In this study we evaluated possible differences in metabolomic profiles of spent embryo culture media (SECM) of human embryos with distinct morphology, karyotype, and implantation outcomes. A total of 153 samples from embryos of patients undergoing in vitro fertilization (IVF) programs were collected and analyzed by HPLC-MS. Metabolomic profiling and statistical analysis revealed clear clustering of day five SECM from embryos with different morphological classes and karyotype. Profiling of day five SECM from embryos with different implantation outcomes showed 241 significantly changed molecular ions in SECM of successfully implanted embryos. Separate analysis of paired SECM samples on days three and five revealed 46 and 29 molecular signatures respectively, significantly differing in culture media of embryos with a successful outcome. Pathway enrichment analysis suggests certain amino acids, vitamins, and lipid metabolic pathways to be crucial for embryo implantation. Differences between embryos with distinct implantation potential are detectable on the third and fifth day of cultivation that may allow the application of culture medium analysis in different transfer protocols for both fresh and cryopreserved embryos. A combination of traditional morphological criteria with metabolic profiling of SECM may increase implantation rates in assisted reproductive technology programs as well as improve our knowledge of the human embryo metabolism in the early stages of development.

The aim of this study was to determine the changes of metabolomic profiles in embryonic culture media (ECM) for the evaluation of quality and implantation potential of human embryos. ECM (n=163) were collected on day 5 before transfer or cryopreservation. Some embryos were used in preimplantation genetic screening for detection of aneuploidy karyotypes. Samples were subdivided into groups according to embryo morphological classification (by Gardner), genetic analysis and implantation data. ECM were extracted with methanol, precipitates were separated by centrifugation and metabolite production of individual embryo was analysed by LC-MS (the positive ion mode). After peak detection and retention time alignment, data were analysed using the PCA algorithm. MS fingerprinting analysis of embryo culture medium showed significant differences between morphologically divided groups. Intragroup comparisons did not reveal differences between subclasses. Genetic screening of embryos revealed 33 aneuploid karyotypes. It was shown that chromosome number did not affect the metabolite profiles comparing with the normal group. The culture media of embryos that were positive or negative for successful implantation showed specific signatures that allowed to distinguish embryos with different outcomes.The characterization of ECMs by LC-MS may facilitate more accurate selection of the best embryo for the implantation, improving single-embryo transfer and thus eliminating the risk and undesirable effects of multiple pregnancies.