Cs fbs

Immortalized human endometrial stromal cells (hESC) were purchased from the American Type Culture Collection (ATCC CRL-4003TM) and cultured according to the manufacturer’s instructions^{73 (link)}. Briefly, stromal cells were cultured in DMEM/F12 (Sigma) supplemented with 10% charcoal-stripped FBS (CS-FBS, Biological Industries) at 37 °C in a humidified chamber with 5% CO2. To induce decidualization in vitro, stromal cells were treated with 1 μM Medroxyprogesterone 17-acetate (MPA, Sigma) and 0.5 mM dibutyryl cAMP (db-cAMP, Sigma) in DMEM/F12 with 2% CS-FBS for 6 days. The medium was changed every 48 h. Under in vitro decidualization, stromal cells were treated with 0.032, 0.16, 0.8, 4, and 20 μM P (Sigma) for further analysis, respectively. The highest treatment dose of P has no significant toxic effect on cell viability.

Immortalized HESC line was purchased from the American Type Culture Collection (ATCC CRL-4003) and cultured according to the manufacturer’s instructions (Krikun et al., 2004 (link)). The identity of the cell lines has been authenticated by ATCC, and we have confirmed that the cell lines tested negative for mycoplasma contamination. Briefly, HESC was cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Gibco) supplemented with 1% penicillin and 1% streptomycin, 10% charcoal-stripped fetal bovine serum (CS-FBS, Biological Industries), 3.1 g/l glucose, 1 mM sodium pyruvate, 1% Insulin-Transferrin-Selenium (ITS) (Gibco), and 500 ng/ml puromycin at 37°C in a humidified chamber with 5% CO₂. To induce decidualization in vitro, HESC was treated with differential medium (DMEM/F12 with 2% CS-FBS) containing 1 μM medroxyprogesterone 17-acetate (MPA, Sigma, M1629), and 0.5 mM dibutyryl cAMP (dbcAMP, Sigma, D0627). The medium was changed every 48 hr. Other reagents including inhibitors, activators are listed in Supplementary file 1B.

The immortalized human endometrial stromal cell line (HESCs) was a gift from Professor Wang Haibin (ATCC® CRL-4003™) (Jiang et al. 2020 (link), Wei et al. 2022 (link)). The cells were grown in phenol red-free Dulbecco’s modified Eagle’s medium: nutrient mixture F-12 (DMEM/F12, Gibco) with 10% charcoal-stripped fetal bovine serum (CS-FBS, Biological Industries, Beit Haemek, Israel), 100 μg/mL streptomycin, 100 IU/mL penicillin, 1% insulin–transferrin–selenium solution (ThermoFisher Scientific), and 500 ng/mL puromycin (Sigma-Aldrich) at 37°C with 5% CO₂. The cell culture medium was exchanged every 48 h. For passaging, the cells were detached using trypsin with EDTA (Sigma) at 37°C for 3 min.

Immortalized HESCs cell line, a telomerase immortalized benign endometrial stromal cell line, was obtained from the American Type Culture Collection (ATCC, USA) (Krikun et al. 2004 (link)). HESCs were then cultured as described previously (Liao et al. 2015 (link)). Specifically, cells were cultured at 37 °C with 5% CO₂ in a humidified chamber in DMEM/F12 (Sigma, USA) containing 1 mM sodium pyruvate and 3.1 g/l glucose, supplemented with 10% charcoal-stripped fetal bovine serum (CS-FBS, Biological Industries, Israel), 1% insulin-transferrin-selenium (ITS, Gibco, UK), 500 ng/ml puromycin (Sigma, USA), and 1% penicillin streptomycin (PS, Gibco, UK). As for induction of decidualization, HESCs were cultured in 2% DMEM/F12 media containing 2% CS-FBS as well as 1% PS overnight before treatment with differentiation medium supplemented with 1 mM medroxyprogesterone acetate (MPA, Sigma, USA) as well as 0.5 mM dibutyryl cAMP (db-cAMP, MCE, USA) according to previous report (Brighton et al. 2017 ). The medium was replaced every 2 days.

Primary DSCs were obtained from normal pregnancy and RSA. The isolation and culturing of primary decidual stromal cells were performed as follows. The decidual tissues were first cut into pieces as small as possible and subjected to 1 mg/ml type IV collagenase digestion at 37 °C for 1.5 h. After adding 2% medium (DMEM/F12, 1%P/S, 2% charcoal-stripped fetal bovine serum (CS-FBS, Biological Industries)) to terminate digestion, the cell suspension was filtered through 100 µm and 40 µm cell strainers, respectively, and then was centrifuged at 1000 rpm for 5 min. After using red blood cell lysis, cells were seeded in a 10 cm dish with 10⁷ cells and cultured in DMEM/F12 complete medium supplemented with 1% P/S and 10% CS-FBS overnight. DSCs were collected the next day to extract mRNA and protein for subsequent experiments.

Immortalized HESCs were maintained in DMEM/F12 without phenolic red (Gibco) in the presence of 10% charcoal stripped fetal bovine serum (CS-FBS, Biological Industries), glucose (3.1 g/l), sodium pyruvate (1 mM, Sigma); sodium bicarbonate (1.5 g/l, Sigma), penicillin–streptomycin (50 mg/ml, Solarbio); insulin–transferrin–selenium (1%, Thermo Fisher) and puromycin (500 ng/ml). The primary cultured cells were maintained in DMEM/F12 (Gibco) without phenolic red and 10% CS-FBS. For decidualization, HESCs were cultured in medium at the present of estrogen (E2, 10 nM, Sigma), medroxyprogesterone acetate (MPA, 1 mM, Sigma), and dibutyl cyclophospsinoside (db-cAMP, 0.5 mM, MCE) in 2% CS-FBS with different days. All the cells were cultured in 5% CO₂, 95% air, 100% humidity at 37℃ and culture medium was replaced every 48 hr. The endometrial stromal cell line was purchased from ATCC and authenticated using STR profiling by ATCC, and tested to be free from mycoplasma contamination.

Immortalized HESC line was purchased from the American Type Culture Collection (ATCC^R CRL-4003^TM) and cultured according to the manufacturer’s instructions22 (link). Briefly, HESCs were cultured in DMEM/F12 (Sigma) supplemented with 10% charcoal-stripped FBS (CS-FBS, Biological Industries) at 37 °C in a humidified chamber with 5% CO₂. Synchronization of HESCs in the G0/G1 phase was achieved by serum starvation overnight as described previously23 (link). To induce decidualization in vitro, HESCs were treated with differential medium (DMEM/F12 with 2% CS-FBS) containing 10 nM E2 (Sigma), 1 μM Medroxyprogesterone 17-acetate (MPA, Sigma), and 0.5 mM dibutyryl cAMP (dbcAMP, Sigma). The medium was changed every 48 h. Thiostrepton (Enzo) and pyridone 6 (Merck Millipore) were used to inhibit FoxM1 and the Janus kinase-STAT3 pathway in culture, respectively.