Patients

Patients diagnosed with AM through imaging, and with a history of no pregnancies after at least 3 embryo transfers (including a total of ≥ 4 good-quality embryos), were recruited for the AM group (n = 25). Patients with tubal obstruction or unexplained infertility who achieved a clinical pregnancy after the first embryo transfer were assigned to the control group (n = 25). The inclusion and exclusion criteria for all patients were as follows: the patients were 25–40 years old with regular menstrual cycles and normal endocrine profiles. Patients who used any intrauterine device or contraceptive drug within the last 6 months, or with any of the following conditions were excluded from the study: intrauterine pathology, hydrosalpinx, salpingitis, polycystic ovary syndrome, endometriosis, chromosome abnormalities, or autoimmune disease (Table 1).

Table 1 Clinical characteristics of women in the control and AM groups

Endometrial specimens were collected at day LH + 7 (days 20–23 of the menstrual cycle, mid-secretory phase) using pipe suction curettage (LILYCLEANER, China). For isolation of primary endometrial cells, endometrium samples (days 11–14 of the cycle, late proliferative phase) were collected from the patients.

Samples are collected during the mid-secretory phase to study the optimal receptivity of the endometrium. Therefore, collecting cells at this stage allows for the verification of gene expression associated with these important functional alterations.

In contrast, samples are collected during the late proliferative phase (days 11–14) for primary cell culture due to practical considerations, making sample collection easier and more efficient.

For detailed information about the experimental design, see Additional file.

Isolation of human endometrial stromal cells (HESCs) and cell cultures

Endometrial tissues were washed, minced, and digested with collagenase IV (17104019, Gibco, Waltham, MA, USA). All endometrial stroma cells were collected from the mixture after passing through a 40 μm sieve. HESCs were obtained by seeding the stromal cells on cell culture dishes, and passage 2–3 stromal cells were used to ensure the purity of the cell.

All isolated cells were cultured in DMEM/F12, 1% penicillin/streptomycin (Gibco), and 10% fetal bovine serum (Gibco), and T-HESCs were cultured in 89% DMEM/high glucose, 1% penicillin/streptomycin, and 10% fetal bovine serum in a 37 °C incubator containing 5% CO2.

cDNA preparation and quantitative real-time polymerase chain reaction (RT-qPCR)

RNA was extracted from endometrial biopsy tissues, HESCs, or TERT-immortalized human endometrial stromal cells (T-HESCs) and then reverse-transcribed into cDNA according to the manufacturer’s instructions (R222, Vazyme, China). RT-qPCR was performed with the SYBR Premix Ex Taq kit (Q711, Vazyme, China) and Prism 7500. Fold change in gene expression was calculated using the 2–ΔΔCt method with GAPDH as the reference gene. The primer pairs used in this study are listed in Additional file.

Western blotting

Proteins from endometrial tissues and HESCs were extracted by RIPA buffer (89900, Thermo Fisher Scientific, Waltham, MA, USA) containing a protease inhibitor cocktail (5892970001, Roche, Switzerland). Proteins were electroblotted onto 0.22 μm polyvinylidene difluoride membranes (ISEQ00010, Millipore, Burlington, MA, USA) after being separated by 10% and 12.5% SDS-PAGE (PG112, PG113, Epizyme, China). Membranes were successively incubated with primary and secondary antibodies (listed in Additional file), followed by blocking with 3% skim milk. Enhanced chemiluminescence analysis (E412-01, Vazyme, China) was performed to obtain the images.

Immunohistochemical staining (IHC)

The tissues were preserved using 4% paraformaldehyde (BL539A, Biosharp, China) and then embedded in paraffin blocks. Each 5 μm-thick section underwent deparaffinization, rehydration, antigen retrieval, and a 10-min submersion in 3% hydrogen peroxide. The sections were then washed with Tris-buffered saline and blocked using 5% normal goat serum for 30 min. Following this, they were incubated with either anti-LEF1, anti-IL-11, anti-HAND2, anti-LIF, or rabbit /rat monoclonal IgG isotype control at 4 °C for 12 h. Sections were then exposed to secondary antibodies, stained with diaminobenzidine, and counterstained with hematoxylin. Visualization was achieved using a Nikon Eclipse 80i microscope (Japan). The intensity of immunostaining was evaluated using the following H-Score (Zhou et al. 2020):

Each section was scored by two independent pathologists, and the results were then analyzed.

Enzyme-linked immunosorbent assay (ELISA) analysis

IL-11, insulin-like growth factor-binding protein 1 (IGFBP1), and prolactin (PRL) expression levels were measured using ELISA kits (EK111, EK1255, and EK1304, Multisciences, China). The process involved adding 50 µl of culture medium to the samples, as per the instructions provided by the manufacturer. After the addition of the samples and the creation of standard curves, the optical density was measured at a wavelength of 450 nm using a Multiskan GO instrument.

For endometrial specimens, 50–100 mg of tissue samples were weighed. Five times the mass volume of 1X PBS buffer containing a 1% protease inhibitor cocktail (5892970001, Roche) was added to the samples. Scissors or similar tools were used to finely chop the tissue samples. The samples were subjected to ultrasonic cell disruption while being kept on ice. Following this, they were incubated at 4 °C or maintained on ice for a duration of 30 min. Thereafter, the samples were centrifuged for 10 min at 12,000 × g, and the supernatant was collected. The levels for each sample were normalized to the amount of the total protein.

Immunofluorescence

Slices of human endometrial tissues and cultured cells were fixed with 4% PFA, permeabilized with 0.3% Triton X-100, blocked with 5% bovine serum albumin, and incubated with primary antibodies. The sections were incubated with secondary antibodies for 1 h, followed by Hoechst 33342 staining for 15 min at room temperature. Images were captured using a fluorescence microscope (Zeiss-Axio Vert.A1, Germany; Leica Thunder DMi8, Germany) or a confocal laser scanning microscope (Leica, sp8). Information on the antibodies used is provided in Additional file.

For cytoskeleton staining, TRITC-phalloidin (G1041, Servicebio, China) was used in accordance with the manufacturer’s guidelines.

Separation and preparation of cytoplasmic and nuclear extracts of HESCs

Proteins from the cytoplasm and nucleus of HESCs were extracted using a nuclear and cytoplasmic protein extraction kit (78833, Thermo Fisher Scientific) following the manufacturer’s instructions. GAPDH and LAMIN-B1 were used as markers for the cytoplasmic and nuclear fractions, respectively.

Lentiviral inhibition and plasmid overexpression in cells

Cells were transduced with either scramble shRNA, LEF1-shRNA, or IL-11-shRNA (2 × 107 transducing units/mL) using polybrene (10 µg/ml). Following transduction, the cells underwent selection with G418 (1000 µg/ml) and were subsequently cultured in the presence of G418 reagent (500 µg/ml). The lentiviruses were purchased from Hanbio (China). The scramble and target sequences of gene interference are provided in Additional file. IL-11 and negative plasmids purchased from GeneCopoeia (Rockville, MD, USA) were transfected into cells using X-tremeGENE HD DNA transfection reagent (06366236001, Roche), according to the manufacturer’s instructions.

In vitro decidualization assay

HESCs and T-HESCs were cultured in serum-free, phenol red-free DMEM/F12 (LD1230, Bioagrio, China) supplemented with medroxyprogesterone acetate (MPA, 1 µM, HY-B0469, MCE) and 8-bromoadenosine 3’,5’-cyclic monophosphate (8-Br-cAMP, 1 mM, ab141448, Abcam, USA) for 3 and 5 days, respectively. Markers of decidualization (PRL and IGFBP1) were analyzed using RT-qPCR, ELISA, and western blotting.

Embryo outgrowth analysis

HESCs were harvested from the endometrium during the late proliferative phase and then seeded in a 24-well plate. After a period of 72 h, these cells once transfected with scramble shRNA, LEF1-shRNA, IL-11-shRNA, negative plasmids, or IL-11 overexpression plasmids underwent the decidualization process as previously described. Mouse blastocysts, which exhibited a standard morphology and had successfully hatched, were co-cultured alongside the confluent monolayers of the decidualized HESCs using a DMEM/F12 complete medium. The areas of trophoblast outgrowth were meticulously marked, and their measurements were computed using the ImageJ software (NIH, Bethesda, MD, USA).

Whole-genome expression profile and bioinformatics analysis

T-HESCs transfected with scramble shRNA or LEF1-shRNA for three days were collected using the Trizol reagent (Thermo Fisher Scientific). The sequencing library was then sequenced on the NovaSeq6000 platform (Illumina) with the assistance of Genekinder Medicaltech (Genekinder Medicaltech Co., Ltd., China). Paired-end sequence files (fastq) were mapped to the reference genome using Hisat2 (v2.0.5). The TPM algorithm was used for normalization. Differential expression analysis for mRNA was performed using the R package DESeq2. An illustration of differentially expressed genes (DEGs) between the two samples was shown in volcano plots generated by the R package (v4.1.0) on the Hiplot Platform (https://hiplot.com.cn).

Dual-luciferase reporter gene assay

The interaction between LEF1 and the IL-11 promoter region was explored using a dual-luciferase reporter assay. Both wild-type IL-11 promoter luciferase and its mutant counterpart were synthesized by Genomeditech Co., Ltd. (China). HEK293T cells were cultured in 24-well plates. For each well, 300 ng of luciferase reporter constructs were co-transfected with 600 ng of either pcDNA-NC or pcDNA- LEF1 using Lipofectamine 2000 (Invitrogen, Waltham, MA, USA). After an incubation period of 48 h, the Dual-Luciferase Reporter Assay kit, sourced from Genomeditech, China, was employed to gauge the relative luciferase activity.

Chromatin immunoprecipitation (ChIP) assay

ChIP was conducted using T-HESCs. Cells were cross-linked by adding 37% formaldehyde to a final concentration of 1% for 10 min at room temperature. The subsequent steps followed the manufacturer’s instructions for the ChIP assay kit (17-10086, Merck, Rahway, NJ, USA). The enriched DNA was then analyzed using specific primers via RT-qPCR.

Animal models and methods for adenomyosis induction

In our study, we induced adenomyosis in female neonatal CD-1 mice (AM-model group) through oral administration of 1 mg/kg tamoxifen (HY-13757 A, mce), dissolved in a mixture of peanut oil, lecithin, and condensed milk (in a 2:0.2:3 volume ratio), with a dosage of 5 µl/g of body weight, from the first to the fifth day post-birth (Bourdon et al. 2023). Meanwhile, control female neonatal CD-1 mice (vehicle group) received an equivalent volume of the solvent mixture without tamoxifen. Upon reaching 21 days of age, all mice were weaned and separated from their mothers.

All CD-1 mice purchased from Vital River Laboratory Animal Technologies Co. Ltd. were housed in SPF with regular diet and diurnal rhythm.

Evaluation of pregnancy outcomes

Female mice at 10 weeks of age were mated with fertile wild-type males, with the detection of a vaginal plug marking day 1 of pregnancy. The day before mating, TAM treatment mice received IL-11 (200 mg/kg) intravenously, whereas control mice were injected with a comparable volume of saline solution. Females showing evidence of mating (plug-positive) were isolated for pregnancy-related studies. On the seventh day of pregnancy, implantation sites in these mice were identified through intravenous administration of 200 µL of 1% Evans Blue in saline, with the sites indicated by clear blue bands. For each experimental condition in every mouse model, a minimum of three mice were used.

Statistical analysis

For normally distributed data with homogeneity of variance, Student’s t-test was used to compare data between two groups, and one-way analysis of variance (ANOVA) with Bonferroni’s test or Dunnett’s test was used to compare data between more than two groups. For nonparametric data, appropriate nonparametric tests were applied. All statistical analyses were performed using SPSS software (version 22.0; IBM Corp., Armonk, NY, USA) and GraphPad Prism software (version 6.0; GraphPad Inc., La Jolla CA, USA). All experiments were conducted at least three times, and statistical significance was defined as P < 0.05.