Medroxyprogesterone 17 acetate mpa

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.

Medroxyprogesterone 17-acetate (MPA) (Sigma-Aldrich, St. Louis, MO), Levonorgestrel (LNG) and Norethisterone (NET) and progesterone (Calbiochem, Gibbstown, NJ) were dissolved in 100% ethanol for an initial concentration of 1 × 10⁻³ M, evaporated to dryness and suspended in immune cell complete media to a concentration of 1 × 10⁻⁵ M. Further dilutions were made to achieve a final working concentration of 1 × 10⁻⁷ M. As a control, an equivalent amount of ethanol without dissolved hormone was initially evaporated.

The endometrial tissues were processed for stromal cell culture as described previously (Fernandez-Shaw et al., 1992 (link)). Briefly, the samples were suspended in PBS after washing twice in 0.9% normal saline. The tissues were then washed twice in DMEM/F12, chopped and digested with 0.2% collagenase type I (c0130; Sigma, St. Louis. MO, United States) for 50 min at 37°C and finally digested with 0.1% deoxyribonuclease (DN25; Sigma) for 20 min at 37°C. The suspension was filtered through a 40 μm griddle (CLS431750-50EA; Sigma, United States), which permits only stromal cells to pass. Primary ESCs were cultured in Phenol Red-free Dulbecco’s modified Eagle’s medium (DMEM)/F-12 (Gibco, Grand Island, NY, United States) plus 10% charcoal-stripped fetal bovine serum (Biological Industries, US origin) and 1% penicillin-streptomycin-neomycin antibiotic mixture (Thermo Fisher Scientific) at 37°C with a 5% CO2 atmosphere. Cell purity was tested routinely by immunofluorescence staining for cytokeratin and vimentin.
To induce decidualization in vitro, 3 × 10⁵ ESCs were incubated with DMEM/F12 containing 2% charcoal-stripped FBS, 1 μmol/L medroxyprogesterone-17-acetate (MPA) (Sigma) and 0.5 mmol/L N6, 20-O-dibutyryladenosine cAMP sodium salt (db-cAMP) (Sigma) for 4 days, and the medium was changed every other day.

EnSCs were subjected to treatment with PlGF (#P1588, Sigma) at a concentration of 20 ng/ml for 6 days^{68 (link)}. For decidualization, EnSCs were treated with 0.5 μM 8-Bromoadenosine-3’,5’-cyclic monophosphate sodium salt (cAMP) (#1140, Tocris) and 1 μM Medroxyprogesterone 17-acetate (MPA) (#M6013, Sigma) for 6 days^{69 (link)}. The cell culture medium was replaced every 48 h with fresh treatment media. The experimental groups are indicated as control (untreated EnSCs), PlGF, cAMP+MPA, and cAMP+MPA+ PlGF.
Where indicated, EnSCs were treated with pravastatin (Pravastatin sodium salt hydrate, #P4498, Sigma) with or without PlGF treatment at 10 µM for 24 h^{70 (link)}. The experimental groups are classified as control (untreated EnSCs), PlGF, pravastatin (Prav), and pravastatin + PlGF (Prav + PlGF). Epidermal Growth factor (EGF) was used as a positive control for Rac1 activation. (#324831, Sigma) at a concentration of 100 ng/ml for 24 h^{71 (link)}.
For gene silencing, EnSCs were treated with siRNAs^{72 (link)}, such as siRac1 (50 nM, #L-003560-00-0010, Dharmacon), siPAK1 (20 µM, #L-003521-00-0010, Dharmacon), and siWAVE2 (5 nM, #s55787, ThermoFisher Scientific). The siRNAs were transfected with Lipofectamine RNAiMAX (#13778075, ThermoFisher Scientific) for 48 h with and without combination with PlGF treatment.

Decidualization of MenSCs was induced in phenol red-free DMEM-F12 supplemented with 2% charcoal-stripped FBS (Gibco) and penicillin–streptomycin by 5 days treatment with 0.5 mM 8-Bromoadenosine 3′,5′-cyclic monophosphate (8-Br-cAMP) and 1 µM medroxyprogesterone 17-acetate (MPA) (all from Sigma). Control culture plates received the same culture mediun as decidulaized cells except 8-Br-cAMP and MPA. In some settings, 25 ng/mL IFN-γ was added in the last 48 hrs of decidualization process. The expression of prolactin (PRL) and IGFBP-1 transcripts in decidualized and control cells was assessed by quantitative real-time PCR (qPCR) experiments using the primer set and the protocol published elsewhere^{57 (link)}.

The hESC line was immortalized by the retroviral transfection of human telomerase (ATCC CRL-4003) as described by Krikun et al [28 (link)] and was a kind gift from Dr. Haibin Wang (Institute of Zoology, Chinese Academy of Sciences, Beijing, China). The cells were cultured in Dulbecco’s modified Eagle’s medium/F-12 without phenol red (Life Technologies Inc., Grand Island, NY, USA) containing 10% (v/v) fetal bovine serum (Biological Industries, Beit Haemek, Israel), 1% (v/v) insulin-transferrin-selenium solution (Invitrogen, Carlsbad, CA, USA), 5 × 10⁻⁴ g/L puromycin (Gibco, Grand Island, NY, USA), 5 × 10⁴ U/L penicillin, and 5 × 10⁻² g/L streptomycin (Gibco).
To induce in vitro decidualization, the concentration of fetal bovine serum was reduced to 2% (v/v), the cells were treated with 10⁻³ mM medroxyprogesterone-17-acetate (MPA) (Sigma Chemical Co., St. Louis, MO, USA), and 0.5 mM N6, 2′-O-dibutyryladenosine cAMP sodium salt (db-cAMP) (Sigma Chemical Co.) for 7 days, while control samples were treated with 0.1% (v/v) ethanol. As for siRNA transfection, Lipofectamine RNAiMAX Reagent (Invitrogen) was mixed with 5 × 10⁻⁵ mM NR5A2 siRNA (Invitrogen), or with a pool of non-targeting control siRNAs. The mixtures were then added to hESCs at 60–80% confluency in 6-well culture plates. After 24 h, the siRNA was removed and the cells were induced to decidualization for 4 days.