Abstract Study question Is there any relationship between blastocyst spent culture medium metabolite levels and reproductive outcomes? Summary answer Metabolomic analysis of 173 metabolites revealed differences in pregnancy, implantation, and live birth rates, highlighting specific metabolites' associations with reproductive outcomes. What is known already Identifying embryos with higher implantation potential poses a significant challenge in assisted reproductive technology (ART). Currently, morphological criteria are utilized for this selection; however, this method does not consistently offer precise predictions of successful implantation. In recent years, there are several hypothesis to employ metabolomic approaches to assess developing embryos and discriminate preferable profiles associated with embryo viability. Metabolomics involves the non-targeted identification and quantification of low molecular weight end-products of metabolism, referred to as metabolites. However, there is limited knowledge regarding the impact of metabolomic profile on implantation rates. Study design, size, duration This prospective case control study enrolled 70 patients who underwent intracytoplasmic sperm injection (ICSI) and single blastocyst transfer. Spent culture mediums (SCM) were collected in fifth day of each embryo. Metabolite levels of SCMs were measured by Liquid Chromatography-Mass Spectrometry. Metabolite levels were compared according to pregnancy, implantation and live birth rates. Participants/materials, setting, methods Normo-responder patients aged between 21 and 39, experiencing primary infertility, whose embryos progressed to the blastocyst stage and underwent single blastocyst transfer. Human Metabolome Database and Compound Discoverer 3.3 was used for the identification of the metabolites. Statistical Package for the Social Sciences (SPSS) software were used for statistical analysis. p<.05 was considered statistically significant. Main results and the role of chance The baseline parameters between groups were not significantly different between pregnant and non-pregnant patient groups. A total of 173 metabolites were analysed. When the metabolomics compared according to pregnancy results(68.5%), 1-(4-Methoxyphenyl)-3-pentanone, morpholine propane sulfanoic acid, naproxen were found higher in pregnant group while 4-indolecarbaldehyde, eicosapentaenoic acid, hepes were significantly higher in non-pregnant group (p<.05). According to the implantation (65.7%) rates, 1-(4-Methoxyphenyl)-3-pentanone was significantly higher in the implantation group. According to the live birth rates (54.2%), 1-(4-Methoxyphenyl)-3-pentanone, 15,16-Dihydroxyoctadecanoic acid, ALA-PRO, HEPES, Naproxen, pyroglutamylglycine, lactamide (p<.05) was higher in the live birth group. 1,2,3,4-Tetrahydro-β-carboline-3-carboxylic acid, diethyl ether and L-Glutamine was higher in non-livebirth group. Limitations, reasons for caution These results can not be generalized to the entire population due to small sample size of our study. More work is needed on the subject. Wider implications of the findings We detected significant differences in some of the metabolites in patients with different reproductive outcomes. The metabolomic analysis of blastocyst spent culture medium can be used as an adjunt method for improving non invasive embryo evaluation in clinical practice after evidence based results. Trial registration number none