Isolation, purification and assessment of viability of spermatogenic cells from testicular biopsies of azoospermic men |
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Authors: | Aslam I; Robins A; Dowell K; Fishel S |
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Institution: | CARE, The Park Hospital, Arnold, Nottingham, UK. |
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Abstract: | The success of spermatid microinjection has generated many concerns. In
particular, there is a lack of appropriate methodology for the isolation 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 centrifugation
(DPC), and separation with fluorescent-activated cell sorter (FACS) using
light in the visible range -- to determine the most suitable method for the
isolation of spermatids. Total mixed cell count/gram of testicular
parenchyma was significantly higher in obstructive azoospermic 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 produced by the two techniques was
not significant, but for round and elongating spermatids the FACS
separation proved to be the better method (P < 0.001). Similarly, in
non-obstructive azoospermic patients, FACS separation proved to be
superior, giving increased yields of primary spermatocytes and round and
elongating spermatids compared with VSUG combined with DPC method (P <
0.001). More than 99 % of the separated cells retained their viability
after FACS separation. As large homogeneous populations of viable
spermatids can be separated with FACS in a relatively short period of time,
FACS separation is the most suitable method for the isolation of spermatids
from testicular biopsy tissue.
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