Alterations in the interaction between the follicle and the matrix must occur before follicular rupture and ovulation can take place. Such interactions are controlled in part by the delicate balance between the matrix metalloproteinases (MMPs) and their inhibitors (TIMPs). We hypothesize that the TIMPs would increase prior to ovulation in order to keep MMP activity in check to provide localized degradation at the apex and prevent wholesale destruction of the follicle wall. In the present study we focused on expression and localization of TIMP3, which is the only TIMP bound to the extracellular matrix, during the periovulatory period in the human. Follicles were collected from patients undergoing elective surgery at different stages of the ovulatory process. Patients were divided into four groups (preovulatory, early ovulatory, late ovulatory, and post-ovulatory phase). Surgery was performed at the preovulatory stage without administering hCG. The remaining women received an injection of 250 µg hCG (Ovitrelle, s.c.) when the diameter of their dominant follicle was ≥14mm and ≤ 20mm on trans-vaginal ultrasound. These patients had surgery during the following time intervals after hCG-injection; early ovulatory phase 12h to ≤18h, late ovulatory phase >18h to ≤ 34h and the postovulatory phase 44h to 70h. For the localization studies, the intact follicle with the apical orientation was embedded and processed for immunohistochemistry (IHC) using a TIMP3 antibody. For the quantitative studies, the follicle was bisected and the granulosa and theca cells separated and collected for real time PCR. Real time PCR analysis for Timp3 mRNA in granulosa and theca cells revealed a significant increase in Timp3 gene expression in granulosa cells from the early to the late ovulatory stage (n=5). Thecal Timp3 expression was constitutive across the periovulatory period. TIMP3 protein was localized by IHC to the granulosa cells and theca cell layers. In the preovulatory follicle there was positive staining in the granulosa cells, no staining in the theca. In the early ovulatory stage there was positive staining in both the granulosa and theca interna. The staining was most intense in the granulosa and theca cells; interna and externa of 2 follicles which were in the late ovulatory group. There was staining of TIMP3 in the lumen of some of the blood vessels and it was particularly intense in one early ovulatory follicle that had a large vascular bed surrounding the follicle. Several primordial follicles were seen in the sections and these were positively stained by the TIMP3 antibody. By using human follicles collected throughout the periovulatory period, we have been able to validate our hypothesis that Timp3 gene expression increases as the follicle progresses toward ovulation. We have shown that localization of TIMP3 protein appears first in the granulosa cell layer and then steadily accumulates in the granulosa-thecal layers as ovulation approaches. This localization places TIMP3 in the appropriate cellular layer to control the degradation of the follicular wall by the MMPs. (poster)