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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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Zona drilling
Assisted insemination by zona pellucida drilling is one of the microsurgical
techniques widely used to facilitate sperm penetration (Ng. et al, 1990).
Zona drilling was first reported by Gordon and Talansky (1986) in which
the mouse zona was punctured with acid Tyrode's solution through a fine
micropipette. The zona can also be punctured with enzymes , e.g trypsin
or pronase (Gordon et al ,1988). As the zona is an acellular glycoprotien
layer, it doesn't "heal" (Walton et al, 1987) but probably may "seal"
off with time. Mouse zygotes that resulted from zona drilling, when transferred
to the oviducts of psoudopregnant foster female mice, gave rise to normal
live young.
Application of chemical zona drilling to clinical ART was not successful
and demonstrated that the human oocyte differed from the mouse in its
response to acid Tyrode's solution. It was noted that microvilli in the
human oocyte flatten upon contact with acidic solution. This may lead
to interfere with the ability of spermatozoa to interact with the oocyte
surface (Alikani et al, 1993).
Moreover, even when fertilization occurred, embryonic development was
poor, likely due to detrimental effects of acidic Tyrode's solution on
unfertilized oocytes (Garrisi , et al, 1990).
Zona drilling c an also be achieved through mechanical force performing
minute holes in the zona using micromanipulator. The breach in the zona
after acid-drilling is 5-10 times wider than after mechanical perforation
. As this breach is larger, there is possibility that sperm that enter
in, may also find their way out. (Sathananathan et al, 1989).
The resulting presence of many sperm in the perivitelline space is not
an indication of fertilization. The lack of fertilization after drilling,
despite the presence of numerous spermatozoa in the perivitelline can
be attributed to the inability of gametes to fuse (Jean et al ,1992).
It's well known that only acrosome-reacted spermatozoa are capable of
fertilizing the oocyte (Yanagimachi R., 1988). In fact, an absence of
fertilization after perivitelline sperm transfer has already been related
to a lack of acrosome reaction (Sathananathan et al, 1989) .
Jean et al, (1992) reported that, after zona drilling, the inability of
sperm in the perivitelline space to fuse with the oocytes could be related
to an absence of acrosome reaction or to a global deficiency of these
gametes.
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Gordon et al, (1988) reported their results of zona drilling of 47 human
oocytes with acid Tyrode's , chemotrypsin and mechanical perforation after
zona softening with chymotrypsin. 31 of the 47 oocytes (66%) survived
the procedure, and of these, 10 were fertilized ( 5 monospermic and 5
polyspermic). Three patients had embryos replaced; with no resulting pregnancies.
Acid PBS for zona drilling may result in arrest of the second meiotic
division of the oocyte, possibly at anaphase II (Ng et al, 1989).
Jean et al, (1992) reported their results of mechanical zona-drilling
in 20 infertile couples with severe semen defect and previous IVF failure.
Fertilization rates were compared to assess the advantages of zona drilling;
93% (80/86) drilled oocytes survived and 18.75% (15) were fertilized,
whereas only 3% (3/100) control oocytes were fertilized. The polyspermy
rate for fertilized drilled oocytes was high 66.6% (10/15). The normal
fertilization rate after zona drilling remained 5.8% (5/86), and was statistically
different from that of control oocytes 3% (3/100).
One implantation occurred after replacement of 5 drilled embryos in three
patients but resulted in early miscarriage. Replacement of the single
control embryo led to a progressive pregnancy and normal male birth.
Technique of mechanical zona drilling
After ovarian stimulation and oocyte retrieval, oocytes enclosed in
their cumulus masses are transferred into 0.1% hyaluronidase type IV from
bovine testes in special culture medium e.g B2 medium, placed in 5% CO2
incubator at 37C for 5 min. To remove the cumulus cells. After washing,
the remaining corona cells are carefully removed with hypodermic needles.
Oocytes are placed in a special medium e.g B2 medium, containing sucrose
to shrink the ooplasm and widen the perivitelline space. This will allow
insertion of a microneedle without damaging ooplasm. While oocyte collection
is going on, semen is obtained in the lab and semen preparation is done.
Oocytes to be drilled are placed under the microscope, fitted with two
micromanipulators. Two types of microneedles are attached to micromanipulators,
one has a hemispherical tip to ensure safer and more precise oocyte handling
during manipulation, the tip of the second microneedle has an external
diameter of 2-4 um.
Zona glycoproteins are dissolved first with AT medium ( ph 2- 4). The
tip of the microneedle is broken and acid solution is expelled by positive
pressure in the microneedle system i.e. ZP softening before mechanical
drilling.
The shrunken oocyte is clamped onto the holding pipette by negative pressure
delivered by the automatic injection system. Negative pressure is kept
very low to prevent oocyte damage.
The microneedle is applied tangentially to the oocyte, and the pinched
up part of ZP is pierced through both sides. The micropipette is then
withdrawn horizontally and removed from ZP in which holes could be observed
immediately after drilling. The entire procedure usually takes less than
2 minutes per oocyte, while oocytes are drilled one after the other.
Oocytes are washed in B2 medium with 0.05 M sucrose for 15 minutes followed
by B2 medium without sucrose to regain their original ooplasm. This is
followed by transfer of oocytes into insemination medium at 37C in 5%
CO2.
Fertilization and polyspermia are assessed 12-24 hours later. The presence
of 2 pronuclei in the ooplasm is evidence of normal fertilization. Presence
of 3 or more pronuclei indicates polyspermic fertilization. The procedure
when done is evaluated by comparison with control oocyte group. Differences
are compared by X test and Fishers exact test.
Laser assisted zona drilling
The observation that UV radiation clearly removes polymeric material
and biological tissues has suggested that its use could be extended to
zona pellucida surgery.
A study was done by Blanchet et al, (1992 ) to introduce a new method
with the 248-nm line of Krypton Fluoride (KrF) excimer laser using cryopreserved
two-cell mouse embryo creating a 2-4 um opening in the zona pellcida.
2-3 pulses are required to cut through the zona without holding the embryo
in place with a pullet pipette. Also, the low level of energy used helps
to avoid any deformation of the embryo , it ranges between 0.6-3 j/cm
in BPS medium.
In conclusion, laser-assisted micromanipulation has the potential for
becoming the method of choice in Z.P. surgery without interrupting blastocyte
formation. The KrF laser 248nm appears to be a feasible method to future
studies with micromanipulation (Blanchet et al, 1992).
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