Abstract
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.MethodsTwo 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 mm3) were diluted in glycerol containing freeze solutions, slow cooled to 4 °C and then rapidly frozen in LN2 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.ResultsMore 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.ConclusionsBy 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.
Highlights
The use of human testicular sperm as a male infertility treatment was enabled by the development of ICSI over 2 decades ago [1,2,3,4]
By 24 h In vitro culture (IVC), there was a significant rise in total motility (50 %) and progressive motility (8 to 15.7 %), which increased (p < 0.05) further at 72 to 96 h
A practical solution rests in adopting a simple and effective testicular biopsy tissue processing procedure, which incorporates pre-freeze IVC motility enhancement and whole tissue cryopreservation
Summary
The use of human testicular sperm as a male infertility treatment was enabled by the development of ICSI over 2 decades ago [1,2,3,4]. The ability to process, evaluate, and properly use surgically acquired testicular sperm involves a steep and challenging learning curve [5,6,7]. Verheyen and coworkers [8] evaluated four different mechanical methods (control shredding, fine mincing, vortexing, and crushing) to dissociate testicular sperm from. There was little difference in the amount and quality of sperm dispersed by shredding or mincing before Percoll density gradient separation. Since the quantity of sperm found in a testicular biopsy may be limited, fine needle or glass slide shredding is a common mechanical method for tissue dispersion used by embryologists [9]. The compression squeezing of individual tubules with a bent, smooth needle surface can be performed to push cellular contents into the culture medium [10, 11]. Many urologists have adopted the fine tissue mincing technique that employs dissecting scissors to generate small tubular segments and free spermatozoa in solution [8, 12, 13]
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