Science against microbial pathogens: communicating current research and technological advances
A. Méndez-Vilas (Ed.)
Separation of human immunodeficiency virus type 1 (HIV-1) from motile sperm
using a continuous density gradient and swim- up
Naoaki KUJI1, Shingo KATO, Hideji HANABUSA32 and Yasunori YOSHIMURA1
1
Department of OB/GYN and 2 Department of microbiology, Keio University School of medicine, 35 shinanomachi,
shinjuku-ku, TOKYO160-8582, JAPAN
3
General Ogikubo Hospital, 3-1-24Imakawa, Suginami-ku, TOKYO167-0035, JAPAN
Abstract
While it has been documented that the density gradient / swim- up method substantially reduces the titers of HIV-1
in semen specimens obtained from infected males, approximately 5% of all motile sperm fractions prepared from
HIV-1 -seropositive males remain positive for HIV-1 following processing, and significant numbers of motile
sperm are lost during the two-step processing procedure. This is especially disadvantageous in cases of infertility
with male factor problems.
We had previously reported that the estimated bouyant density of HIV-1 was 1.042 after isopycnic centrifugation in
density gradient media. Also, sedimentation velocities of HIV-1 in both of these two gradient media seemed to be
rather small compared with those of sperm, since most HIV-1 particles resided in the layers above that of estimated
HIV-1 buoyant density (1.042) even after 40min centrifugation. Interestingly, small amount(0.05%) of HIV-1 was
detected from the bottom fraction (S.G. 1.250) in Pureception density gradiet, and was not detected in Percoll
gradient.
Based on those experimental information, we have developed the semen processing procedure using a continuous
density gradient and the swim- up technique. The continuous density gradient was prepared by the tilted tube
rotation method ; in brief , Percoll (80%; 2ml) was overlaid with 2 ml of culture medium in a sterilized disposable
centrifuge tube and rotated by tilting the tube, using the computer- regulated apparatus. Two aliquots of the semen
specimen from the infected males were overlaid on the prepared continuous gradient and centrifuged at 600xg for
20 min, and sperm were recovered from the small hole made at the bottom. The swim- up method has also been
improved since, with the use of a double- tube procedure to avoid direct contact between the raw semen adhering to
the wall of the tube and the swim-up fraction.
With written informed consent, the frozen sperm suspension that was negative for HIV-1 was used for ICSI with
the oocytes originating from the HIV-1 -seronegative partner. The presence/absence of HIV-1 in the sperm
suspension was assessed by nested PCR targeting the gag gene. The lower limit of detection was 50 copies / ml of
sperm suspension. Among the 68 semen specimens obtained from seropositive males, of whom 11 were diagnosed
to have oligoasthenozoospermia according to WHO criteria, motile sperm could be recovered from the HIV-1negative sperm suspensions for all the patients (i.e. at least one swim- up suspension was negative for HIV-1). The
correlation coefficient of the recovered sperm count between two processed sperm suspensions from the same
semen sample was 0.95. Only 3%（5/151） of the semen suspensions tested was positive for HIV-1. Among 69
IVF-ICSI trials in 30 couples, 16 pregnancies were achieved, and no evidence of horizontal or vertical transmission
was observed in 18 babies from 14 deliveries.
In conclusion, processing of sperm with a continuous density gradient prepared using the tilted tube rotation
method combined with an improved swim- up method appeared to be reproducible and also effective for recovering
motile sperm, even from HIV-1-seropositive males with reduced semen quality. The method was also found to
yield sperm suspensions that were clinically effective for the IVF-ICSI procedure for HIV-1 serodiscordant
couples.
Keywords HIV-1; Spermatozoa; Semen processing; Infertility; Assisted Reproductive Technology