Determining the optimal time interval between sample acquisition and cryopreservation when processing immature testicular tissue to preserve fertility

The cryopreservation of immature testicular tissue (ITT) prior to gonadotoxic therapy is crucial for fertility preservation in prepubertal boys with cancer. However, the optimal holding time between tissue collection and cryopreservation has yet to be elucidated. Using the bovine model, we investigated four holding times (1, 6, 24, and 48 h) for ITTs before cryopreservation. Biopsies from two-week-old calves were stored in transport medium and cryopreserved following a standard slow-freezing clinical protocol. Thawed samples were then assessed for viability, morphology, and gene expression by haematoxylin and eosin (H&E) staining, immunohistochemistry and real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR). Analysis failed to identify any significant changes in cell viability when compared between the different groups. Sertoli (Vimentin+) and proliferating cells (Ki67+) were well-preserved. The expression of genes related to germ cells, spermatogenesis (STRA8, PLZF, GFR alpha-1, C-KIT, THY1, UCHL-1, NANOG, OCT-4, CREM), and apoptosis (HSP70-2) remained stable over 48 h. However, seminiferous cord detachment increased significantly in the 48-h group (p < 0.05), with associated cord and SSC shrinkage. Collectively, our analyses indicate that bovine ITTs can be stored for up to 48 h prior to cryopreservation with no impact on cell viability and the expression levels of key genes. However, to preserve the morphology of frozen-thawed tissue, the ideal processing time would be within 24 h. Testicular tissues obtained from patients for fertility preservation often need to be transported over long distances to be cryopreserved in specialist centres. Our findings highlight the importance of determining optimal tissue transport times to ensure tissue quality in cryopreservation.