OBJECTIVE: To evaluate the effect of oocyte vitrification in the metabolomic profile of embryos developed from vitrified and fresh oocytes in our ovum donation program. DESIGN: Analysis of the metabolic profiles of spent culture medium samples corresponding to embryos developed from vitrified and fresh oocytes. SETTING: In vitro fertilization (IVF) unit/metabolomic facility. PATIENT(S): Oocyte donors between the ages of 18 and 35 years. INTERVENTION(S): Metabolomic profile liquid chromatography coupled with mass spectrometry (LC-MS) of spent media samples. MAIN OUTCOME MEASURE(S): Identification of spent media components and metabolites present and absent in vitrified and fresh day-3 embryos. RESULT(S): We obtained a total of 190 spent media samples: vitrification group (65), fresh group (59), and a matched control media group (66). Multivariate data analysis was performed after global metabolomic and amino acid profiles did not reveal any statistically significant differences in day-3 embryos derived from fresh and vitrified oocytes, indicating that other metabolic differences between the samples (e.g., patient-to-patient variability, analytical variation) are greater than those between the vitrified and fresh sample groups. Univariate statistical analysis revealed a series of possible biomarkers, such as tryptophan, phenylalanine, and 2,5,7,8-tetramethyl-2-(2'-carboxyethyl)-6-hydroxychroman (alpha-CEHC), although only alpha-CEHC was statistically significant after correction for multiple testing. CONCLUSION(S): Multivariate data analysis did not reveal statistically significant differences between the analyzed groups, suggesting that oocyte vitrification does not disturb embryonic metabolomic profiles.