Abstract
BackgroundCryopreservation of prepubertal testicular tissue preserves spermatogonial stem cells (SSCs) that may be used to restore fertility in men at risk of infertility due to gonadotoxic treatments for either a malignant or non-malignant disease. Spermatogonial stem cell-based transplantation is a promising fertility restoration technique. Previously, we performed xenotransplantation of propagated SSCs from prepubertal testis and found human SSCs colonies within the recipient testes six weeks post-transplantation. In order to avoid the propagation step of SSCs in vitro that may cause genetic and epigenetic changes, we performed direct injection of single cell suspension in this study, which potentially may be safer and easier to be applied in future clinical applications.MethodsTestis biopsies were obtained from 11 infant boys (median age 1.3 years, range 0.5-3.5) with cryptorchidism. Following enzymatic digestion, dissociated single-cell suspensions were prelabeled with green fluorescent dye and directly transplanted into seminiferous tubules of busulfan-treated mice. Six to nine weeks post-transplantation, the presence of gonocytes and SSCs was determined by whole-mount immunofluorescence for a number of germ cell markers (MAGEA, GAGE, UCHL1, SALL4, UTF1, and LIN28), somatic cell markers (SOX9, CYP17A1).ResultsFollowing xenotransplantation human infant germ cells, consisting of gonocytes and SSCs, were shown to settle on the basal membrane of the recipient seminiferous tubules and form SSC colonies with expression of MAGEA, GAGE, UCHL1, SALL4, UTF1, and LIN28. The colonization efficiency was approximately 6%. No human Sertoli cells were detected in the recipient mouse testes.ConclusionXenotransplantation, without in vitro propagation, of testicular cell suspensions from infant boys with cryptorchidism resulted in colonization of mouse seminiferous tubules six to nine weeks post-transplantation. Spermatogonial stem cell-based transplantation could be a therapeutic treatment for infertility of prepubertal boys with cryptorchidism and boys diagnosed with cancer. However, more studies are required to investigate whether the low number of the transplanted SSC is sufficient to secure the presence of sperm in the ejaculate of those patients over time.
Highlights
During the last decades, improved diagnostics and cancer treatments of children have resulted in more long-term survivors [1]
Spermatogonia were located on the basal membrane, while the gonocytes were present in the center of the tubules
According to the formula of germ cell density, we found that the initial mean germ cell density was from 1513 to 28399 cells per mm3 (Figure 2C)
Summary
During the last decades, improved diagnostics and cancer treatments of children have resulted in more long-term survivors [1]. In post pubertal boys and men, semen cryopreservation prior to gonadotoxic therapy is the standard method for fertility preservation. This is not an option for prepubertal boys who are unable to provide a semen sample [4]. Cryopreservation of testicular tissue before gonadotoxic treatment, malignant and non-malignant diseases, is the only clinical method available to potentially preserve the fertility for prepubertal boys [5–7]. Prepubertal testicular tissue cryopreservation (TTC) is an experimental method that preserves spermatogonial stem cells (SSCs), which are sperm progenitors that can potentially be used to restore spermatogenesis and produce spermatozoa in adulthood [8]. Cryopreservation of prepubertal testicular tissue preserves spermatogonial stem cells (SSCs) that may be used to restore fertility in men at risk of infertility due to gonadotoxic treatments for either a malignant or non-malignant disease. In order to avoid the propagation step of SSCs in vitro that may cause genetic and epigenetic changes, we performed direct injection of single cell suspension in this study, which potentially may be safer and easier to be applied in future clinical applications
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