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RESEARCH QUESTION Can IVF outcomes be predicted from the steroid profile generated by liquid chromatography-mass spectrometry (LC-MS/MS) from follicular fluid collected from a single dominant follicle and serum after ovarian stimulation. DESIGN Prospective observational cohort study in which serum and follicular fluid were collected from women and used to generate steroid profiles by LC-MS/MS. A total of 93 consecutive women enrolled for IVF treatment were recruited at the Fertility Unit, Royal Prince Alfred Women and Babies Hospital, Sydney between September 2014 and July 2015. Baseline and serum levels at oocyte retrieval, as well as follicular fluid samples from the largest single antral follicle, were collected. All samples underwent steroid analysis within a single batch to measure progesterone (P4), oestradiol (E2), oestrone (E1), dehydroepiandrosterone (DHEA), androstenedione (A4), testosterone (T), dihydrotestosterone (DHT), and 3 α, 5α androstanediol (3α-diol) and 3β, 5α androstanediol (3β-diol). RESULTS P4, E2, E1, A4, T, DHEA and A4 were detectable in all baseline serum levels, at oocyte retrieval and in follicular fluid samples, whereas DHT, 3α-diol and 3β-diol were only detectable in a minority of samples. The most consistent predictor of pre-transfer (number of follicles >14mm in diameter, oocytes retrieved or fertilized, day-5 blastocysts) outcomes was baseline serum anti-Müllerian hormone. In follicular fluid, E2 was a negative predictor of the number of oocytes retrieved and the number of day-5 blastocysts but no follicular fluid steroids predicted pregnancy outcome. CONCLUSIONS None of the nine steroids measured in follicular fluid predicted pregnancy outcome in women undergoing IVF.