Freezing of testicular tissue as a minced suspension preserves sperm quality better than whole‐biopsy freezing when glycerol is used as cryoprotectant

Abstract

Frozen-thawed testicular spermatozoa have been used successfully for ICSI, especially in cases of obstructive azoospermia with normal spermatogenesis. Fewer attempts, however, have been made to check whether these rather immature spermatozoa, in a different environment with several other cell types present, have cryobiological requirements other than those of ejaculated spermatozoa. This is the reason why the freezing protocols and cryoprotectants (glycerol) used for freezing testicular tissue are based on experience with semen freezing. This study aimed to assess whether cryosurvival and/or motility was influenced by freezing of testicular tissue either as an intact biopsy or as a shredded tissue suspension, when glycerol was used as cryoprotectant. Freezing of testicular tissue as a suspension preserved motility (type B + C) significantly better than freezing of whole biopsies (9.2% vs. 4.0%). Similar observations have been made for vitality (39.3% vs. 25.4%). Centrifugation on 50% Percoll in order to remove the cryoprotectant resulted in a huge loss of spermatozoa (or late spermatids) and should therefore be especially avoided in cases of testicular failure. On the basis of these observations, mincing of the testicular biopsies before freezing may be advocated. Testicular spermatozoa seem to be better preserved when frozen in suspension, at least when slowly permeating glycerol is used as a cryoprotectant.

The effects of tissue sample size and media on short-term hypothermic preservation of porcine testis tissue

“…We expected to have higher number of cells survive in smaller size testis fragments compared to larger fragments or intact testes. It has been reported that spermatozoa could be better cryopreserved in minced tissues than in larger tissue biopsies (Crabbé et al 1999). Tissue sizes ranging from 0.5 to 1.5 mm 3 are routinely used for testis cryopreservation (Abrishami et al 2010;Zeng et al 2009).…”

Prepubertal testis hücre süspansiyonun dondurulmasında seeding etkisi

“…Fazla kriyoprotektan kullanımının ise hücrelerde toksik etkinin artmasına neden olduğu bildirilmektedir [28]. Ayrıca, testisin hücre süspansiyonu şeklinde dondurulmasının doku olarak dondurulmasına kıyasla, hücre canlılığını daha iyi koruduğu da belirtilmektedir [29]. Bu nedenle, çalışmamızda prepubertal testis dokusunu hücre süspansiyonu olarak dondurmayı tercih ettik.…”

Testicular tissue cryopreservation and spermatogonial stem cell transplantation to restore fertility: from bench to bedside

“…The median age of spermarche is estimated to be around 13 to 14 years, with a range between 11 and 17 years [36,37]. Thus, we recommend searching for testicular sperm in specimens from all boys aged 10 years or older, since freezing testicular sperm in glycerol-based medium [38] for use in ICSI is available in most ART laboratories [39]. Protocols for freezing small samples (2 to 4 mm 3 ) of immature human testicular tissue using dimethyl sulfoxide (DMSO) as the main cryoprotectant showed good structural integrity of testicular tubules, and pre-tubular and intra-tubular cells after thawing [11,40].…”