Context Young boys treated with high-dose chemotherapy are often confronted with infertility once they reach adulthood. Cryopreserving testicular tissue before chemotherapy and autotransplantation of spermatogonial stem cells at a later stage could theoretically allow for restoration of fertility. Objective To establish in vitro propagation of human spermatogonial stem cells from small testicular biopsies to obtain an adequate number of cells for successful transplantation. Design, Setting, and Participants Study performed from April 2007 to July 2009 using testis material donated by 6 adult men who underwent orchidectomy as part of prostate cancer treatment. Testicular cells were isolated and cultured in supplemented StemPro medium; germline stem cell clusters that arose were subcultured on human placental laminin-coated dishes in the same medium. Presence of spermatogonia was determined by reverse transcriptase polymerase chain reaction and immunofluorescence for spermatogonial markers. To test for the presence of functional spermatogonial stem cells in culture, xenotransplantation to testes of immunodeficient mice was performed, and migrated human spermatogonial stem cells after transplantation were detected by COT-1 fluorescence in situ hybridization. The number of colonized spermatogonial stem cells transplanted at early and later points during culture were counted to determine propagation. Main Outcome Measures Propagation of spermatogonial stem cells over time. Results Testicular cells could be cultured and propagated up to 15 weeks. Germline stem cell clusters arose in the testicular cell cultures from all 6 men and could be subcultured and propagated up to 28 weeks. Expression of spermatogonial markers on both the RNA and protein level was maintained throughout the entire culture period. In 4 of 6 men, xenotransplantation to mice demonstrated the presence of functional spermatogonial stem cells, even after prolonged in vitro culture. Spermatogonial stem cell numbers increased 53-fold within 19 days in the testicular cell culture and increased 18 450-fold within 64 days in the germline stem cell subculture. Conclusion Long-term culture and propagation of human spermatogonial stem cells in vitro is achievable.

Multipotent cells can be established from adult human testis. Their easy accessibility and ethical acceptability as well as their non-tumorigenic and autogenic nature make these cells an attractive alternative to human ES cells for future stem cell therapies.

Y chromosome gr/gr deletions significantly reduce sperm counts and are thus associated with low semen quality.

The azoospermia factor c (AZFc) region harbors multi-copy genes that are expressed in the testis. Deletions of the AZFc region lead to reduced copy numbers of these genes. Four (partial) AZFc deletions have been described of which the b2/b4 and gr/gr deletions affect semen quality. In most studies, (partial) AZFc deletions are identified and characterized using plus/minus sequence site tag (STS) polymerase chain reaction (PCR). However, secondary duplications increase the gene copy number without re-introducing the STS boundary marker. Consequently, the actual copy number of AZFc genes cannot be determined via STS PCR. In the current study, we first set out to determine by quantitative real-time PCR the actual copy number of all AZFc genes in men with (partial) AZFc deletions based on STS PCR. We then analyzed whether reduced gene copy numbers of each AZFc gene family were associated with reduced total motile sperm count (TMC), regardless of the type of deletion. We screened 840 men and identified 31 unrelated men with (partial) deletions of AZFc based on STS PCR. Of these 31 men, 6 men (19%) had one or more secondary duplications. For all AZFc genes, we found an association between a reduction in the copy number of each individual AZFc gene and reduced TMC. In gr/gr-deleted men, restoration of reduced gene copy numbers restored their TMC to normal values. Our findings suggest that the gene content of the AZFc region has been preserved throughout evolution through a dosage effect of the AZFc genes on TMC safeguarding male fertility.