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Submitted by Dr. Hesham Al-Inany, M.D. Lecturer, Gynaecology
& Obstetrics dept. Kasr El-Aini hospital, Cairo University, Egypt.
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During Gamete Micromanipulation sperms are injected into an ovum to assist in
union of the gametes.
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Sperm preparation
The most common method still in use for sperm preparation is washing
in medium 3 times the volume of semen, centrifugation, discarding the
supernatant, resuspension of the sperm in the pellet and its layering
by medium for an incubation period of 30-60 minutes (Lopata et al, 1979).
This method is well known as "Swim-up" procedure.
This well known technique has the disadvantage of loosing more than 80%
of the sperm in the pellet. A modification of the swim-up technique is
the addition of Ficoll, a density medium, to the resuspended sperm pellet,
thus creating a density gradient interface allowing to recover a higher
number of motile sperm. However, this technique was shown to be useful
for normal semen samples and also for cases of mid sperm motility (Wood
and Trounson, 1989). Therefore, other methods are used in cases with oligozoospermia.
Percoll gradient technique
Percoll, density gradient medium, has been used as a migration medium,
whereby the sperm is layered on an isotonic discontinous Percoll gradient,
centrifuged and collected from the pellet. In this way cell debris, bacteria
and malformed sperm are removed. The normal motile sperm are selected
according to their specific gravity
Recently, instead of using several Percoll gradients, a minipercoll gradient
of only two different concentrations of Percoll in Ham's F10 has been
described (Ord et al, 1992 ). The gradient consists of 0.3 ml each of
95% , 70% and 50% isotonic Percoll. The gradients are centrifuged at 300xg
for 20 to 45 minutes. After centrifugation, the 95% layer is removed,
diluted in 1ml of HEPES-HTF, and centrifuged at 200xg for 10 minutes.
This last step can be repeated more than once (Ron-EL et al, 1993)
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The sperm pellet is resuspended in a culture tube with 1ml of medium and
incubated at 37C in a humidified atmosphere of 5% CO2 and air until the
time of oocyte insemination (Ord et al, 1992 ).
Van Der Zwalmen et al, (1991) have shown that with the Percoll separation
technique, there was a significant increase in morphologically normal
spermatozoa which led also to a higher pregnancy rate compared to the
swim-up technique. However, in severe teratozoospermia cases, the morphology
remains unchanged after Percoll work up.
Methods like albumin gradient separation and storage in TEST yolk buffer
are less common and still doubtful in their capability to select better
sperm.
To obtain one or more spermatozoa for micromanipulation, it is aspirated
by a micropipette so simply if it is immotile, but if motile it's needed
to touch the tail of the spermatozoa by the tip of a microneedle. This
will produce temporary paralysis of the sperm then it can be aspirated
(Palermo et al, 1993).
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