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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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Subzonal injection of sperm [SUZI]
Single sperm transfer in the human was first done by Metka et al (1985
), the sperm transferred was treated as in routine IVF of 9 oocytes manipulated,
one pronuclear oocyte cleaved to 4 cells. After embryo replacement there
was no pregnancy .
This was achieved by Laws-King et al (1987) where they used a calcium
depleted strontium-based medium ( Mortimer et al, 1986 ) to incubate the
sperm over night. Of 7 human oocytes cultured 6-9 hours after collection
which had single sperm transfers, 5 fertilized. Two out of 3 that were
cultured under went normal cleavage while the rest were examined by TEM
or were karyotyped (Laws-King et al, 1987 ).
Single Sperm Transfer
Lassalle et al, (1987) first reported multiple sperm transfer into
human oocytes 3-5 sperms were transferred into 7 oocytes and fertilized,
all monospermic. When the number of sperm transferred was increased to
10-12 sperm per oocyte, 2 of 3 oocytes fertilized but they were polyspermic
.
It is possible that with high sperm number, more than one sperm could
attach onto their receptors on the oolemma before the receptors are inactivated
possibly by cortical granule release.
The first pregnancy after SUZI has been reported (Ng et al, 1988) . She
has delivered a healthy baby boy weighing 3.3kg by LSCS. This was the
first delivery by micro-insemination.
In the second pregnancy, 2 monospermic zygotes and 1 dispermic zygote
were obtained after SUZI. The monospermic zygotes were transferred into
the fallopian tube at pronuclear stage. Unfortunately, she developed a
left ectopic pregnancy and an intra-uterine pregnancy which was aborted.
Ng et al, (1990) studied incorporation of multiple transferred human spermatozoa
into hamster oocytes. Of 49 hamster oocytes, 43 (87.8%) had spermatozoa
incorporation ( decondensed spermatozoal heads ) following SUZI, compared
to 88 of 121 (72.7%) control zona- free hamster oocytes.
when the human spermatozoa were micro-centrifuged before introduction
to the hamster oocytes , there was reduced sperm incorporation 22 of 41
MIST and 79 of 130 for control zona-free hamster oocytes. Thus, in both
groups, MIST resulted in significantly better sperm incorporation. However,
when results were compared between micro-centrifuged spermatozoa and sperm
that were not , sperm incorporation into hamster oocytes was better when
spermatozoa were not centrifuged.
There is no evidence of increased chromosomal abnormality following MIST
. Kola et al (1990) karyotyped 18 fertilized human oocytes after transfer
of single sperm from patients with severe male factor. Two were monosomic
and two were trisomic, these rates were not significantly different from
30 oocytes fertilized by conventional IVF.
Cohen et al ( 1992 ) , suggest that both SUZI and PZD should be practiced
simultaneously , as fertilization is not affected by the severity of semen
profiles. The effects of both methods of microsurgical fertilization are
additive in term of fertilization and implantation. A comparison of the
results before and after the introduction of SUZI has demonstrated that
the overall pregnancy rate would be considerably reduced if one of the
methods would be discontinued (Behrman, et al,1994).
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Technique
Because usually the semen quality is very poor , so the entire semen
sample is used and spermatozoa are collected by the Ficoll entrapment
procedure (Cummins and Breen, 1984). This procedure improves sperm samples
from W.H.O motility grade I to II with concomitant improvement in fertilization
(Bongso et al ,1989 ) .
After the motile spermatozoa are collected from the supernatant , they
are concentrated in a micro-centrifuge for 10 minutes and the supernatant
is removed .
Such micro-centrifigation does reduce the fertilizability of such sperm
after MIST (as explained before ). Ficoll solution (5% in PBS, 0.1 ml
) is added and the suspension is kept at 4C until use.
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