HORMONE-RELEASE FROM CULTURED LUTEINIZED-GRANULOSA CELLS MIMICS DIFFERENCES SEEN IN-VIVO IN PATIENTS UNDERGOING IVF-ET

Objectives: Previous research from this laborator,y has suggested that a relationship exists between the increase in circulating progesteron e concentrations at the time of hCG administration and cycle outcome i n patients undergoing IVF. Progesterone (P) increases of threefold or better within the 24-h period surrounding hCG administration appeared to be associated with a higher pregnancy rate. These data suggest a fu nctional difference in the luteinized-granulosa of patients undergoing IVF, To rest this hypothesis: Design: A split-split plot arrangement of treatments with two cell sources under five hormonal stimulations a t four time points. Methods: Luteinized-granulosa cells (LGC) were col lected from patients with either a normal increase (greater than or eq ual to threefold = NC) in circulating P (n = 4) or those with lower P increases (AC; n = 4). The cells were isolated by Ficoll gradient cent rifugation and then cultured in 24-well culture plates using a modifie d media 199 containing 100 mIU/ml of hMG, FSH, LH, hCG, or a nonhormon al control to stimulate steroid-hormone production. At rime points 2, 4, 6, and 8 days, media from each well was frozen for later hormone as say and replaced with fresh media containing the same stimulating fact or. After all the media had been collected, P and estradiol( (E(2)) re leased into the media were measured using radioimmunoassay. Results: R esults indicate a higher media concentration of P (P < 0.001), but not E(2) (P = 0.254), from NC, regardless of hormone stimulation or time in culture, when compared to the media from AC. Media concentrations o f E(2) were affected by a cell source by hormone stimulation by time i nteraction (P < 0.001) with varying effects, Media from NC maintained a constant E(2) of between 1000-3000 pg/ml over the 8-day period (P = 0.163). However, media from AC demonstrated a stimuli-dependent E(2) r elease (P < 0.001) ranging from < 1000 to over 11,000 pg/ml. Conclusio ns: These data appear to support the existence of functionally differe nt populations of luteinized-granulosa cells from patients undergoing IVF-ET.