line (KGN) via oestrogen receptor beta (ERβ) The optimal incubation concentration and period of E2 was screened by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay in KGN cells. Among the different incubation concentrations and time, both 10−6 mol/L and 10−8 mol/L E2 inhibited cell viability at 48 h and 72 h (p < 0.05). Thus, to avoid the cytotoxicity, and to mimic a relatively low E2 concentration as the state of endometriosis, we chose 10−10 mol/L for 72 h as final experimental condition for E2 (E2 group) during the following experiments (). PHTPP, a selective ERβ antagonist, was used to treat KGN cells alone (PHTPP group) or in combination with E2 (E2 + PHTPP group). Regarding the MTT assay, there were no significant differences in cell viability among the control cells, the E2 group, the E2 + PHTPP group, and the PHTPP group (p > 0.05; ). Cell apoptosis measured by flow cytometry found no significant difference in apoptosis rate among the 4 groups (p > 0.05; ). In the wound-healing assay and transwell migration assay, E2 significantly reduced the migration rate (15.8 ± 2.3%; p < 0.001) as well as the cell migration number (44 ± 3; p < 0.001), respectively, compared with other treatments. PHTPP (migration rate 51.1 ± 1.3%, p < 0.001; cell migration number 70 ± 3, p < 0.001) reversed these effects of E2. PHTPP alone could increase both migration rate (67.0 ± 1.6%, p = 0.003) and cell migration number (116 ± 4; p = 0.006) compared with control cells (migration rate 61.2 ± 0.2%; cell migration number 100 ± 4; ). As for transwell invasion assay, the number of invaded cells were lowest in E2 group (23 ± 3; p < 0.005), while PHTPP reversed the effect of E2 (37 ± 3; p = 0.003). Compared with the control cells (number of invaded cells 45 ± 4), PHTPP alone could significantly increase the number of invaded cells (58 ± 2; p = 0.006; ). E2 regulated AMH/Smad signalling pathway in KGN cells via ERβ The AMH/Smad signalling pathway was then assessed in KGN cells after treatments. By rt-qPCR, it was found that E2 significantly reduced the gene expressions of AMH, AMHR2, SMAD1, SMAD5, SAMD9, ALK2, ALK3, ALK6, and BMP15, compared with other treatments (p < 0.05; ). By WB, E2 significantly reduced the protein expressions of AMH, AMHR2, SMAD1/5/9, ALK2, ALK3, ALK6, and BMP15 (p < 0.05; ). These effects of E2 were reversed by PHTPP (p < 0.05; ). Moreover, compared with the control cells, PHTPP alone could activate AMH/Smad signalling pathway (p < 0.05; ), which showed opposite effects to E2.

Endometriosis is an inflammatory and oestrogen-dependent gynaecological disorder characterised by endometrial-like tissue outside the uterus, mainly in the pelvic peritoneum, and ovaries. It affects approximately 5–10% of women of reproductive age, and it is associated with pelvic pain and infertility (Zondervan et al., Citation2018). It is estimated that 35–50% women with endometriosis are infertile (Giudice & Kao, Citation2004). Many studies focused on the pathogenesis of endometriosis in terms of hormonal dysregulation, oestrogen dominance, inflammatory response, and immune dysregulation (Ochoa Bernal & Fazleabas, Citation2024). Moreover, infertility is also attributed to suboptimal oocyte quality resulting from compromised ovarian function in endometriosis. The mechanism of detrimental effect of endometriosis on oocyte quality needs further investigation. We have shown for the first time that AMH/Smad signalling pathway was altered in ovarian granulosa cells in patients with ovarian endometriosis. More importantly, we have shown that E2 inhibited migration and invasion of KGN via ERβ, and E2/ERβ regulated AMH/Smad signalling pathway in KGN cells. Granulosa cells are important for follicular differentiation, influencing the optimal conditions for oocyte development, ovulation, fertilisation and implantation. A study confirmed that ERβ isoform mRNAs were predominant over that of ERα in human granulosa cells and overexpression of ERβ in HGrC1 cells increased cell apoptosis (Pierre et al., Citation2021). It is well known that oestrogen plays an important role in the growth of oocytes mediated by oestrogen receptors. Research showed decreased serum E2 and follicular E2 levels, in patients with endometriosis (Sreerangaraja Urs et al., Citation2020). However, ERβ expression is elevated in patients with endometriosis (Chen et al., Citation2022; Eldafira et al., Citation2021). The hyperactivation of ERβ was found to promote cell proliferation and tissue invasion of ectopic lesions in endometriosis (Tang et al., Citation2019). In this study, we found E2 inhibited migration and invasion, and reduced the expression of AMH of KGN via ERβ, indicating the detrimental effects of E2/ERβ on follicular granulosa cells in endometriosis. As a member of the TGFβ superfamily produced by follicular granulosa cells, AMH is thought to inhibit aromatase expression and decrease the conversion of androgens to oestrogens, especially in small antral follicles before dominance is achieved (Mamsen et al., Citation2021). The effects of TGF-β superfamily members are known to be mediated by Smad intracellular signal transducer proteins. Various studies investigated the effects of Smad on human granulosa cells, especially SMAD5 (Prapa et al., Citation2015). In a study focused on polycystic ovarian syndrome, AMH inhibited proliferation and did not increase apoptosis in KGN cells. AMH treatment produced diametrically opposed effects on Smad protein expressions in granulosa cells from normal ovaries and those from polycystic ovarian syndrome (Dilaver et al., Citation2019). These results indicate that AMH has different effects on granulosa cells and Smad protein expressions in different diseases. Until now, there have been few studies investigating the effects of E2 on AMH and Smad, especially in endometriosis. Our study confirms AMH/Smad signalling is inhibited in ovarian granulosa cells in patients with ovarian endometriosis. Moreover, E2/ERβ down-regulated AMH/Smad signalling pathway in KGN. In conclusion, this study confirms that activation of E2/ERβ compromised the function of follicular granulosa cells, which may be mediated by AMH/Smad signalling pathway, and this is associated with a decline of ovarian function and damage to oocyte quality in patients with endometriosis. Supplemental material Supplemental Material Download MS Word (18.1 KB)Supplemental MaterialSupplemental Material Download MS Word (18.2 KB)Supplemental MaterialSupplemental Material Download MS Word (18.4 KB)Supplemental MaterialDisclosure statement No potential conflict of interest was reported by the authors. Data availability statement Data is available upon reasonable request from the corresponding author. Additional information Funding

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