Effect of pre-chilling on ovarian tissue

Th e centralized 'Danish model' of ovarian tissue cryopreservation (OTC) aims to improve patient access by consolidating cryobank facilities.However, transport and processing logistics oft en delay the cryopreservation of ovarian cortical tissue collected for future fertility and hormone restoration.Th e eff ects of these delays on tissue quality post-thaw remain poorly understood, leading to uncertainty and potential variability in patient outcomes.However sheep ovarian tissue has been shown to have improved follicle survival post-thaw when subjected to a 48hour period at 4 C prior to cryopreservation, compared to immediate processing.We hypothesized that prechilling may precondition tissue by reducing metabolic activity, thereby enhancing their resilience to cryoinjury and ischemia.We investigated the impact of pre-chilling on mouse ovaries, assessing mitochondrial metabolism via TOM20 (translocase of the outer mitochondrial membrane 20) as a marker of oxidative phosphorylation.Ovaries from 19-day-old F1 (C57B/6xCD1) mice were either immediately fi xed (0h) or stored at 4 C for 12, 24, 36, or 48 h before fi xation.TOM20 expression was detected by DAB immunohistochemistry, and staining intensity in granulosa cells and oocytes of secondary follicles was quantifi ed using ImageJ.Data are presented as mean SD, with signifi cance of p 0.05.TOM20 levels in granulosa cells signifi cantly decreased aft er 24 h (32.15 11.91, n = 21), 36h (33.38 10.72, n = = 15), and 48 h (32.52 15.83, n = 17) of cooling compared to 0 h controls (56.54 11.97, n = 19; p < 0.001).In contrast, TOM20 expression in oocytes remained stable across all time points.Th ese results suggest that pre-chilling alters mitochondrial metabolism in granulosa cells from 24h onwards, while oocyte metabolism appears unaff ected within this timeframe.Our preliminary fi ndings provide new insight into follicular metabolism under lowtemperature conditions and highlight the potential impact of processing delays on ovarian tissue quality.Further studies are needed to explore whether similar metabolic changes occur in other follicular stages and to determine long-term eff ects on oocyte health.Th ese results could inform the optimization of clinical protocols for fertility preservation.