Led explanation of the study protocol with particular emphasis on the
Led explanation of the study protocol with particular emphasis on the risks associated with the endometrial biopsy and the use of steroids during their luteal phase. A written informed consent was obtained at that time. Oocyte donors were stimulated with a GnRH antagonist protocol. Briefly, all donors had a baseline measurement of FSH and E2 serum concentrations on the second day of their menstrual cycles after the discontinuation of oral contraceptive pills. In addition, a transvaginal sonogram was performed to rule out early follicular development and any anatomic anomalies. Providing that serum FSH was less than 10 mIU/ml and E2 was less than 60 pg/ml, ovarian stimulation was initiated with 225 IU recombinant FSH (Follitropin Alfa, Gonal-F; Serono Laboratories, Norwell, MA). A daily evening dose ofganirelix acetate (Antagon; Organon, West Orange, CA), 0.25 mg sc, was started either 6 d after the initiation of gonadotropins or at the time of identification of a leading follicle with mean diameter more than 13 mm and continued through the day of human chorionic gonadotropin (hCG). Thereafter, the dose of gonadotropins was adjusted PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28506461 in a step-down fashion according to follicular development by serial transvaginal ultrasound and serum E2 response. When at least three follicles reached a mean diameter of 18 mm, ovulation was triggered with a single im dose of 10,000 IU hCG (Profasi; Serono) or 20 U of a GnRH agonist administered in two doses 24 h apart. Transvaginal oocyte retrieval was performed under iv sedation 34-36 h after hCG or the initial dose of GnRH agonist. On the day of oocyte retrieval, the study participants were randomized into three treatment categories: The “none” group did not receive any luteal phase support; the “P” group received micronized P in the form of vaginal suppositories (200 mg every 6 h starting from the day after retrieval); the “P + E” group received a daily oral dose of 2 mg 17b-E2 in addition to the micronized P. (Figure 1) Up to that point, all donors had been stimulated according to the same protocol and had received a comparable amount of medication. An endometrial biopsy on the PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28388412 day of oocyte retrieval was performed on all study participants to define a baseline status for the endometrium. Endometrial biopsies were performed using a Pipelle catheter (Unimar, Wilton, CT) on the day of oocyte retrieval (cycle day [CD] 14 of the benchmark cycle) and then 3, 5, and 10 days after oocyte retrieval corresponding to ideal cycle days 17, 19, and 24. At least 2-4 endometrial biopsies were obtained from each donor. The tissue in the endometrial samples were identified as either luminal epithelium, the surface epithelium lining the uterine cavity, ARA290 chemical information glandular epithelium, or stromal tissue. The specimens were then fixed in 10 formalin, and subsequently embedded into paraffin for tissue microarray sectioning.Microarray analysisTissue microarrays (TMA) were assembled from paraffin embedded endometrial tissues in a 1.5-mm diameter for each tissue sample. Three representative tissue samples were obtained for each specimen. The arrays encompassed 108 tissue cores derived from 12 donors (Table 1). All tissue cores were sectioned in 5-m thickness and affixed to the TMA slides. (Figure 2) The expression of ERa and PR-B were examined using Confirm antiER (6F11) and Confirm anti-PR [16] mouse monoclonal antibodies (Ventana Medical Systems Inc, Tucson, Arizona) directed against the human ER and PR molecules. The tissue section.
