Dehydroepiandrosterone Supplementation and the Impact of Follicular Fluid Metabolome and Cytokinome Profiles in Poor Ovarian Responders

Background: Poor ovarian responders (POR) are women undergoing in-vitro fertilization who respond poorly to ovarian stimulation, resulting in the retrieval of lower number of oocytes, and subsequently lower pregnancy rates. The follicular fluid (FF) provides a crucial microenvironment for the proper development of follicles and oocytes through tightly controlled metabolism and cell signaling. Conversely, dysregulated FF metabolism and cytokine production in POR could impart or reflect detrimental effects on oocytes. Androgens such as dehydroepiandrosterone (DHEA) have been proposed to alter the POR follicular microenvironment and induce metabolic changes but its utility in improving pregnancy rates remain uncertain, partially due to the unknown impact DHEA imposes on the FF metabolome and cytokine profiles. Methods: FF samples were collected from 52 POR patients who underwent IVF with DHEA supplementation (DHEA+) and without (DHEA-). The FF samples were split for profiling analyses using untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) metabolomics and a large-scale multiplex suspension immunoassay covering 65 cytokines, chemokines and growth factors. Multivariate statistical modelling by partial least squares-discriminant regression (PLSR) analysis was performed for revealing metabolome-scale differences. Further, differential metabolite analysis between the two groups was performed by PLSR β-coefficient regression analysis and Student’s t-test. Results: Untargeted metabolomics identified 118 FF metabolites of diverse chemistries and concentrations which spanned three orders of magnitude. They include metabolic products highly associated with ovarian function – amino acids for regulating pH and osmolarity, lipids such fatty acids and cholesterols for oocyte maturation, and glucocorticoids for ovarian steroidogenesis. The FF metabolomic profiles of DHEA+ and DHEA- groups were notably distinct as classified by multivariate partial least squares regression analysis. Specifically, glycerophosphocholine, linoleic acid, progesterone, and valine were significantly lower in DHEA+ relative to DHEA-. Using the large-scale cytokine profiling assay, we observed significantly lower MCP1, IFNγ, LIF and VEGF-D levels in DHEA+ relative to DHEA. Conclusions: Collectively, our large-scale study characterized a diversity of metabolites and cytokines in the FF of POR individuals and identified several of these compounds associated DHEA supplementation.