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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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Partial zona dissection
A new method introduced by Cohen et al, (1990) in the field of ART to
assist zona pellucida (ZP) penetration . It is a non-traumatic method using
mechanical force to open the ZP by piercing it in two opposite positions
by means of micromanipulators.
Method
The cumulus oophorus cells of non inseminated oocytes are removed with
a solution of 0.1% hyaluronidase for 1-2 min. at 37C and the remaining cells
are removed manually with hypodermic needles.
To facilitate successful insertion of microneedle without rupturing the
vitellus, a large perivitelline space is needed. This is achieved by using
a 0.05mol solution of sucrose in culture medium leading to cytoplasm shrinkage.
This concentration is suitable for 1-day old oocytes, but, freshly collected
oocytes require a higher concentration of sucrose (0.1mol).
Oocytes are clamped by suction onto a holding pipette, so that the largest area of perivetilline space is at
the "12 O' clock" position and the holding pipette at 9 O'clock. A microneedle
tangentially pierce one side of the zona pellucida between 1 O'clock and
2 O'clock positions.
The oocyte is moved upwards slightly using the microneedle in order to avoid
contact between the holding pipette and the microneedle.
The ZP is pierced a second time between the 10 O'clock and 11 O'clock positions.
The suction is now discontinued and the microneedle holding the released
oocyte is brought parallel to the holding pipette.
The ZP is rolled gently across the suction side of the holding pipette in
order to force the ZP to slice open.
The microneedle is now removed and the opening thus created will never exceed
20 um in size. The whole procedure can be repeated on different sites of
the ZP. Sucrose removal is achieved by washing the oocytes four times in
fresh culture medium.
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The oocytes are deposited in a dish with sperm concentration of 5-10 million/ml
which are normally difficult to achieve with severe male factor. The oocytes
are assessed for being fertilized and for the number of pronuclei.
Monospermic zygotes should be separated from multispermic ones, then cultured
and checked daily until blastocysts formation is well established. At this
moment, embryo transfer can be done.
Biochemical pregnancy is confirmed using a serum B-HCG assay 12 days following
replacement. Fetal sonography is performed 2-4 weeks following biochemical
confirmation of pregnancy.
The incidence of monospermic fertilization of freshly harvested oocytes
( in cases of male infertility) at insemination and one-day old oocytes
following reinsemination is double that with conventional IVF techniques.
This doubled incidence provides evidence that actual movement of spermatozoa
through the holes occurs in at least half the time .
It should be remembered that (PZD) is a relatively non-traumatic, easily
achieved procedure and damage less than 10% of the oocyte when sucrose is
used to shrink the cytoplasm.
Cohen et al, 1990 , reported replacement of 13 PZD and 6 untreated embryos
in 7 patients resulted in three pregnancies with six fetal sacs. Absolute
proof of the implanting capacity of PZD embryos occurred in one patient.
In addition , two other twin pregnancies were confirmed following replacement
of two PZD and single control embryo in each patient. It is thus highly
probable that a PZD embryo produced at least one of the four gestational
sacs.
All sex fetal sacs were derived from oocytes treated with hyaluronidase
and sucrose. Exposure to these substances apparently does not jeopardize
fertilization and implantation. Also piercing and opening of the ZP didn't
inhibit the implantation of at least two PZD embryos ( Cohen et al ,1990)
.
Though the clinical use of partial zona dissection appears promising , true
success, measured as an increase in monospermic fertilization as well as
a normal ability of the embryos to implant, has been reported by only a
limited number of IVF programs (Cohen et al , 1992) .
While there is little doubt that partial zona dissection improves sperm-egg
fusion, excessive rate of polyspermy may lessen clinical efficiency. Monospermic
fertilization is controlled by the size of the hole, number of spermatozoa
used for insemination and patient selection criteria. These factors must
be considered carefully in order to apply PZD as a clinically valid tool
(Alikani et al, 1993) .
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