Klinefelter Syndrome (KS) is characterized by a masculine phenotype, supernumerary sex chromosomes (47, XXY), and impaired fertility due to loss of spermatogonial stem cells (SSCs). Early testicular cryopreservation could be an option for future fertility treatments in these patients, including SSCs transplantation or in vitro spermatogenesis. It is critically essential to adapt current in vitro SSCs propagation systems as a fertility option for KS patients. KS human testicular samples (13,15- and 17-year-old non-mosaic KS boys) were donated by patients enrolled in an experimental testicular tissue banking program. Testicular cells were isolated from cryopreserved tissue and propagated in long-term culture for 110 days. Cell-specific gene expression confirmed the presence of all four main cell types found in testes: Spermatogonia, Sertoli, Leydig, and Peritubular cells. A population of ZBTB16+ undifferentiated spermatogonia was identified throughout the culture using digital PCR. Flow cytometric analysis also detected an HLA-/CD9+/CD49f+ population, indicating maintenance of a stem cell subpopulation among the spermatogonial cells. FISH staining for chromosomes X and Y showed most cells containing an XXY karyotype with a smaller number containing either XY or XX. Both XY and XX populations were able to be enriched by magnetic sorting for CD9 as a spermatogonia marker. Molecular karyotyping demonstrated genomic stability of the cultured cells, over time. Finally, single-cell RNAseq analysis confirmed transcription of ID4, TCN2, and NANOS 3 within a population of putative SSCs population. This is the first study showing successful isolation and long-term in vitro propagation of human KS testicular cells. These findings could inform the development of therapeutic fertility options for KS patients, either through in vitro spermatogenesis or transplantation of SSC, in vivo.

No abstract

Lenvatinib, a multi-kinase inhibitor, is used in the treatment of solid malignancies. Lenvatinib belongs to a family of tyrosine kinase inhibitors and targets VEGF receptors 1-3, FGF receptors 1-4, PDGF receptor alpha, RET and KIT. However, it is not known whether Lenvatinib like other chemotherapeutic drugs affects spermatogenesis. The objective of this study was to examine whether Lenvatinib induces damage to spermatogenesis in mice. Twenty adult mice (C57BL/6) were randomly divided into 2 groups to receive daily gavage of either water (as control) or Lenvatinib (10 mg/kg) for 6 weeks. All mice were euthanized at the end of the study. We identified that Lenvatinib significantly (p<0.05) decreased testis weight (TW: 91.75±1.49mg) compared to control mice (TW: 111.9±3.07mg). This difference in testis weight however, became non-significant after correcting for body weight. The cauda epididymal sperm count was significantly (p<0.01) decreased in the Lenvatinib treated (0.82±0.04 million/mg cauda) as compared to control (1.26±0.07 million/mg cauda) mice. There were no differences in plasma testosterone concentrations between Lenvatinib treated (29.76±7.67ng/dl) and control (31.72±6.89ng/dl) mice. Lenvatinib did not induce notable morphological changes in testicular histology. We conclude that 6 weeks of Lenvatinib treatment had minimal effect if any on mouse spermatogenesis. The long-term treatment effect of Lenvatinib on spermatogenesis remains to be determined.