OBJECTIVE To characterize nuclear and mitochondrial DNA (mtDNA) in spent culture media from normally developing blastocysts to determine whether it could be used for noninvasive genetic assessment. DESIGN Prospective embryo cohort study. SETTING Academic center and private in vitro fertilization (IVF) clinic. PATIENT(S) Seventy patients undergoing intracytoplasmic sperm injection (ICSI) and 227 blastocysts. INTERVENTION(S) Culture media assessment, artificial blastocoele fluid collapse and DNA analysis using digital polymerase chain reaction (dPCR), long-range PCR, quantitative PCR (qPCR), and DNA fingerprinting. MAIN OUTCOME MEASURE(S) Presence of nuclear and mtDNA in three different commercial culture media from Vitrolife and Irvine Scientific, spent embryo media assessment at the cleavage and blastocyst stages of development, and analysis of the internal media controls for each patient that had been exposed to identical conditions as embryo media but did not come into contact with embryos. RESULT(S) Higher levels of nuclear and mtDNA were observed in the culture media that had been exposed to embryos compared with the internal media controls. Nuclear DNA (∼4 copies) and mtDNA (∼600 copies) could be detected in spent media, and the levels increased at the blastocyst stage. No increase in DNA was detected after artificial blastocoele fluid collapse. Mixed sex chromosome DNA was detected. This originated from contamination in the culture media and from maternal (cumulus) cells. Due to the limited amount of template, the presence of embryonic nuclear DNA could not be confirmed by DNA fingerprinting analysis. CONCLUSION(S) Currently DNA from culture media cannot be used for genetic assessment because embryo-associated structures release DNA into the culture medium and the DNA is of mixed origin.