Non-Invasive Prediction of Blastocyst Formation by Day Three Embryo Culture Medium Mass Spectrometry Lipid Fingerprinting.

Objective: To identify lipid markers of blastocyst formation by day three culture medium mass spectrometry (MS) fingerprinting.

Methods: For this study, 50 embryo samples from culture media were harvested on day three, from patients undergoing embryo transfers on day five. Samples were split into groups based on their degree of expansion and hatching status on day five (Complete-Blastocyst, n=25 and No-Blastocyst, n=25) and its secretomes were analysed by MS. Mass spectra fingerprinting was acquired using a Q-Tof spectrometer (LC-MS, Agilent 6550 iFunnel Q-TOF) equipped with an automated injector. The data was analysed using the principal component analysis (PCA) followed by a partial least square discrimination analysis (PLS-DA), combined with variable influence in the projection (VIP) scores.

Results: In total, there were 1,657 ions found, in which 165 ions were differently expressed between groups, with a fold chance ≥ 4x and P<0.001, in the t-test. PLS-DA showed a clear separation between the groups and among 15 VIPs selected by the program, 13 of them were highly expressed in the Complete-Blastocyst Group and two were expressed in the No-Blastocyts Group. Besides embryo status on day five, the PLS-DA was also able to classify samples according to patients' age. Lipids supposedly highly expressed in the Complete-Blastocyst Group included: isoprenoids, diacylglycerols, sterols, fatty esters, secosteroids, phosphosphingolipids, glycerophosphates and diacylglycerophosphates, while fatty amides were suggested to be highly expressed in the No-Blastocysts Group.

Conclusions: Day three culture medium MS is a promising approach for the identification of embryos that should be cultured until day five.