Human Reproduction, Vol. 17, No. 1, 161-172,
January 2002
© 2002 European Society of Human Reproduction and Embryology
Developmental potential of human spermatogenic cells co-cultured with Sertoli cells
1 Department of Medical Genetics, Faculty of Medicine, 2 Centre for Reproductive Genetics, Porto and 3 Laboratory of Cell Biology, Institute of Biomedical Sciences Abel Salazar, State University of Porto, Porto, Portugal
BACKGROUND: Development of an in-vitro culture system capable of supporting human early germ cell differentiation would be important for treatment of azoospermic patients. METHODS: Sertoli cells, spermatogonia and spermatocytes were isolated from testicular biopsies of 61 non-obstructive azoospermic patients, and co-cultured using Vero cell conditioned medium only or supplemented with recombinant (r)FSH or rFSH plus testosterone. Germ cell purity was checked by fluorescent in-situ hybridization (FISH) analysis. RESULTS: Best results were achieved with both hormones, which elicited 6.9% of meiosis index and 22.7% of differentiation into normal late spermatids after 2–3 weeks of culture. In-vitro matured spermatids were microinjected into oocytes to study their developmental potential. Round spermatids elicited 37.5% of fertilization and 28.6% blastocyst rates. Abnormal elongating and elongated spermatids enabled 8.3 and 27.3% fertilization rates respectively, but none achieved the blastocyst stage. Normal elongating and elongated spermatids elicited 30.5% fertilization and 42.9% of blastocyst rates. FISH analysis showed sex chromosome anomalies in all embryos, except in the case of morulae from normal late spermatids. CONCLUSIONS: Results suggest that meiosis and spermiogenesis can be resumed in vitro, with normal differentiated spermatids showing a low fertilization potential but regular rates of blastocyst formation. However, most of the embryos did not reach the morula stage and showed major sex chromosome abnormalities.
Key words: in-vitro maturation/meiosis/non-obstructive azoospermia/spermatogenesis
4 To whom correspondence should be addressed at: Laboratory of Cell Biology, Institute of Biomedical Sciences Abel Salazar, State University of Porto, Lg. Prof. Abel Salazar 2, 4099-003 Porto, Portugal. E-mail: msousa{at}icbas.up.pt
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