ISOLATION, PURIFICATION AND ASSESSMENT OF VIABILITY OF SPERMATOGENIC CELLS FROM TESTICULAR BIOPSIES OF AZOOSPERMIC MEN

The success of spermatid microinjection has generated many concerns. I n particular, there is a lack of appropriate methodology for the isola tion of large homogeneous populations of spermatids, with minimum loss of viability, from the testicular tissue of azoospermic men. Here we have compared two different isolation methods - velocity sedimentation under unit gravity (VSUG) combined with discontinuous Percoll centrif ugation (DPC), and separation ,vith fluorescent-activated cell sorter (FACS) using light in the visible range - to determine the most suitab le method for the isolation of spermatids, Total mixed cell count/gram of testicular parenchyma was significantly higher in obstructive azoo spermic men compared with non-obstructive azoospermic men (P < 0.001). The results of the comparison showed that in obstructive azoospermic patients the difference in the yields of primary spermatocytes produce d by the two techniques was not significant, but for round and elongat ing spermatids the FAGS separation proved to be the better method (P < 0.001). Similarly, in non-obstructive azoospermic patients, FAGS sepa ration proved to be superior, giving increased yields of primary sperm atocytes and round and elongating spermatids compared with VSUG combin ed with DPC method (P < 0.001). More than 99% of the separated cells r etained their viability after FAGS separation. As large homogeneous po pulations of viable spermatids can be separated with FAGS in a relativ ely short period of time, FAGS separation is the most suitable method for the isolation of spermatids from testicular biopsy tissue.