Non-invasive prediction of blastocyst implantation, ongoing pregnancy and live birth, by mass spectrometry lipid fingerprinting

OBJECTIVE To identify lipid markers of blastocyst implantation and ongoing pregnancy by day three culture medium mass spectrometry (MS) fingerprinting. METHODS For this study, 33 culture media samples were harvested on day three, from 22 patients undergoing day five embryo transfers. All embryos achieved the blastocyst stage and were split into groups based on their implantation (Negative Implantation, n= 14 and Positive Implantation, n= 19). The positive implantation cycles resulted in successful ongoing pregnancies. The lipid extraction was performed by the Bligh-Dyer protocol and mass spectra were obtained with a direct infusion into a Q-Tof mass spectrometer. The data obtained was analyzed by Principal Component Analysis (PCA) and Partial Least Square Discrimination Analysis (PLS-DA). The statistical analysis was performed using the Metabo-Analyst 2.0. RESULTS The variable importance in the projection (VIP) plot of the PLS-DA provided a list of four ions, in the positive mode, with an area under the curve (AUC) of 73.5%; and eight ions, in the negative mode, with and AUC of 72.0%. For both positive and negative modes, possible biomarkers for the negative implantation were identified by the lipidmaps: phosphoethanolamine, dicarboxylic acids, glycerophosphoglycerol, glycerophosphocholine, glicerophosphoinositol, phosphoethanolamine and unsaturated fat acids. The other ions were not identified. These lipids are involved in the GPI anchor biosynthesis and synthesis of lycerophospholipids and phosphate inositol. CONCLUSION MS fingerprinting is useful to identify blastocysts that fail to implant, and therefore this technique could be incorporated into the laboratory routine, adjunct to morphology evaluation to identify embryos that should not be transferred.