JBRA Assist. Reprod. 2025;29(Suppl 1):31-31
POSTER PRESENTATION
doi: 10.5935/1518-0557.20250085
1CONICET. Universidad de Buenos Aires, Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales (IQUIBICEN). Laboratorio de Inmunofarmacología y Laboratorio de Inmunología Reproductiva de la Fertilidad. Buenos Aires, Argentina
2Fertilis Medicina Reproductiva, Buenos Aires, Argentina
Objective: The concept of ‘inflammaging’ represents the inflammation associated to aging, which is systemic, chronic and linked to the increase of pro-inflammatory mediators. Particularly in the ovary, inflammaging leads to a decrease in the ovarian reserve and oocyte quality, causing infertility. Platelet-rich plasma releasates (PRPr) therapy was proposed to improve ovarian function, increasing the chances of pregnancy, however the PRP effects on ovarian immune cells are still unknown. Here, we investigate the mechanisms underlying the exacerbated inflammatory response during ovarian aging and their impact in oocyte quality. Since oocytes develop in the context of ovarian follicles, we obtained follicular fluid (FF) from women with different ages to investigate ovarian immune microenvironment and its modulation after in vitro PRPr treatment.
Methods: We studied FF (n=102) from women undergoing ovodonation (21-29 years old) and assisted reproductive treatment indication (36-42 years old). Mononuclear cells were isolated using Ficoll-Hypaque and macrophages isolated by adherence during 90 minutes. Follicular macrophages profile (CD68+) was tested by flow cytometry. 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 the number of M2 oocytes and atretic follicles. Finally, macrophages were treated in vitro with PRPr (obtained from fertile women) and, after 5 days, reprogramming of macrophages profile was tested.
Results: Follicular macrophages from aged women (36-42 years old) showed a significant increase in IL-1β production in comparison with the youngest group (21-29 years old), IL-1β production also correlates negatively with the number of M2 oocytes recovered from each patient. Focusing on macrophage characterization, FF from aged women displayed an increased frequency of CD68+ macrophages in comparison with young women. Since CD68 shuttles between the cytoplasm and the plasmatic membrane to incorporate lipids, we evaluated lipid accumulation in FF macrophages to generate foamy cells. Interestingly, we found an increase in lipid droplets accumulation in follicular macrophages from aged women in comparison with the youngest group. Therefore, we discriminated between surface and intracellular CD68 detection and found higher intracellular CD68 expression in aged macrophages, correlating with lipid droplets accumulation. The accumulation of lipid droplets in the cytoplasm indicates a dysregulation in the immunometabolism of aged follicular macrophages. Taking this into account, we continued characterizing follicular macrophages metabolism and we observed a hyperpolarization of the mitochondrial membrane and, as we expected, 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 in vitro with PRPr from fertile women to evaluate its modulatory effects and we observed a reduction in the frequency of CD68+IL-1β+ macrophages in comparison with those cultured without treatment.Also, treated macrophages showed a reduction in the levels of ROS and lactate production.
Conclusion: We demonstrated that, during aging, follicular macrophages acquired an altered metabolism related to a proinflammatory profile, impacting on 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.