Follicular steroidogenesis, involving proteins including insulin-like growth factor 1 (IGF-1), progesterone (P4), androstenedione, aromatase, and estrogen (E2), may influence the success of in vitro fertilization (IVF) treatments. This research aimed to 1) compare levels of these proteins in follicular fluid (FF) between subjects with successful (n=10) and unsuccessful (n=20) outcomes; 2) compare these proteins between FF and serum within each group; and 3) investigate correlations of these proteins with reproductive hormones. Classification into successful and unsuccessful groups was based on the detection of fetal heartbeat. Blood samples were collected to measure anti-Müllerian hormone (AMH) and follicle-stimulating hormone (FSH) at baseline as well as IGF-1, FSH, P4, androstenedione, aromatase, and E2 in FF and serum on the day of ovum pick-up (OPU). In unsuccessful pregnancies, FF and serum IGF-1 levels and FF aromatase levels were significantly higher, whereas FF and serum androstenedione levels were significantly lower when compared to successful pregnancies ( P<0.05 all). There were no significant differences in FF E2 and P4 levels between groups; however, there was a non-significant trend towards decreased FF E2 levels in unsuccessful pregnancies. Levels of IGF-1, P4, androstenedione, and E2 were significantly higher in FF than in serum in both successful and unsuccessful groups ( P<0.01 all). The FF/serum ratios for successful and unsuccessful subjects were 2.28 and 2.06 for IGF-1, 7,920.61 and 1,919.26 for P4, 5.49 and 4.00 for androstenedione, and 193.45 and 225.60 for E2, respectively. For correlation analyses, basal AMH showed significant positive correlations with serum androstenedione in all subjects and a significant negative correlation with FF E2 in unsuccessful subjects ( P<0.05 all). Basal FSH levels were significantly negatively correlated with serum androstenedione levels in all subjects ( P<0.05). On OPU day, FF androstenedione levels showed significant positive correlations with FF and serum E2 levels in unsuccessful subjects ( P<0.05 all). Serum androstenedione levels were significantly positively correlated with FF E2 levels in successful subjects and with serum E2 levels in all and unsuccessful subjects ( P<0.01 all). FF aromatase levels had significant positive correlations with serum FSH levels in all and unsuccessful subjects ( P<0.01 all). FF E2 levels were significantly positively correlated with FF androstenedione levels in unsuccessful subjects and with serum androstenedione levels in successful subjects ( P<0.01 all). In unsuccessful pregnancies, elevated aromatase levels, despite similar or insignificantly lower FF E2 levels, could likely result from high FSH levels, potentially indicating defective FF E2 production processes that may be due to inadequate substrate availability or decreased aromatase activity. Lower androstenedione levels in unsuccessful pregnancies suggest that there may be a defect in substrate production; however, we could not determine whether there is a defect in aromatase function in these subjects. Lower FF and serum androstenedione levels in unsuccessful pregnancies might be due to poor ovarian response, as indicated by lower basal AMH and higher FSH levels observed in these subjects in our previous study, findings further supported by a positive correlation between androstenedione and basal AMH, and a negative correlation with basal FSH. The elevated IGF-1 levels in the unsuccessful group might be attributable to high FSH levels. Higher levels of IGF-1, P4, androstenedione, and E2 in FF than in serum indicate that these hormones might be primarily secreted into FF before being released into circulation. In conclusion, unsuccessful pregnancies exhibited lower FF and serum androstenedione levels, but higher FF aromatase and IGF-1 levels in both FF and serum, indicating a decreased substrate availability for E2 production in subjects with poor ovarian response. IGF-1, P4, androstenedione, and E2 were likely synthesized primarily in FF rather than serum. This research was funded by the Faculty of Medicine Siriraj Hospital Research Fund ((IO) R016433005 and Grant to support Strategic Driving Project for pushing the World University Rankings by Subject, Fiscal Year 2021 ((IO) R016421007). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.