J. Auger et al., FLOW CYTOMETRIC SORTING OF LIVING, HIGHLY MOTILE HUMAN SPERMATOZOA BASED ON EVALUATION OF THEIR MITOCHONDRIAL ACTIVITY, The Journal of histochemistry and cytochemistry, 41(8), 1993, pp. 1247-1251
We investigated the applicability of flow cytometric (FCM) sorting to
select, with no deleterious effects, fractions of living, highly motil
e spermatozoa after staining with rhodamine 123 (Rh123) and propidium
iodide (PI) for assessment of their mitochondrial activity and viabili
ty, respectively. Sperm cells were subjected to FCM sorting according
to their Rh123 fluorescence intensity, and computer-aided sperm analys
is (CASA) for percentage motility and movement characteristic measurem
ents was carried out on the entire sperm populations and on the Rh123-
positive (Rh123+) sorted fractions. A first experiment on five sperm s
amples from fertile donors pre-selected by either swim-up or simplifie
d Percoll gradient indicated no detrimental effect of the FCM sorting
procedure because: (a) the numbers of Rh123+ motile sperm were not dec
reased by FCM sorting; (b) data on the sorted fractions showed a tende
ncy (not significant) for an increase in movement parameters rather th
an a drop; and (c) a significant decrease in the percentage of PI-posi
tive (PI+) sperm (13% vs 3%; P<0.05) was measured. A second experiment
was performed on sperm samples from four patients, only washed and re
-suspended in B2 medium. This demonstrated a significant increase in s
ome characteristics of movement quality related to a substantial and s
elective immobilization of the less motile sperm. The significant drop
in the percentage of PI+ sperm after FCM sorting (p<0.01) was less pr
onounced than after FCM sorting of pre-selected sperm (12% vs 3%, resp
ectively), indicating a lethal effect of FCM sorting on a small propor
tion of presumably moribund sperm. These preliminary data indicate a d
ifferential effect of FCM sorting on sperm according to their function
characteristics and suggest the potential importance of these methods
for the characterization in vitro of sperm subpopulations on the basi
s of functional criteria.