The number of infertile couples has increased in recent years and is now
estimated at 1520% of all couples worldwide. Since its introduction
into reproductive medicine in 1978 
, in vitro fertilization
has been considered a successful method of treating tubal sterility. With
increasingly intensive diagnosis of both partners, certain aspects of male
subfertility are coming to the forefront. Nowadays almost 50% of insterility
in couples can be attributed to male subfertility 
sperm injection (ICSI) has raised hopes that these couples can have children
of their own. In the meantime this method of treating male subfertility
has achieved a breakthrough 
and is gaining in importance.
It is establishing itself as the preferred method of treatment in the field
of assisted reproduction, following numerous pregnancies and births
Preparation for ICSI involves obtaining an oocyte by means of follicle
aspiration, removal of the cumulus cells and of the corona radiata and preparation
of semen in accordance with the Mini Swim-Up process 
preparation procedures 
. Thereafter, one sperm is injected
into this oocyte with a thin glass capillary (pipette). If fertilization
has occurred, the embryo is transferred into the uterus 48 hours after microinjection.
For the microinjection process described, Eppendorf
ideal tools for easing the workload: the micromanipulator TransferMan, used
for moving the holding capillary, and a second micro-manipulator TransferMan
for transferring the sperm.
The actual holding of the oocyte is carried out using CellTram Air, and
CellTram Oil is used for transferring the sperm.
Together with an inverted microscope, combined with Hoffmann contrast
optics or differential interference contrast optics, microinjection can
be effected quickly and simply.
Operating TransferMan, by means of four self-explanatory keys, is extremely
simple. For every TransferMan, two spatial coordinates are defined and stored.
The capillary can be moved easily in any direction (x y z)
by means of a joystick. By simply pressing the joystick button, the capillary
can repeatedly assume one of the pre-programmed positions. Sensibly, a point
in the focal plane is chosen for the holding side at which the oocyte can
be easily held (position 1 H) and the sperm can be easily injected with
the second TransferMan (position 1 T). For both manipulators/ capillaries,
a point outside the drops in the overlay oil (Fig. 1) is advisable for position
2 (H and T).
The method of procedure is as follows:
The transfer capillary and the holding capillary are directed at the focal
plane and the positions are stored as Pos. 1 (T and H). Similarly, the positions
in the overlay oil are defined as Pos. 2 (T and H). Sperm and ova are put
onto the petri dish in several drops of oil. By simply pressing the joystick
button, the transfer capillary (inner diameter approx. 45 m)
is lowered to position 1 T. Magnified x20 times, a sperm cell is immobilized
by means of a quick movement of the transfer capillary across the tail,
or by pressing the tail of the sperm cell against the bottom of the dish
until it stop
s moving. The sperm cell is aspirated, tail first into the
transfer capillary by rotating the knob of the CellTram Oil. By pressing
the joystick button, the transfer capillary, which now contains the sperm
cell, is moved up into the overlay oil. The petri dish is moved in order
to visualize an oocyte in one of the drops surrounding the sperm medium.
The oocyte is brought into focus. With the second TransferMan in position
1 H and simultaneous rotation of the knob of the CellTram Air, the oocyte
is held with the holding capillary (inner diameter approx. 20 m).
Magnified x10 times, the oocyte is turned with the aid of the transfer capillary,
which has been lowered again (position 1 T), until the polar body comes
to rest either above (12 oclock) or below (6 oclock). This requires
the under-pressure on the CellTram Air to be slightly varied. In this position,
the oocyte is held with the holding capillary by turning the knob of the
CellTram Air. Then the capillaries and the oocyte, (magnification x40) are
sharply focussed, the sperm cell in the transfer capillary is brought to
the tip by rotating the knob of the CellTram Oil. By moving the joystick
slightly, the transfer capillary is carefully pushed through the zona pellucida
and the oolemma into the ooplasm at 3 oclock. The oocyte should be
pricked in the middle, so that the oolema membrane is broken gently and
atraumatically. If this is not immediately successful, it is aspirated until
the membrane perforates. The sperm cell is then injected. In order to introduce
only one minimal volume of the medium and PVP solution, or none at all,
into the cytoplasm, the transfer capillary is withdrawn gently after the
head of the sperm cell has left the pipette tip and is no longer injected.
The injected oocyte is released from the holding capillary. Both capillaries
are moved to position 2 by pressing the joystick button.
If several oocytes are obtained, only 34 oocytes are injected as
a rule. They are then placed into Hams F-10-medium 
and the remaining oocytes are then injected. Further methods of procedure
are described in commonly-used literature, e.g. .
 Steptoe PC, Edwards RG (1978) Birth after reimplantation of a human
embryo. Lancet ii:366
 Kpker W, Al Hasani S, Bauer O, Diedrich K (1994) New technologies
of assisted fertilization. Fertilitt 10:216-220.
 Palermo G, Joris H, Devroey P, Van Steirteghem AC (1992) Pregnancies
after intracytoplasmic injection of single spermatozoon into oocyte. Lancet
 Van Steirteghem AC, Liu J, Joris H, Nagy Z, Janssenswillen C, Tournaye
H, Derde M-P, Van Assche E, Devroey P (1993) Higher success rate by intracytoplasmic
sperm injection than subzonal insemination. A report of a second series
of 300 consecutive treatment cycles. Hum Reprod 8:1055-1060
 Al Hasani S, Kpker W, Felberbaum R, Diedrich K, Sturm R, Bauer
O , Diedrich K (1995). Intracytoplasmatic sperm injection using the Mini
Swim-Up process. Fertility 11:111-114
 Van Steirteghem AC, Nagy Z, Joris H, Liu J, Staessen C, Smitz J, Wisanto
A, Devroey P. (1993) High fertilization and implantation rates after intracytoplasmic
sperm injection. Hum Reprod 8, 1055-1060.
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