The Metabolomic Profile of Spent Culture Media from Day-3 Human Embryos Cultured under Low Oxygen Tension

Abstract

Despite efforts made to improve the in vitro embryo culture conditions used during assisted reproduction procedures, human embryos must adapt to different in vitro oxygen concentrations and the new metabolic milieu provided by the diverse culture media used for such protocols. It has been shown that the embryo culture environment can affect not only cellular metabolism, but also gene expression in different species of mammalian embryos. Therefore we wanted to compare the metabolic footprint left by human cleavage-stage embryos under two types of oxygen atmospheric culture conditions (6% and 20% O2). The spent culture media from 39 transferred and implanted embryos from a total of 22 patients undergoing egg donation treatment was analyzed; 23 embryos came from 13 patients in the 6% oxygen concentration group, and 16 embryos from 9 patients were used in the 20% oxygen concentration group. The multivariate statistics we used in our analysis showed that human cleavage-stage embryos grown under both types of oxygen concentration left a similar metabolic fingerprint. We failed to observe any change in the net depletion or release of relevant analytes, such as glucose and especially fatty acids, by human cleavage-stage embryos under either type of culture condition. Therefore it seems that low oxygen tension during embryo culture does not alter the global metabolism of human cleavage-stage embryos.

Fig 1. Principal components analysis (PCA) of untargeted global metabolomic profiles of the spent culture media from embryos cultured under 6% oxygen (red) and 20% oxygen (blue) concentrations.

The first two principal components, t[1] and t[2], which represent the most important metabolic variation in the samples, are shown and were captured by Positive (R²X = 0.662 and Q² = 0.519) (A) and Negative (R²X = 0.838 and Q² = 0.492) analysis modes (B).

Fig 2. Volcano plots representing the relationship between fold change and statistical significance for all the spent culture media metabolites metabolites analyzed.

The log2 average fold-change between the two groups is represented on the x-axis: 6% oxygen vs. 20% oxygen. The y-axis represents the -log10 p-value.

Fig 3. Box plots for the three metabolic features which were found to be significantly different after univariate analysis.

Each metabolic feature is labeled based on their retention time and mass-to-charge ratio (termed Rt-m/z), 0.6267_671.7923 (A) 0.4981_151.0359 (B) and 0.583_92.9273 (C).

Fig 4. Box plots for selected metabolic features related to lipid, amino acids and carbohydrate metabolism.

No significant differences were observed after univariate analysis. Araquidonic acid (20:4n-6) (A); alpha-linolenic acid (18:3n-6) (B); lysophosphatidylcholine LPC(18:1) (C); tryptophan (D); phenylalanine (E) and glucose and related hexoses metabolites (F).