032 Cryobanking of testicular tissue in men of reproductive age

Abstract

Cryopreservation of ejaculated or testicular spermatozoa is recommended for fertility preservation in adolescence and adult men. However cryobanking of the properly preserved human testicular tissue in men of reproductive age is an essential source for further clinical application and additional examination for diagnostic purposes. During the last three years 107 infertile patients were referred for diagnostic testicular biopsy to the Andrology Laboratory, Center of Andrology and Sexual Medicine at Karolinska University Hospital, Stockholm, Sweden. All patients had azoospermia with a testis volume of 1–15 mL. An open biopsy was performed in all cases. The primary section of the biopsy was disaggregated for testicular spermatozoa retrieval for cryopreservation and subsequent use for intracytoplasmic sperm injection (ICSI). Two fresh pieces were fixed for histological evaluation and early cancer detection. As a part of our routine clinical protocol, we typically assess spermatogenesis in semi-thin plastic embedded sections and cryopreserve a further piece of each testis for an additional diagnostic examination. The following three protocols – conventional cooling in two different devices and vitrification, were applied and adjusted for a proper cryopreservation of the testicular tissue as a multicellular system: 1. Small 1–2 × 2 × 2 mm pieces were washed in basic solution, equilibrated in cryoprotectant medium with 0.7 M Me 2 SO for 30 min at +4 °C and cryopreserved by slow programmed cooling protocol, established for cryopreservation of prepubertal testicular tissue in 1.8 mL Nunc cryovials according Keros et al., Hum Reprod., 2007. 2. To preserve tissue for possible clinical application and avoid leaking of LN2 when storing in a liquid faze nitrogen, cryovials were replaced by CBS™ High Security tissue 2 mL straw (CryoBioSystem) and cryopreserved in a closed system using the same protocol. This resulted in a visible structural damage of the tissue. To achieve an appropriate survival of all types of testicular cells, the protocol was optimised, 1.1 M Me 2 SO was used as a cryoprotectant (CPA) and the temperature of initiated seeding was changed to −5 °C. 3. It is well established that cryopreservation of the tissue in pieces as a multicellular system is more complicated and conventional cryopreservation results in harmful ice formation in the cells and in the surrounding extracellular matrix. As an alternative to conventional cryopreservation we applied the vitrification protocol developed for human ovarian tissue (Keros et al., 2009, Hum Reprod ). For this combination of Me 2 SO, Propanediol, Ethylene glycol, PVP was applied. Ice crystal formation in human testicular tissue at ultra rapid cooling and warming rates was analysed by freeze substitution technique and transmission electron microscopy. On the basis of our experience, we summarize that human testicular tissue can be successfully cryopreserved by programmed slow freezing using cryovials or tissue straws as well as by vitrification. All types of testicular cells, including cells of different stages of spermatogenesis, motile spermatozoa, Leydig cells and Sertoli cells can survive cryopreservation and long-term storage in liquid nitrogen when an optimized cryopreservation protocol is used. Source of funding: The National council for human tissues and cells (Nationella Vävnadsrådet) at The Swedish Association of Local Authorities and Regions (SKL). Conflict of interest: None declared. Victoria.Keros@ki.se