Medroxyprogesterone acetate mpa

Human endometrial stromal cells were isolated from mid-secretory phase endometrial tissues collected from fertile control and RIF patients in accordance with the standards described above. These cells were isolated and cultured as previously described. To induce decidualization, the hESCs were cultured in phenol red-free DMEM/F12 medium (HyClone, Thermo Scientific, South Logan, UT, USA) containing 2.5% (v/v) charcoal/dextran-treated fetal bovine serum (FBS; HyClone, Thermo Scientific, South Logan, UT, USA), 100 IU/ml penicillin, and 100 μg/ml streptomycin supplemented with 0.5 mM 8-Br-cAMP and 1 μM medroxyprogesterone-acetate (MPA) (Sigma, St. Louis, MO, USA) for the indicated durations.

Confluent monolayers on a BioFlex six‐well plate (Flexcell International Corporation, Hillsborough, NC, USA) were treated with or without 0.5 mmol/L 8‐bromo‐cAMP (8‐br‐cAMP; Sigma‐Aldrich) and 10⁻⁶ M medroxyprogesterone acetate (MPA; Sigma‐Aldrich) as decidual stimulation for three days. Decidualized or non‐decidualized HESCs were then subjected to cyclic sinusoidal equibiaxial tensile strain (amplitude, 0% to 8%; frequency, 0.1 Hz: 6 cycles/min; sine waveform) for 24 hours using the Flexcell FX‐4000™ Tension System (Flexcell^® Tension Plus System; Flexcell Corporation, McKeesport, PA, USA) according to the manufacturer's instructions. Culture medium had not been changed after the start of decidual stimuli. All experiments were performed before the fourth cell passage.

Retro-2 (2-([(5-Methyl-2-thienyl)methylene]amino)-N-phenylbenzamide; C₁₉H₁₆N₂OS; R2) was purchased from EMD Millipore (Temecula, CA, USA) and stored at 50 mg/mL in DMSO at −20 °C. R2 spontaneously cyclizes to Retro-2^cycl [52 (link)], the mixture is referred to as R2 herein. The purified Retro-2.1 (6-Fluoro-1-methyl-2-(5-(2-methylthiazol-4-yl)thiophen-2-yl)-3-phenyl-2,3-dihydroquinazolin-4(1H)-one; C₂₃H₁₈FN₃OS₂; R2.1) S-enantiomer ((S)-R2.1) and R-enantiomer ((R)-R2.1) were purchased from ChiroBlock (Wolfen, Germany). Heregulin β1 (HRGβ1) was purchased from R&D Systems Inc (Minneapolis, MN, USA). Medroxyprogesterone acetate (MPA) and Dulbecco’s modified Eagle’s medium: Ham’s F12 1:1 (DMEM) were purchased from Sigma Aldrich (Saint Louis, USA). RPMI 1640 was from Gibco (Thermo Fisher Scientific, CA, USA), trastuzumab was from Hoffmann-La Roche Ltd Genentech Inc. (Basel, Switzerland), and dimethyl sulfoxide (DMSO) was from Merck (USA). The antibodies are listed under Table S2.

Dulbecco's modified Eagles medium (DMEM) and phenol red-free DMEM, glutamine, penicillin, streptomycin, Fetal Bovine Serum (FBS), charcoal stripped FBS and TaqMan probes were purchased (Life Technologies, Carlsbad, CA). 17β-estradiol (E₂), R5020, RU486, progesterone and medroxyprogesterone acetate (MPA) were purchased from Sigma Aldrich (Saint Louis, MO). Growth factor reduced matrigel (Cat# 356231) and calcein AM fluorescent dye (Cat# 354216) were purchased from BD Biosciences (San Jose, CA). PR-B directed siRNAs [60 (link), 61 (link)] and control non-silencing siRNA (Cat# SIC001) were ordered from Sigma Aldrich (St. Louis, MO). siRNAs targeting TM4SF1 (Cat# S8367), HES1 (Cat# S6920), PRKCH (CAT#S1107), and ELF5 (CAT# S4629) were purchase from Life Technologies (Carlsbad, CA).

Human endometrial stromal cells (hESCs) were isolated from endometrial tissue by collagenase digestion as previously described [30] (link), [31] (link). hESCs were maintained in phenol red–free RPMI-1640 medium (Gibco, Grand Island, NY) containing 0.1 mM sodium pyruvate (Gibco, Grand Island, NY), 10% fetal bovine serum (FBS; Gibco, Grand Island, NY) depleted of steroids by pre-treatment with dextran-coated charcoal (Sigma Aldrich, St. Louis, MO) (Charcoal-stripped FBS; CS-FBS), and 1% penicillin streptomycin (P/S; Gibco, Grand Island, NY). To induce in vitro decidualization, hESCs were transferred to OPTI-MEM medium (Gibco, Grand Island, NY) containing 2% CS-FBS, 10 nM estradiol (E2, Sigma-Aldrich, St. Louis, MO), 1 mM medroxyprogesterone acetate (MPA; Sigma-Aldrich, St. Louis, MO), 50 µM cAMP (Sigma-Aldrich, St. Louis, MO), and 1% P/S. Decidualization medium was changed every two days and treatment lasted for 6 days. All results from in vitro decidualization were confirmed in hESCs obtained from at least three independent biological replicates.

We used previously isolated Human endometrial stromal cells (hESCs) for this study^{34 (link), 62} . hESCs were then maintained in phenol red–free RPMI-1640 medium (Gibco, Grand Island, NY) containing 0.1 mM sodium pyruvate (Gibco, Grand Island, NY), 10% fetal bovine serum (FBS; Gibco, Grand Island, NY) depleted of steroids by pre-treatment with dextran-coated charcoal (Sigma Aldrich, St. Louis, MO) (Charcoal-stripped FBS; CS-FBS), and 1% penicillin streptomycin (P/S; Gibco, Grand Island, NY). Cells were cultured in monolayer at 37 °C in 5% CO₂. The induction of in vitro decidualization has been previously described^{46 , 62} . To induce in vitro decidualization, cells were washed with PBS and incubated to OPTI-MEM medium (Gibco, Grand Island, NY) containing 2% CS-FBS, 10 nM estradiol (E2; Sigma-Aldrich, St. Louis, MO), 1 mM medroxyprogesterone acetate (MPA; Sigma-Aldrich, St. Louis, MO), 50 μM cAMP (Sigma-Aldrich, St. Louis, MO), and 1% P/S. Differentiation medium was changed every 48 hours for a total of 6 days. For GPR64 knockdown, small interfering RNA (siRNA) was obtained from Dharmacon (Lafayette, CO). Human GPR64 siRNA was transfected using Lipofectamine 2000 reagent (Invitrogen Crop., Carlsbad, CA) prior to in vitro decidualization.

We previously generated primary human endometrial stromal cell lines and human endometriotic stromal cell lines from ovarian endometrioma [49 (link)]. These human endometrial stromal cells were cultured in six-well plates (1 × 10⁵ cells per well in triplicate) with DMEM/F-12 media containing 10% FBS, 100 units/ml penicillin, and 0.1 mg/ml streptomycin. At 90% confluence, the human endometrial cells were cultured with 1 × Opti-MEM I reduced-serum medium containing 2% FBS, 100 units/ml penicillin, and 0.1 mg/ml streptomycin. After 24 h, the human endometrial cells were treated with 1 × Opti-MEM I reduced-serum media with 2% FBS plus decidualization hormone cocktail [EPC; estradiol (100 nM), medroxyprogesterone acetate (MPA: 10 µM, Sigma–Aldrich) and cAMP (50 µM, Sigma–Aldrich)]. The day that the decidualization medium was added to the human endometrial cells was designated Day 0. For these studies, the decidualization medium was renewed every other day. Cells were harvested on the 3rd day after adding the decidualization hormone cocktail. Total RNA was isolated to assess the transcript levels of the decidualization markers prolactin (PRL) and insulin-like growth factor binding protein-1 (IGFBP-1) [12 (link)].