Establishment of a controlled slow freezing-based approach for experimental clinical cryopreservation of human prepubertal testicular tissues

Doron Kabiri; Myriam Safrai; Michal Gropp; Guy Hidas; Talya Mordechai-Daniel; Karen Meir; Ariel Revel; Tal Imbar; Benjamin Reubinoff

doi:10.1016/j.xfre.2021.11.001

Establishment of a controlled slow freezing-based approach for experimental clinical cryopreservation of human prepubertal testicular tissues

F S Rep. 2021 Nov 5;3(1):47-56. doi: 10.1016/j.xfre.2021.11.001. eCollection 2022 Mar.

Authors

Doron Kabiri¹, Myriam Safrai¹, Michal Gropp², Guy Hidas³, Talya Mordechai-Daniel⁴, Karen Meir⁵, Ariel Revel¹, Tal Imbar^{1

4}, Benjamin Reubinoff^{1

2

4}

Affiliations

¹ Department of Obstetrics and Gynecology, Hadassah Hebrew University Medical Center, Jerusalem, Israel.
² The Sydney and Judy Swartz Embryonic Stem Cell Research Center of The Goldyne Savad Institution of Gene Therapy, Hadassah Hebrew University Medical Center, Jerusalem, Israel.
³ Department of Pediatric Urology, Hadassah Hebrew University Medical Center, Jerusalem, Israel.
⁴ IVF unit, Hadassah Hebrew University Medical Center, Jerusalem, Israel.
⁵ Department of Pathology, Hadassah Hebrew University Medical Center, Jerusalem, Israel.

Abstract

Objective: To develop an efficient, clinical-grade, freezing protocol toward experimental clinical cryopreservation of testicular tissues in prepubertal boys suffering from cancer.

Design: Experimental cryopreservation of testicular tissue.

Setting: University Medical Center.

Patients: Adult patients undergoing orchiectomy for various tumors and prepubertal boys scheduled for gonadotoxic treatment.

Interventions: None.

Main outcome measures: Histopathological analysis of tissue architecture, structural integrity, and cellular morphology was performed for control and frozen-thawed cryopreserved tissues.The number of seminiferous tubules per testicular section was calculated. The survival of spermatogonial stem cells (SSCs) and Sertoli cells of the control and frozen-thawed cryopreserved tissues was analyzed by immunofluorescence staining.

Results: Uncontrolled Slow Freezing, Controlled slow freezing, and vitrification similarly preserved the integrity of the adult testicular tissues and the survival of SSCs and Sertoli cells. Controlled slow freezing of prepubertal testicular tissues effectively preserved their architecture, the number of tubules, SSCs, and Sertoli cells. In addition, we observed SSC loss after chemotherapy in prepubertal boys, reemphasizing the importance of fertility preservation before gonadotoxic treatment.

Conclusions: Future fertility restoration for male survivors of pediatric cancers depends on the development of an optimal prepubertal testicular tissue cryopreservation method. Our findings demonstrate the effectiveness of controlled slow freezing for cryopreservation of human prepubertal testicular tissues and may contribute to more effective banking of these tissues and potential fertility restoration.

Clinical trial registration number: NIH research clinical trials number: NCT02529826.

Keywords: Fertility preservation; controlled slow freezing; human testicular cryopreservation; male infertility; prepubertal boys.

Associated data

ClinicalTrials.gov/NCT02529826