P-629 Dysregulation of the immunometabolic profile in the aging human ovary

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Abstract Study question Is ovarian aging associated with the generation of inflammatory macrophages with a dysregulated immunometabolism and be ameliorated after treatment with platelet-rich plasma-releasate (PRPr)? Summary answer Aged follicular macrophages show an inflammatory profile associated with an altered metabolism that can be reprogrammed with an alternative therapeutic treatment. What is known already Inflammaging is a systemic and chronic inflammatory response, linked with advanced age. Particularly in the ovary, inflammaging leads to a decrease in the ovarian reserve, oocyte quality and infertility. PRPr therapy is being widely explored to improve ovarian function and increase the chances of pregnancy. Here, we investigate the mechanisms underlying the exacerbated inflammatory response during ovarian aging, focusing on the metabolic profile of follicular macrophages and a novel T lymphocyte regulatory subpopulation, CD3+CD4-CD8- (DNT), that is emerging as effector cells capable of mediating immune tolerance in the female reproductive system. Study design, size, duration Macrophages and lymphocytes were recovered from follicular fluid (FF) from women of different ages and tested by immune-staining and FACS analysis. In macrophages (CD68+), we evaluated lipid accumulation, reactive oxygen species (ROS), lactate and IL-1β production, and mitochondrial membrane potential. Immune cells profiles were correlated with clinic parameters such as number of M2 oocytes and atretic follicles. Finally, macrophages were treated in vitro with PRPr and, after 5 days, reprogramming of macrophages profile was tested. Participants/materials, setting, methods We studied FF (n = 102) from women from ovodonation (21-29y) and ART indication (36-42y). The San Isidro Ethics Committee, Buenos Aires, Argentina, approved this study. Mononuclear cells were isolated using Ficoll-Hypaque and adherent macrophages profile (CD68+ IL-1β+) and DNT cell subpopulation (CD3+CD4-CD8-) tested by flow cytometry. Lipid droplets accumulation, ROS and mitochondrial potential were measured using their respective probes. Follicular macrophages were treated with PRPr (obtained from fertile women) for 5 days. Main results and the role of chance FF macrophages from aged women (35-42y) showed a significant increase in IL-1β production in comparison with the youngest group (21-29y), that also correlates negatively with the number of M2 oocytes recovered. Focusing on macrophage (CD68+) metabolic profile we found an increase in lipid droplets in follicular macrophages from aged women in comparison with the younger group. Moreover, these macrophages had hyperpolarization of the mitochondrial membrane and, as we expected, a higher ROS and lactate production. This leads us to infer that these M1 macrophages undergo glycolysis. Then, the isolated follicular macrophages from aged women were treated with PRPr from fertile women to evaluate its modulatory effects and we observed a reduction in the frequency of CD68+IL-1β+ cells in comparison with those cultured without treatment. Also, treated macrophages showed a reduction in the levels of ROS and lactate production. Finally, we focused on a novel DNT lymphocyte regulatory subpopulation, CD3+CD56-CD4-CD8-, that is emerging as effector cells capable of mediating immunoregulation in the female reproductive system. We found that FF from aged women displayed a reduced frequency of DNT, in comparison with FF from younger women. Limitations, reasons for caution The present results were obtained using FF samples from women from different ages, using a pool of cells from all the follicles in each patient. Even though the samples represent the immune ovarian microenvironment, further studies are necessary to elucidate whether these mechanisms and PRPr treatment operate similarly in vivo. Wider implications of the findings We demonstrated that, during aging, follicular macrophages acquired an altered metabolism related to pro-inflammatory profile, accompanied by a reduction of DNT cells, impacting in oocyte quality. Notably, we showed for the first time that in vitro PRPr treatment reprogrammed macrophage’s profile, suggesting its relevance as a potential therapeutic treatment. Trial registration number No

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