Endometrium was collected from 10 normal-cycling reproductive-age women by endometrial biopsy with informed consent, under an approved Human Investigations Committee protocol. Tissue was immediately frozen in liquid nitrogen and stored at -80°C. Some of the tissue was fixed in formalin for histological examination or in paraformaldehyde for in situ hybridization. Menstrual-cycle dating was determined by menstrual history and confirmed by histological examination by using the criteria of Noyes et al.
Endometrial samples were obtained from four different normal-cycling women in the proliferative phase. Endometrial epithelium and stromal cells were separated as described previously. Briefly, endometrial tissue was digested by incubation of tissue minces in Hanks Balanced Salt Solution (HBSS) (Sigma, St. Louis, MO) that contained Hepes (25 mmol), penicillin (200 U/ml), streptomycin (200 mg/ml), collagenase (1 mg/ml, 15 U/mg), and deoxyribonuclease (0.1 mg/ml, 1500 U/mg) for 30 min at 37°C with agitation. The dispersed endometrial cells were separated by filtration through a wire sieve (73-^m diameter pore, Sigma). The stromal cells passed through the sieve into the filtrate, whereas the endometrial glands were retained by the sieve. The stromal cells were pelleted, washed, and suspended in phenol red-free charcoal stripped Hams F12/Dulbeccos minimal essential medium (1:1 vol/vol; Sigma) containing antibiotics and fetal bovine serum (FBS; 10% vol/vol; GIbCo BRL, Rockville, MD).