Lipid metabolism provides a potent source of energy and has an important role in the acquisition of oocyte competence. However, there are conflicting reports about how lipid exposure during invitro maturation (IVM) impacts the gamete and further embryo development. In this study, we performed IVM of oocytes in the presence of lipid-rich culture media and used a broad lipid screening to accurately map the impact on the lipid profile and developmental potential. For that, nonpolar lipids were extracted from fetal bovine serum (FBS) with organic solvents (Bligh-Dyer method) and then used to supplement IVM medium (TCM-199 bicarbonate+10% FBS, hormones, pyruvate and antibiotics). COCs obtained from abattoir ovaries were submitted to IVM (4 biological replicates) in 2 groups: OC (control; IVM medium) and OHL (high lipid; IVM medium supplemented with extra 10% FBS nonpolar lipids). After 24h, we collected mature oocytes and those remaining followed to IVF and then to IVC (synthetic oviductal fluid with amino acids, SOFaa, with 5% FBS) for 7 days at 38.5°C, 20% O2, and 5% CO2 in air in high humidity. Expanded blastocysts were collected (BC and BHL) and blastocyst rates were assessed. Lipid extracts of individual oocytes and embryos (n=10/group) were analysed by multiple reaction monitoring (MRM)-profiling mass spectrometry. A total of 379 lipids from 10 classes were investigated [triacylglycerol (TAG), cholesteryl esters (CE), free fatty acids (FFA), acyl-carnitine, sphingomyelin (SM) and phospholipids derived from choline (PC), ethanolamine (PE), glycerol (PG), serine (PS), and inositol (PI)]. Exploratory data analysis was performed by principal component analysis (PCA; Metaboanalyst 4.0), and fold-change (FC) values were calculated based on the relative intensity of lipid ions (FC > 2 and P<0.05). IVC rates were compared by t-test (α=5%). PCA revealed a clear distinction in the lipid content for both oocytes and blastocysts (control vs. treated). More specifically, there was 2-fold enrichment for total TAG and CE in control groups and a 1.5-fold enrichment for total FFA in the treated groups at the oocyte and the blastocyst stages. Surprisingly, the average blastocyst rate was higher in the group derived from oocytes exposed to a high-lipid environment (41.56±7.73 vs. 22.62±1.67; P=0.003), which led us to investigate specific lipid ions. Groups OHL and BHL had increased contents of structural and signalling phospholipids (PC, SM, PE, and PS) and up to 3 times more oleic and linoleic acids, which have been associated with improved oocyte maturation and blastocyst development. Here, we demonstrate how distinct lipid exposures during IVM can robustly alter the lipid profile of oocytes. But more interestingly, it is clear that these are long-term effects, still observed at the blastocyst stage. More studies are required to verify the metabolic impact of this alternative lipid supplementation; however, these results indicate that high lipid exposure is not necessarily detrimental and, at a certain point, may even counteract lipid accumulation commonly observed during invitro embryo production.
Comparison of two methods for the separation of polar from nonpolar lipids
