Testicular organoids formation from leukaemia-infiltrated prepubertal testicular tissue: implications for fertility preservation

Acute lymphoblastic leukaemia (ALL) in children and adolescents may affect the testes at diagnosis or relapse. Highly aggressive treatments such as haematopoietic stem cell transplantation are recommended for early testicular relapse and T-cell ALL. Patients with late isolated testicular relapse of B-cell ALL can generally be cured by salvage chemotherapy combined with scrotal irradiation or orchiectomy [ 1 ]. These treatments frequently lead to severe gonadal dysfunction and azoospermia, highlighting the need for fertility preservation. While postpubertal males can preserve fertility through semen cryopreservation, prepubertal boys do not yet produce sperm. As a result, fertility preservation for this group relies on cryopreservation of testicular tissue. Currently, autotransplantation appears to be the most promising approach for the maturation of immature testicular tissue, as offspring have been obtained in non-human primates after testicular tissue transplantation [ 2 ]. However, when leukaemic cells infiltrate the testicular tissue, alternative fertility restoration methods are needed. In vitro spermatogenesis, including explant tissue and testicular organoid culture, shows promise but remains experimental. Further research is required before clinical application. To optimise the cryobanking of prepubertal testicular tissue for patients with leukaemic testicular relapse, further research on the impact of leukaemic cells contamination on spermatogonia and the testicular somatic cell function is required. Reference values for spermatogonial quantity in human testes throughout healthy prepuberty have been established [ 3 ], and patient-derived testicular organoids and explant tissue cultures have proven valuable for studying somatic cell function [ 4 , 5 ]. This study applied these advanced methods to assess the effects of overt and microscopic leukaemia contamination on the cellular composition and functionality of prepubertal testicular tissue. The study material comprised bilateral testicular samples from a prepubertal boy experiencing a combined bone marrow and testicular relapse of ALL. The patient had completed anti-leukaemia therapy one year before the relapse and received a three-week re-induction therapy course (Supplementary information) prior to a testicular biopsy for fertility preservation on normal-sized prepubertal testes measured volume: 1.8 ml, testicular volume Z-score: +0.5 SD, and an orchiectomy of an overtly enlarged testis (volume: 13.0 ml, Z-score: +4.4 SD) [ 6 ]. Two-thirds of the tissue obtained from the normal-sized testis was cryopreserved for clinical fertility preservation, while the remaining one-third, along with the entire orchiectomy specimen, was used in an experimental study under the NORDFERTIL fertility preservation programme (Fig. 1A , Supplementary Fig. 1 ). The patient and his parents received verbal and written information about the research project and provided written consent. Further details are provided in the Supplementary Information. Fig. 1: Characterisation of human prepubertal testicular tissue microscopically and overtly infiltrated with leukaemic cells. Full size image A Schematic illustration of the experimental conditions and characterisation analysis (created with BioRender®, Toronto, Canada). B Immunofluorescent staining of a tissue section depicting localisation of germ cell marker DDX4 (red staining), the basement membrane component LAMA1 (green staining), Sertoli cell marker SOX9 (red staining), and leukaemic cell marker CD79a (green staining). C Representative PAS staining images showing the histology of tissue samples from microscopically and overtly leukaemic cell infiltrated testes (white arrowheads: germ cells). Scale bars = 100 µm (insets = 20 µm). DDX4 DEAD-box helicase 4, LAMA1 laminin alpha 1, SOX9 SRY-Box transcription factor 9, WT1 Wilms’ Tumour 1, PAS Periodic Acid Schiff.