pathways, the nuclear receptors known to regulate the pathway, and the end product bile acids. Furthermore, we provide functional evidence that bile acids are produced by the human follicular granulosa cells in response to cholesterol presence in the culture media. These findings establish a novel pathway present in the human ovarian follicle that has the capacity to compete directly with sex steroid synthesis. Results Bile Acids are Present in Human Ovarian Follicular Fluid Follicular fluid (FF) from mature follicles from 93 patients was obtained during in vitro fertilization (IVF). The individual FF samples were analyzed in duplicate for content of total bile acids by enzymatic assay. The mean bile acid concentration in FF was measured to be 17.301 mM (STD 6 5.41) (Figure 1, a). The origin of bile acids in FF is not known. They could enter the follicle through the blood, or they could be synthesized in the follicular compartment. In the case of diffusion from blood, their content in FF and serum is expected to be nearly identical. Alternately, active bile acid synthesis within the follicle could result in a higher follicular content compared to serum. In order to investigate the bile acids origin we compared content in blood and FF in 19 of the patients. The mean concentration of bile acids in serum obtained within 30 minutes of FF collection was found to be 7.672 mM Ovarian Bile Acid Synthesis (STD 6 2.548) (Figure 1, b). This dramatic difference of bile acid content in FF and serum is surprising and statistically significant (p,0.000001). The IVF procedure includes treatment with human chorionic gonadotropin (hCG). It is likely that the observed difference is a result of the hCG treatment. Therefore, we compared bile acid content in blood collected 24 hours before and 36 hours after hCG administration in 13 of these patients to evaluate the impact of hCG administration on bile acid content (Figure 1, c). The mean pre-hCG bile acid concentration in the serum of the 13 samples was measured to be higher (13.61 mM, STD 6 5.47) than in the post-hCG serum (7.672 mM) (p = 0.00064). The mean pre-hCG bile acid concentration in the serum (13.61 mM ) was lower than in the corresponding FF (17.301 mM) but this difference was not statistically significant (p = 0.185). The highly significant difference in the bile acid content between FF and the post-hCG serum obtained within 30 minutes of FF collection supports the proposal for active intrafollicular ovarian synthesis. This is the first demonstration of the presence of bile acids in this compartment. The high cholesterol concentration in the follicle is likely to be supporting not only active steroidogenesis but also synthesis of bile acids. This finding raises the obvious question of whether the enzymes responsible for bile acid synthesis are present in the ovarian follicle. The intrafollicular synthesis of bile acids could be facilitated by activities within the granulosa cells, the oocyte, or both. Figure 1. Presence of bile acids in human ovarian follicular fluid. Bile acid concentration was measured in FF (Panel a), in FF compared to post-HCG serum (Panel b), and in pre-HCG serum compared to post-HCG serum (Panel c). doi:10.1371/journal.pone.0007333.g001 PLoS ONE | www.plosone.org 2 October 2009 | Volume 4 | Issue 10 | e7333