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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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Semen factors and limitations of micromanipulation
Results of the microsurgical fertilization cycles were studied by Cohen
et al, to investigate the prognosis of patient with severely abnormal
semen analysis and to determine whether low cut-off limits should be implemented
for application of assisted fertilization. (Behrman S.J et al, 1994)
Results of microsurgical fertilization expressed as an analysis of the
percentage of normal sperm forms shows that the severity of teratozoospermia
can not be used as a prognostic factor for predicting the outcome of micromanipulative
study (Behrman S.J et al, 1994).
It is obvious from these results that the outcome of microsurgical fertilization
can not be predicted on the basis of World Health Organization criteria.
The presence of one, two, or three abnormalities does not affect the rates
of fertilization and pregnancy. A substantial number of viable pregnancies
were established with semen specimens well below the normal cut-off values
for regular assisted conception procedures (Behrman et al, 1994).
Fertilization and pregnancy occurred following the use of spermatozoa
without progressive motility and without normal morphology. These finding
provide evidence that spermatozoa from extremely oligoasthenoteratozoospermic
men can produce normal offspring after the application of micromanipulative
techniques even when fertilization previously failed following standard
IVF (Behrman et al, 1994).
Generally speaking it is wise not to employ restrictive criteria for semen
used in micromanipulative studies (Behrman et al, 1994).
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Hemizona assay
A spermatozoon's ability to fertilize an egg is dependent on sperm
structure , the quality of sperm motility and capacitation (Bedford, 1973).
The HZA was developed to estimate sperm fertilizing potential based on
the relative binding of spermatozoa from a test patient versus a known
fertile control using the matching halves of a non living, bisected (hemi)
human ZP (Burkman et al, 1988).
The HZA offers statistically reliable data due to the use of matched zona
halves from a single egg (the penetration assay typically uses two to
four eggs per test) (Overstreet et al,1983). Also, inadvertent fertilization
of a human egg can not occur, since viability is destroyed during bisection.
In male factor cases of infertility, the HZA may be a useful tool by which
to monitor the progress of therapeutic courses or to select a "better"
sperm for microsurgical injection, than would a random choice (Brooks
& Bobby, 1991). The HZA is important for detecting sperm function changes
during infertility treatment, or during male contraceptive therapy or
following chemotherapy.
Technique
Human oocytes used in HZA can be obtained from ovarian tissue either
surgically excised or removed at autopsy. Another source is the excess
oocytes obtained beyond the need in ART programs. Under the microscope,
using two micromanipulators the oocyte is bisected, so that the two halves
are equal in size.
Semen is obtained from test subject and from proven fertile control. After
washing with culture medium, motile sperm is allowed to "swim-up" during
one hour of incubation at 37C.
The supernatant sperm are first diluted with medium; two sperm drops prepared
under oil 250,000 motile/ml. One hemizona from a matching pair added to
each drop.
After coincubation of 4hs, each hemizona is rinsed 5 times to dislodge
loose sperm. Direct count of sperm bond to outer surface is done. An assay
is rejected if the hemizona that was coincubated with control sperm bound
only (Franken , et al, 1993 ).
Calculation of the hemizona assay index from the resulting numbers =
(number of test sperm bound to hemizona)
____________________________________ x100
(number of control sperm bound to hemizona)
It should be noted that the duration of coincubation should not deviate
much from the standard 4hs. A very short incubation period 1 to 2hs fall
in the early part of the zona binding kinetics curve and may not be reliable
for diagnostic purposes (Burkman et al, 1988).
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