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Submitted by Dr. Hesham Al-Inany, M.D. Lecturer, Gynaecology & Obstetrics dept. Kasr El-Aini hospital, Cairo University, Egypt.

Micromanipulation
 

During Gamete Micromanipulation sperms are injected into an ovum to assist in union of the gametes.

 
 

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Gametogenesis: a basic review
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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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