Abstract Abstract Ovarian tissue cryopreservation is currently the only fertility preservation option for young girls, yet little is known about follicle health and development after freezing in previously treated young cancer patients. This study investigated the effect of cryopreservation on follicle health and developmental potential in ovarian tissue from paediatric and adolescent cancer patients by comparing fresh and cryopreserved tissue from six girls aged 10–16, five of whom had received prior cancer treatment. Tissues were cultured in vitro for 2 or 8 days, and follicle morphology, health, developmental stage, and density were assessed by histological analysis. Data were analysed using advanced statistical models, including generalized linear mixed models, to account for patient variability. Fresh ovarian tissue contained approximately 15% healthy follicles by Day 8 of culture, whereas cryopreserved tissue showed complete follicular depletion, with 0% healthy follicles remaining. These findings demonstrate the detrimental effects of both the freeze–thaw process and extended in vitro culture on follicle health and development in paediatric and adolescent ovarian tissue. Statistical analyses confirmed the significant impact of cryopreservation, as cryopreserved tissue failed to maintain any healthy follicles beyond one week in culture, and substantial inter-patient variability was observed (p<0.05), highlighting the influence of individual patient factors. Overall, these findings help resolve discrepancies in the literature regarding follicle survival after ovarian tissue cryopreservation in young cancer patients and underscore the importance of personalised fertility preservation strategies, given the significant inter-patient variability observed. The insights gained offer potential avenues for refining cryopreservation and culture protocols. Lay Summary Young girls undergoing cancer treatment often rely on ovarian tissue freezing as their only fertility preservation option, yet it remains unclear how well this approach maintains follicle health. Follicles are functional units of the ovary that contain immature eggs and are fundamental to future fertility. In this study, we compared fresh and frozen ovarian tissue from girls aged 10–16, most of whom had received chemotherapy, and examined follicle health and developmental potential during in vitro culture. Fresh tissue retained a small proportion of healthy follicles after eight days, whereas frozen tissue showed a complete loss of healthy follicles, with considerable differences observed between patients. These findings show that the freeze–thaw process and subsequent culture have a severe impact on follicle health in paediatric and adolescent ovarian tissue. Our work highlights the importance of improving and tailoring fertility preservation methods for young cancer patients and provides important evidence that current approaches to ovarian tissue freezing may not adequately protect follicle health in young cancer patients.