Age-Effect On The Fatty Acid And Lipidomic Profile Of The Follicular Fluid During In-Vitro Fertilization Cycles.

Compelling evidence suggests that the metabolic profile and lipidomic composition of the human follicular fluid (FF) may play a role in reproductive outcomes. However, there are gaps in current knowledge regarding the follicular environment of aging, which is the main factor affecting female reproductive capacity. Thus, we aimed to evaluate if there is any variability in the fatty acid and lipidomic composition of the FF between oocyte donors and infertile patients with advanced age. Prospective observational case-control and experimental study. Non-targeted lipidomic and fatty acid analysis of the FF from two groups: 17 oocyte donors aged < 35 years versus 17 infertile women aged ≥ 38 years that underwent controlled ovarian stimulation with an antagonist protocol between September and October 2018. Women with diseases that could potentially impair ovarian function were excluded. Pooled follicular aspirate from each patient was centrifuged at 800g for 20 min to isolate FF from cells. The supernatant was sterile-filtered using a 0.22 mm pore size membrane filter and stored at -80ºC. Lipids were extracted according to the method of Bligh and Dyer, subjected to liquid chromatography-mass-spectrometry and analysed using MassHunter Qualitative Analysis software. Fatty acid groups were analysed as methyl ester derivates by gas chromatography and compared with authentic standards. We detected 4431 molecular species in the FF. Only those found in at least 70% of samples of the quality-control and presenting a low variability were selected, which resulted in 159 lipid species. Multivariate analysis failed to demonstrate statistically significant differences in the lipidomic profile between groups. Principal component analysis (PCA) and hierarchical clustering revealed that the lipidome does not seem to be an important feature defining the reproductive age. Similarly, partial least square-discriminant analysis (PLS-DA) was applied to identify molecules that could explain variability within samples. Despite the good clusterization between groups, permutation test and cross-validation showed that the model was overfitted. Univariate statistics revealed two lipid species which were substantially increased in the older group. Those were the ones with the highest weight explaining the variability in the PLS-DA model. Unfortunately, only one of them could be identified, the sphinganine. Finally, we analyzed the fatty acid composition of samples, but no differences were found regarding saturated fatty acids, unsaturated fatty acids, monounsaturated fatty acids, and polyunsaturated fatty acids n-3 and n-6 series. Likewise, the double bond index and the peroxidability index were similar between groups. The sphinganine was the only lipid specie showing higher concentrations in FF from older patients. Since the sphinganine is a precursor of ceramide, it could be associated with increased apoptotic metabolism and, ultimately, depletion of ovarian reserve and function as women age. Further molecular studies should confirm our findings.