Assessment of the architecture and integrity of frozen‐thawed testicular tissue from (pre)pubertal boys with cancer

Abstract

Background Testicular tissue freezing is proposed for fertility preservation to (pre)pubertal boys with cancer before highly gonadotoxic treatment. Studies accurately comparing human (pre)pubertal testicular tissue quality before freezing and after thawing are exceptional. No study has reported this approach in a systematic manner and routine care. Objectives To assess the impact of a control slow freezing protocol on testicular tissue architecture and integrity of (pre)pubertal boys after thawing. Materials and methods (Pre)pubertal boys (n = 87) with cancer from 8 Reproductive Biology Laboratories of the French CECOS network benefited from testicular tissue freezing before hematopoietic stem cell transplantation. Seminiferous tubule cryodamage was determined histologically by scoring morphological alterations and by quantifying intratubular spermatogonia and the expression of DNA replication and repair marker in frozen‐thawed testicular fragments. Results A significant increase in nuclear and epithelial score alterations was observed after thawing (p < 0.0001). The global lesional score remained lower than 1.5 and comparable to fresh testicular tissue. The number of intratubular spermatogonia and the expression of DNA replication and repair marker in spermatogonia and Sertoli cells did not vary significantly after thawing. These data showed the good preservation of the seminiferous tubule integrity and architecture after thawing, as previously reported in our studies performed in prepubertal mice and rats. Discussion The current study reports, for the first time, the development of a semi‐quantitative analysis of cryodamage in human (pre)pubertal testicular tissue, using a rapid and useful tool that can be proposed in routine care to develop an internal and external quality control for testicular tissue freezing. This tool can also be used when changing one or several parameters of the freezing‐thawing procedure. Conclusion Control slow freezing protocol without seeding maintains the seminiferous tubule architecture and integrity, the concentration of spermatogonia and the expression of DNA replication and repair marker in spermatogonia and Sertoli cells after thawing.

Spermatogonial Stem Cell-Based Therapies: Taking Preclinical Research to the Next Level

“…However, slow freezing seems to provide greater protection to testicular tissue than SSV. This is in line with other studies reporting the ability of a slow freezing protocol to maintain tubular architecture and integrity of pre-pubertal human testicular tissue fragments ( 21 , 23 – 31 ).…”

A 20-year overview of fertility preservation in boys: new insights gained through a comprehensive international survey

“…Keros et al evaluated a protocol for cryopreservation of immature testicular tissue, using sample sizes 1–3 mm × 1–5 mm × 1–7 mm, and demonstrated maintenance of structural integrity and spermatogonia when compared with fresh tissue ( Keros et al , 2007 ). Similarly, a report from eight French CECOS centres comparing pre-pubertal testicular tissue histologically pre- and post-freezing found maintenance of spermatogonial numbers and seminiferous tubule integrity in tissue frozen in 10–15 mg fragments ( Rives-Feraille et al , 2022 ). While these studies did not assess the fertilization capacity of the tissue, the non-human primate tissue fragments used by Fayomi et al (2019) to ultimately yield live offspring fell within the larger size bracket (9–20 mm 3 ); the graft survival and subsequent spermatogenesis suggest that cryoprotectant penetration was adequate in those tissues.…”

“…Moreover, individual centres have established these analyses also for frozen/thawed testicular tissues, for quality assurance purposes, and have published the results of these assessments ( Keros et al , 2005 , 2007 ; Kvist et al , 2006 ; Gille et al , 2021 ; Kabiri et al , 2022 ; Wang et al , 2022 ). Semi-quantitative analytical techniques have been proposed as a means of standardized quality assessment of freezing techniques and integrity of frozen tissue ( Rives-Feraille et al , 2022 ). However, it should be recognized that the stored material is relatively small and frequent quality assurance during the cryopreservation period will have a significant impact on the amount of material available for future clinical application.…”

Advancements in fertility preservation strategies for pediatric male cancer patients: a review of cryopreservation and transplantation of immature testicular tissue

“…Nevertheless, it is important to highlight that the slow freezing method did not achieve full spermatogenesis and offspring production in human tissue [ 31 ]. Wyns et al discovered that slow freezing can preserve the proliferation capacity of spermatogonial cells even after 6 months of xenografting, which may have beneficial effects on the long-term survival of the tissue.…”

“…In a study conducted by Rives-Feraille et al in 2021, they cryopreserved and subsequently stored ITT obtained from prepubertal boys with cancer by CSF. Their findings indicated that there was no significant difference in seminiferous tubule integrity between fresh tissue and frozen-thawed tissue [ 31 ].…”