Flow cytometry isolation and reverse transcriptase polymerase chain reaction characterization of human round spermatids in infertile patients

Flow cytometry coupled to cell sorting is proposed as a method to isolate r ound spermatids from testicular biopsies in obstructive azoospermic patient s. The cells were separated on the basis of their size and density only. We obtained homogenous populations of alive round spermatids free of lymphocy tes and diploid germ cells. The detection of protamine 1 gene (PRM1) and PR M2 expression in the sorted cells proves that these cells are round spermat ids. On the contrary, neither the expression of CD3-delta, which is specifi c to lymphoid cells, nor that of MAGE1, which has been demonstrated in dipl oid germ cells, could be observed in the round spermatid population even af ter using a nested polymerase chain reaction (PCR) assay. The flow cytometr y procedure failed to isolate round spermatids from ejaculates in non-obstr uctive azoospermic patients. In >39 ejaculates tested by reverse transcript ase-PCR, only nine revealed the presence of some round spermatids, as demon strated by the expression of PRM1. However, these round spermatids did not express PRM2.