dants and fatty acids on the lipid profile of mice blastocysts

Objective: To investigate the impact of oocyte vitrification on lipid profile of mice blastocysts by comparing blastocysts originated from fresh and from vitrified oocytes subjected to in vitro fertilization. Also, to assess the effects of supplementing the vitrification media with antioxidants and unsaturated fatty acids by comparing blastocysts vitrified with Irvine Scientific, a commercial standard vitrification media; Tvitri-4, produced in small scale for research by INVITRA®, based on the standard composition with four modifications including carbohydrate trehalose instead sucrose, reduced nonpermeant cryoprotectant concentration, and addition of two aminoacids; and Tvitri-4 supplemented with L-carnitine (LC) and oleic and linoleic fatty acids (FA). Methods: Experimental study including 23 C57BL/6J mice females superovulated with 5UI eCG followed by 5UI hCG. Oocytes (n=562; 4 replicates) were randomly divided in 4 groups: fresh control group (FC), vitrified using Irvine (IRV), Tvitri-4 (T4), or supplemented Tvitri-4 (T4-LC/FA) media. Fresh or vitrified-thawed oocytes were inseminated with spermatozoa and cultivated for 96 or 120 hours in KSOM (Cosmo bio co., LTD) incubated at 37oC with 5% CO2. Blastocysts of each group were individually fixed in methanol/ water. Lipids were extracted using methanol (9 blastocysts/ group) and flow injected into a triple quadrupole spectrometer equipped with an electrospray ion source. Lipids were analyzed using the multiple reaction monitoring profiling (MRM-profiling) method and values of relative intensities of ions detected in each group were compared using univariate (one-way ANOVA, volcano plot) and multivariate analysis (PLS-DA). Results: One-way ANOVA (p-value ≤0.05) showed that 90 out of the 125 lipids were differently expressed among the four groups, while a comparison between the vitrified groups showed no difference. Two by two comparisons between the control and vitrified groups using volcano plot (p-value ≤0.05, fold-change ≥1.5) detected 55, 17, and 11 significant features between FC vs. IRV, FC vs. T4, and FC vs. T4-LC/ FA, respectively; all of them more abundant in the FC group. Partial least square discriminant analysis (PLS-DA) variables of importance (VIP scores) higher than 1.3 followed the same pattern and identified the phosphatidylinositol containing 36 carbon and one unsaturation in the fatty acyl residues PI(36:1), and phosphatidylcholines PC(38:4), PCo(36:5), PC(34:1), and PC(30:0) among the top features; the exception being the free stearic acid (C18:0), which was the top feature in the FC x T4-LC/FA comparison, being more abundant in the latter. Conclusion: Vitrification changed the lipid profile of mice blastocysts causing an overall reduction on lipid abundances that affected PC lipids the most. This effect was more apparent in IRV, followed by T4, and T4-LC/FA suggesting that supplementation of media with L-carnitine and unsaturated JBRA Assisted Reproduction 2022;26(1):165-196 doi: 10.5935/1518-0557.20210105