Validation-verification of a highly effective, practical human testicular tissue in vitro culture-cryopreservation procedure aimed to optimize pre-freeze and post-thaw motility

PURPOSE: The aim of our paper was to validate a testicular biopsy procedure that simplifies handling, processing, and cryopreservation, while at the same time optimizes sperm motility before freezing and after thawing. METHODS: Two prospective studies were conducted to verify, optimize, and understand the virtues of pre-freeze testicular tissue IVC at different temperatures (21, 30, or 37 °C). Testicular tissue was obtained from clinical specimens designated for whole tissue cryopreservation (i.e., intact mass of tubules) and/or for fresh use in IVF-ICSI cycles. Whole testicular biopsy pieces (1–3 mm(3)) were diluted in glycerol containing freeze solutions, slow cooled to 4 °C and then rapidly frozen in LN(2) vapor. Fresh and post-thaw testicular biopsy tissue were evaluated for changes in the quantity (%) and pattern of motility (I–IV: twitching to rapid progression, respectively) over a 1 week duration. The clinical effectiveness of IVC-cryopreserved whole testicular biopsy tissue was also validated analyzing fresh embryo transfers. RESULTS: More reliable recovery of motile testicular sperm was achieved using whole tissue freeze preservation combined with IVC (24–96 h) post-acquisition at an incubation temperature of 30 °C compared to ambient temperature (21 °C) or 37 °C. Up to 85 % of the pre-freeze motility was conserved post-thaw (+3 h) for easy ICSI selection. Sperm longevity was optimized to fresh tissue levels by implementing testicular biopsy sucrose dilution post-thaw. Favorable clinical outcomes were proven using frozen-thawed testicular biopsy sperm for ICSI. CONCLUSIONS: By employing minimal tissue manipulation, integrating pre-freeze IVC processing at 30 °C and the freezing of whole testicular biopsy tissue, we have reduced the labor and improved the efficacy of processing testicular tissue for freeze-preservation and subsequent ICSI use.