Recent studies have shown that the metabolome of single embryo culture media is linked to successful pregnancy. In this study, the analysis was expanded to compare the metabolomes of viable and non-viable early-stage embryos and to examine metabolomic markers associated with hatching in viable embryos. The authors hypothesized that the metabolomic profiles of high-quality early blastocysts differ from those of non-viable embryos that reach the blastocyst stage but undergo developmental arrest at later stages. The metabolic profile of 43 spent bovine embryo culture medium samples were analyzed using liquid chromatography–mass spectrometry, covering 189 metabolites, including 40 acylcarnitines, 42 amino acids/biogenic amines, 91 phospholipids, 15 sphingolipids, and the sum of hexoses. Embryos were produced from abattoir-derived oocytes, and the culture medium samples were derived from Grade 1 early blastocysts that progressed to hatching (VBL; n = 10), non-viable early blastocysts that developed to the blastocyst stage but failed to hatch (DBL; n = 12), Grade 1 hatched blastocysts (HBL; n = 16), and plain growth media for control (CM; n = 5). It was observed that methionine sulfoxide (Met-SO) and lysophosphatidylcholine (lysoPC) C24:0 concentrations were significantly lower in the culture media from viable blastocysts compared to those from non-viable blastocysts (p < 0.001). Additionally, blastocysts that resulted in successful hatching had significantly lower levels of phospholipid, arginine (Arg), and methionine-related metabolites that significantly differentiated the control and viable blastocyst culture media from the media containing non-viable embryos. Building on previous studies, there appears to be an overlap in metabolites released during hatching that are also associated with successful pregnancy. The identified biomarkers can aid in assessing an embryo’s developmental potential and enhance embryo selection for transfer or cryopreservation.

This study investigated whether metabolomic fingerprints of bovine embryo growth media improve the prediction of successful embryo implantation. In this prospective cohort study, the metabolome from in vitro-produced day 7 blastocysts with successful implantation (n = 11), blastocysts with failed implantation (n = 10), and plain culture media without embryos (n = 5) were included. Samples were analyzed using an AbsoluteIDQ^® p180 Targeted Metabolomics Kit with LC-MS/MS, and a total of 189 metabolites were analyzed from each sample. Blastocysts that resulted in successful embryo implantation had significantly higher levels of methionine sulfoxide (p < 0.001), DOPA (p < 0.05), spermidine (p < 0.001), acetylcarnitine-to-free-carnitine ratio (p < 0.05), C2 + C3-to-free-carnitine ratio (p < 0.05), and lower levels of threonine (nep < 0.001) and phosphatidylcholine PC ae C30:0 (p < 0.001) compared to control media. However, when compared to embryos that failed to implant, only DOPA, spermidine, C2/C0, (C2 + C3)/C0, and PC ae C30:0 levels differentiated significantly. In summary, our study identifies a panel of differential metabolites in the culture media of bovine blastocysts that could act as potential biomarkers for the selection of viable blastocysts before embryo transfer.

Equine in vitro embryo production (IVP) via ovum pick-up (OPU) and intracytoplasmic sperm injection (ICSI) has gained prominence in sport-horse breeding due to high success rates, efficient semen utilization, and year-round embryo production, including from mares with fertility challenges or postmortem. However, assessing viability of early-stage ICSI-produced equine embryos remains challenging due to subjective morphological evaluations and limited visibility of developmental features. Non-invasive metabolomic analysis of culture media offers a promising alternative to enhance embryo selection. This study compared metabolomic profiles of culture media from viable Day 4 ICSI-derived equine embryos progressing to the blastocyst stage with non-viable embryos arrested at the morula stage. Thereby, hypothesizing distinct metabolic signatures associated with developmental competence. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), amino acids, lipids, and other metabolites in individually cultured embryo media were analyzed. Results showed viable Day 4 embryos consumed significantly more dihydroxyphenylalanine (DOPA) than non-viable embryos. Increased secretion of amino acid derivatives (e.g., hippuric acid, glutamate, homoarginine), glycerolipids (e.g., diglyceride DG 18:0_20:0, triglyceride TG 17:0_36:4), phosphatidylcholines (PC O-34:3, PC O-36:3), cholesteryl ester (CE 22:2), ceramides (Hex2Cer d18:1/24:1, Hex2Cer d18:1/20:0, Cer d16:1/23:0), dehydroepiandrosterone sulfate (DHEAS), and glycodeoxycholic acid (GDCA) were observed in viable early embryos. These findings suggest metabolomic profiling can identify biomarkers of equine embryo viability as early as Day 4, potentially enabling early embryo selection, shorter culture period and improved vitrification and transfer outcomes. This study represents one of the first comprehensive metabolomic analyses of equine ICSI embryos, laying a foundation for developing non-invasive, biomarker-based assays to enhance assisted reproductive technologies (ART) efficiency.

We recently demonstrated that the metabolome of single embryo culture media is associated with successful pregnancy [13]. In this study, we expanded the analysis to compare the metabolomes of viable and nonviable early-stage embryos, as well as to examine metabolomic markers associated with hatching in viable embryos. We analyzed the metabolic profile of 43 spent bovine embryo culture media samples using liquid chromatography-mass spectrometry, covering 189 metabolites, including 40 acylcarnitines, 42 amino acids/biogenic amines, 91 phospholipids, 15 sphingolipids, and the sum of hexoses. The embryos were produced from abattoir-derived oocytes, and the culture media samples were derived from Grade 1 early blastocysts that progressed to hatching (n = 10), non-viable early blastocysts that developed to the blastocyst stage but failed to hatch (n = 12), Grade 1 hatched blastocysts (n = 16), and plain growth media for control (n = 5). We found that methionine sulfoxide (Met-SO) and Lysophosphatidylcholine C24:0 concentrations were significantly lower in the culture media from viable blastocysts compared to those from non-viable blastocysts (p < 0.001). Additionally, blastocysts that resulted in successful hatching had significantly lower levels of phospholipid, arginine and methionine-related metabolites that significantly differentiated the control and viable blastocyst culture media from the media containing non-viable embryos. Building on previous studies, there appears to be an overlap in metabolites released during hatching that are also associated with successful pregnancy. The identified biomarkers can aid in assessing an embryo's developmental potential and enhance embryo selection for transfer or cryopreservation.

This study investigated whether metabolomic fingerprints of bovine embryo growth media improve the prediction of successful embryo implantation. In this prospective cohort study, the metabolome from in vitro produced Day 7 blastocysts with successful implantation (n=11), blas-tocysts with failed implantation (n=10), and plain culture media without embryos (n=5) was in-cluded. Samples were analyzed using AbsoluteIDQ® p180 Targeted Metabolomics Kit with LC-MS/MS, and a total of 189 metabolites were analyzed from each sample. Blastocysts that re-sulted in successful embryo implantation had significantly higher levels of methionine sulfoxide (p<0.001), DOPA (p<0.05), spermidine (p<0.001), acetylcarnitine to free carnitine ratio (p<0.05), C2+C3 to free carnitine ratio (p<0.05), and lower levels of threonine (nep<0.001) and phosphati-dylcholine PC ae C30:0 (p<0.001) compared to control media. However, when compared to embryos that failed to implant, only DOPA, spermidine, C2/C0, (C2+C3)/C0 and PC ae C30:0 levels differentiated significantly. In summary, our study identifies a panel of differential metabolites in the culture media of bovine blastocysts that could act as potential biomarkers for the selection of viable blastocysts before embryo transfer.