Follicular fluid of adolescents undergoing fertility preservation is more pro-inflammatory compared to oocyte donors

Oocyte cryopreservation is used to preserve fertility in peri- and postpubertal adolescent patients with diseases and/or anticipated treatments that can jeopardize ovarian function. However, the quality of eggs in very young patients is relatively unknown. Recent studies suggest that oocyte aneuploidy is increased in children and young adults similar to women of advanced reproductive age. The oocyte microenvironment can be sampled to investigate the quality of the associated gamete. For example, a pro-inflammatory cytokine profile of follicular fluid (FF) is associated with advanced reproductive age. Our goal was to analyze the cytokine profile of FF in adolescent patients compared to oocyte donors to evaluate whether similar alterations exist in this younger end of the aging spectrum. This project is IRB approved. At the time of egg retrieval, FF from 2 dominant 18-20mm follicles per participant was collected from 10-19 years old adolescents (average age 16.3 ± 2.5, n=18) undergoing fertility preservation and from oocyte donors 25-30 years old (average age 26.8 ± 1.6, n=16). Halt protease inhibitor cocktail (Thermo Scientific) was added to these samples to preserve cytokines, cells were pelleted via centrifugation, and FF was stored at -80ºC. All FF samples were run in parallel on Human Cytokine Array C5 (RayBiotech). The resulting chemiluminescence data was quantified, and the levels of 80 cytokines were compared between adolescent patients and oocyte donors. A subsequent analysis was performed to compare the levels of the cytokines between patients without cancer diagnoses (n=6) and adolescent patients with cancer (n=12) to ascertain that the observed differences in cytokine levels are likely due to age and not the cancer diagnosis. An unpaired two-sided Student’s t-test or the Mann-Whitney test was used for statistical analysis using GraphPad Prism software. P values < 0.05 were considered statistically significant. The levels of 9 cytokines were significantly different, and all demonstrated higher levels in FF of adolescents compared to donors: IL-1 alpha (2-fold), IL-1 beta (1.7-fold), I-309 (2-fold), IL-15 (1.6-fold), TARC (1.9-fold), TPO (2.1-fold), IGFBP-4 (2-fold), IL-12-p40 (1.7-fold) and ENA-78 (1.4-fold). Interestingly, 7 of these cytokines have known pro-inflammatory roles. The other two, IGFBP4 and TPO, were shown to be important in the regulation of insulin-like growth factor action and in the production of platelets, respectively. None of these 9 cytokines demonstrated significantly different levels in the FF of adolescents with cancer compared to those who underwent fertility preservation for non-cancer diagnoses. The FF of peri- and postpubertal adolescent patients undergoing fertility preservation is more pro-inflammatory compared to oocyte donors. This difference is likely due to age and not the cancer diagnosis.