M. Shekarriz et al., A METHOD OF HUMAN SEMEN CENTRIFUGATION TO MINIMIZE THE IATROGENIC SPERM INJURIES CAUSED BY REACTIVE OXYGEN SPECIES, European urology, 28(1), 1995, pp. 31-35
Current techniques of sperm preparation for in vitro fertilization or
intrauterine insemination require centrifugation of human semen to sep
arate spermatozoa from the seminal plasma. Centrifugation increases re
active oxygen species (ROS) formation in semen. Moreover, high levels
of ROS are associated with sperm membrane injury through spontaneous l
ipid peroxidation, which may alter sperm function. We investigated the
relationship between centrifugation variables (time and g-force) and
ROS production to establish an optimal centrifugation protocol for spe
rm preparation techniques, Semen from 38 men (24 patients and 14 norma
l volunteers) was evaluated for the formation of ROS before centrifuga
tion and after centrifugation at 200 g for 2 or 10 min and after 500 g
for 2 or 10 min. The absence of white blood cells in semen which can
also produce ROS was determined with the myeloperoxidase technique (En
dtz test), All specimens were negative (< 1 x 10(6)/ml) by the Endtz t
est, The formation of ROS was measured by chemiluminescence, ROS forma
tion was regarded as high (positive) when the chemiluminescence respon
se was at least 10 x 10(4) counted photons/min (cpm), The sperm concen
tration in each sample was adjusted to 15-20 x 10(6) cells/ml before a
nalysis. Eight specimens (7 patients and I donor) exhibited high level
s of ROS before centrifugation, All 8 showed further, significant incr
eases in ROS formation regardless of g-force or time. The increase in
ROS was significantly less when semen was centrifuged for 2 as compare
d to 10 min (p < 0.001). Six specimens previously ROS-negative became
ROS-positive after centrifugation for 10 min at 200 and 500 g. We conc
lude that the time of centrifugation is more important than g-force fo
r inducing ROS formation in semen. Based on these results, we recommen
d a shorter centrifugation period in the preparation of sperm for assi
sted reproductive techniques.