Abstract Study question Is the GDF9 concentration in Day 3 human embryo culture medium linked to embryo quality and clinical outcome? Summary answer The embryo quality and clinical outcome appears to be linked to the GDF9 concentration in Day 3 culture medium. What is known already Previous studies have demonstrated that the GDF9 level in follicular fluid and cumulus granulosa cells can be used as biomarker for predicting embryo quality. Study design, size, duration In study 1, a total of 280 embryos from 70 patients who obtained at least 4 embryos with 6-10 blastomeres (2 transferable and 2 non-transferable embryos) at Day 3 were enrolled. The corresponding GDF9 concentration in culture media were compared between transferable and un-transferable embryos and also compared among good, fair and poor quality embryos. In study 2, a total of 119 embryos from 61 patients (29 implanted and 32 non-implanted patients) were enrolled. Participants/materials, setting, methods The expression pattern of GDF9 in human embryos was investigated using Q-PCR and immunofuorescence. GDF9 concentration in embryo culture media was quantified by ELISA assay. Statistical analyses defined correlations between GDF9 concentration and embryo quality, in study 1, and implantation and live birth, in study 2. Main results and the role of chance GDF9 mRNA and protein were detected from human oocytes to eight-cell embryos and displayed a slow decreasing trend. In study 1, GDF9 concentration in culture medium is lower for transferable embryos compared with non-transferable embryos (331 pg/mL (quartiles: 442, 664 pg/mL) vs. 518 pg/mL (quartiles: 328, 1086 pg/mL), P < 0.001), and increased commensurate with the diminution of the embryo quality (P < 0.001). In study 2, signifcantly lower GDF9 concentration was detected for implanted embryos than non-implanted embryos (331 pg/mL (quartiles: 156, 665 pg/mL) vs. 518 pg/mL (quartiles: 328, 1086 pg/mL), P < 0.001). The same trend was found between the embryos that led to live birth and those that failed. Limitations, reasons for caution Further randomized controlled trials are needed for exploring whether GDF9 assay of spent embryo culture medium can improve the clinical outcome in IVF. Wider implications of the findings GDF9 in culture medium has the potential to be a non-invasive biomarker for embryo selection. Trial registration number not applicable