Participants

Human endometrial tissues were obtained from ten women aged 29–36 (32.3 ± 2.7) years undergoing surgery for idiopathic infertility in the First Affiliated Hospital of Harbin Medical University (Harbin, PR China). Eutopic endometrial tissues were obtained from ten patients aged 26–42 (34.3 ± 7.6) years undergoing surgery for endometriosis. None of the patients had received any hormonal therapy prior to surgery within six months. Women suffering from cancers, benign ovarian cysts other than endometriomas, perioperative pelvic inflammatory disease, or endometrial polyps were excluded from this study. All patients signed an informed consent form prior to recruitment and the study protocol was approved by the Ethics Committee of Harbin Medical University (202106). All experimental methods were carried out in accordance with the approved guidelines of Harbin Medical University. All patients gave their written informed consent prior to study inclusion.

Animal experiments

All animal experiments were conducted using female Balb/c nude mice aged approximately 5 weeks and weighing 17–19 g. These mice were purchased from Charles River Laboratories in Beijing (No. 11400700316857), and the animal experiments were performed in strict accordance with the guidelines for the Care and Use of Laboratory Animals of the Harbin Medical University Ethics Committee. All procedures were approved by the Committee on the Ethics of Animal Experiments of Harbin Medical University. All efforts were made to minimize animal suffering. All cell protocols were approved by the Harbin Medical University Ethics Committee.

Isolation and culture of ESCs

Endometrial tissues were collected and washed with ice-cold medium (DMEM/F-12 1:1) (Hyclone, USA) containing 10% fetal bovine serum (Ausbian, USA) and 1% penicillin-streptomycin (Gibco, USA). The samples were transported to the laboratory on ice within 2 h. The endometrial tissues were cut into smaller pieces and digested in type IV collagenase (Life Technologies, Carlsbad, CA, USA) at 37 °C for 60–90 minutes. The cell suspension was passed once through a 70-μM sieve (HEAD, Beijing, China) to remove debris and glandular epithelial cells. The filtrates were then centrifuged at 800 rpm for 5 minutes at room temperature. The isolated cells were maintained in the medium mentioned above at 37 °C and 5% CO2. The medium was replaced after 2–3 days to remove nonadherent cells. Cells were subcultured on new plates at a 1:2 ratio and marked as passage 1 (P1) [16, 17]. Cells from P3-P5 were used for the experiments.

Observation of ESC morphology

The morphology of ESCs from different passages was observed by inverted light microscopy (Olympus, Japan).

Immunofluorescence

Serum-starved ESCs were seeded on cover glass slides, fixed with 4% paraform aldehyde for 15 min, and permeabilized with 0.1% Triton X-100. The purity of ESCs was detected by separately immunostaining for the epithelial marker cytokeratin 7 (CK 7) (33,060 M, Biosis, China) and stromal marker vimentin (VIM) (0756R, Biosis, China). We used 4′,6′-diamidino-2-phenylindole (DAPI) immunofluorescence to identify ESC nuclei. Cells were incubated with fluorescein isothiocyanate (FITC)-phalloidin (for F-actin staining, Sigma, USA) at room temperature for 40 minutes before incubation with the primary antibodies anti-CK 7 and anti-VIM overnight at 4 °C.Cell nuclei were stained with DAPI (Thermo Fisher Scientific). Immunofluorescence signals were observed using an inverted light microscopy (Olympus, Japan) [18]. The areas with CK 7 and VIM were computed using ImageJ software. At least 100 cells were analyzed from triplicate cover slides in each sample, and experiments were repeated with samples from five different individuals.

Cell proliferation assay

Cell proliferation was assessed by the Cell Counting Kit-8 (CCK-8; Dojindo, Japan) assays. To determine whether the SHH signaling pathway effected ESC proliferation, we applied the SHH signaling pathway inhibitor GANT61. After 48 hours, cells were seeded in 96-well plates (4000 cells per well) stimulated with different concentrations of GANT61 (SIGMA, USA) in 100 μL of full culture medium. The cells were tested in the absence (NC) and presence of 10 μmol/L, 20 μmol/L and 30 μmol/L inhibitor of GANT61. Ten microliters of CCK-8 solution was added to each well. Absorbance was read at a wavelength of 450 nm by a microplate reader (ELX800; Bio-Tek, Ameria). The proliferation rate was derived from the cell index, which was calculated as the difference between the well with only cells minus the well with only culture media, divided by the nominal value. Three independent experiments were performed in triplicate.

Wound healing assay

To examine the migratory capacity of ESCs, we conducted a scratch wound assay. Cells cultured with GANT61 (10 μmol/L, 20 μmol/L and 30 μmol/L) were seeded in six-well culture plates with serum-containing medium and cultured until the cell density reached 90–95% confluence. An artificial homogeneous wound was created by scratching the monolayer with a sterile 200 μL pipette tip. After scratching, the cells were washed with PBS and then cultured with serum-free DMEM F12 1:1 media for 48 hours. Images of cells migrating into the wound were captured at 0 and 48 hours using a microscope (EVOS, USA). The assay was performed in triplicate [19]. Three independent experiments were performed in triplicate.

Transwell invasion assay

Transwell assays were used to assess cell invasive capacity. Cell invasion assays were carried out using a BioCoat Matrigel Transwell chamber (BD, Franklin Lakes, NJ, USA) with a pore size of 8.0 μm. The inserts were placed in 24-well plates containing 700 μL of DMEM F12 1:1 medium for 30 minutes in a humidified 37 °C incubator under 5% CO2 before seeding the cells. GANT61 (30 umol/L) was used to block the SHH signaling pathway, and after 48 hours, 5 × 104 cells in each group resuspended in DMEM F12 1:1 medium containing 5% FBS were placed in each chamber. The lower compartment was loaded with full media containing 15% FBS as the nutritional attractant. After incubated at 37 °C for 48 hours, noninvaded cells were scraped off with a cotton swab. The translocated cells on the bottom of the upper chamber membrane were fixed with 5% formaldehyde and stained with 1% Giemsa stain. The number of cells that penetrated the upper compartment of the Transwell chamber was determined under an inverted microscope. Five fields of fixed cells were randomly chosen and counted under a light microscope [19].

Immunohistochemistry

All tissues were fixed in 10% formaldehyde, embedded in paraffin and cut into 4 mm sections. Immunohistological staining was conducted by boiling the sections in 10 mM citric acid, pH 7.0. The slides were incubated with a polyclonal rabbit antibody (1:200 dilution; Biosis) for 2 hours at 37 °C. The sections were washed in phosphate-buffered saline (PBS) three times and then incubated with mouse anti-rabbit secondary antibody for 40 minutes at 37 °C Peroxidase substrate containing 3,3′-diaminobenzidine tetrahydrochloride chromogen was added to the sections for 2 minutes to develop the reaction. All slides were examined and scored by two independent pathologists who were blinded to both the clinical and pathological data. The quantification of the selected proteins was performed using Image-Pro Plus 6.0 (Media Cybernetics). Scoring was carried out for the mean density (ratio of integrated optical density SUM/area) [19].

ESCs and eutopic endometrium tumourigenicity analysis

Mice were kept on a 12 h light/dark cycle and provided sterile food and water. The mice were allowed to acclimate to specific pathogen-free (SPF) conditions before experiments. The mice were randomly separated into 2 tumorigenicity groups (n = 5 per group), the ESC and eutopic endometrium groups. For further study of the function of the SHH signaling pathway in vivo, a mouse model of experimental endometriosis was established by injecting NS with 0.5 cm3 in size of eutopic endometrial fragments into the right subcutaneous scapular tissue and injecting the contralateral side with 0.2 ml of normal saline (NS) as the negative control. Lesions were monitored daily in both groups. After 40 days, the mice were sacrificed by cervical dislocation. Both the left and right subcutaneous scapular tissues were collected. Macroscopic observation and H&E staining were used to assess lesion formation [20].

GANT61 treatment of ESCs in the endometriosis model in vivo

Human eutopic endometrial tissues were obtained from patients with endometriosis as described above. Mice were maintained on a 12 h light/dark cycle and were provided with sterile food and water. The mice were allowed to acclimate to SPF conditions before experiments. Twenty mice received a single subcutaneous injection of a 0.5 cm3 eutopic endometrial fragment in 0.2 ml of NS into their back. Seven days later, when the endometriosis model was confirmed, the mice were randomly divided into two groups (n = 10 per group), the GANT61 group and the control group. GANT61 was subcutaneously injected into experimental group mice, while NS was injected into the controls. In the GANT61 group, 30 umol GANT61 in 0.3 ml of NS was administered intravenously into the tail vein. The mice in the control group were only injected with 0.3 ml of NS. The injections were performed weekly. The animals were sacrificed one week after the third injection, and the endometriotic lesions of the two groups were collected to detect the effect of GANT61 on lesion reduction [20].

Statistical analysis

All statistical analyses were performed using SPSS 19.0 (SPSS, Inc., Chicago, IL). Continuous variables are expressed as the mean ± standard deviation. Differences between groups were evaluated using the independent samples Student’s t test. Completely random design analysis of variance was performed to test the significance of the migration of ESCs in response to different GANT61 doses. Data from the invasion assay were assessed by paired T tests. Differences were considered statistically significant at P < 0.05.