Endometrial Explant Cultures

Proliferative phase endometrial samples were collected from 6 women of reproductive age with no evidence of endometriosis or submucosal fibroids, undergoing hysterectomy for benign reasons (eg, pelvic relaxation). The tissue collection protocol was approved by the institutional review board at University of Texas Southwestern Medical Center. Phenol red-free, serum-free DMEM/F12 medium (Thermo Fisher Scientific, Waltham, MA) with 1% antibiotic/antimycotic was used in all stages of tissue preparation and explant culture. The tissues obtained were first washed in medium and cut into uniform approximately 2 mm³ pieces with a sterile scalpel blade. Then 3 to 4 tissue pieces were immediately transferred onto a triangle sieve covered with sterile filter paper in a 6-well plate. Explants were treated and incubated for 24 hours at 37°C in a humidified 95% air–5% CO₂ environment. Thereafter, explants were immediately placed into RNAlater solution (Thermo Fisher Scientific, Waltham, MA) and stored at −80°C. To isolate RNA, tissues were homogenized in TRIzol reagent (Thermo Fisher Scientific, Waltham, MA).