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Human Reproduction, Vol. 14, No. 2, 379-387, February 1999
© 1999 European Society of Human Reproduction and Embryology

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

Ahmed Ziyyat1, Bruno Lassalle1, Jacques Testart1,2, Pascal Briot2, Edouard Amar2, Catherine Finaz1 and Annick Lefèvre1,3

1 Unité Maturation Gamètique et Fécondation, INSERM U 355 and Institut Fédératif de Recherche sur les Cytokines, IFR 13, 32 rue des Carnets, 92140 Clamart and 2 Centre de Médecine de la Procréation, Hôpital Américain, 63, Boulevard Victor Hugo, 92202 Neuilly, France

Flow cytometry coupled to cell sorting is proposed as a method to isolate round spermatids from testicular biopsies in obstructive azoospermic patients. The cells were separated on the basis of their size and density only. We obtained homogenous populations of alive round spermatids free of lymphocytes and diploid germ cells. The detection of protamine 1 gene (PRM1) and PRM2 expression in the sorted cells proves that these cells are round spermatids. On the contrary, neither the expression of CD3-{delta}, which is specific to lymphoid cells, nor that of MAGE1, which has been demonstrated in diploid germ cells, could be observed in the round spermatid population even after using a nested polymerase chain reaction (PCR) assay. The flow cytometry procedure failed to isolate round spermatids from ejaculates in non-obstructive azoospermic patients. In >39 ejaculates tested by reverse transcriptase–PCR, only nine revealed the presence of some round spermatids, as demonstrated by the expression of PRM1. However, these round spermatids did not express PRM2.

Key words: flow cytometry/obstructive azoospermia/protamine/round spermatids

3 To whom correspondence should be addressed at: INSERM unité 355, 32 rue des Carnets, 92140 Clamart, France


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