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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
Anomalies of the female genital tract

 
   
 
     

Microimplantation and preimplantation diagnosis of the cleaving embryo

Single blastomere can be removed from cleaving embryos, or several trophoblast cells can be taken from blastocysts by microscopic manipulation without impairment of development (Tarin & Handyside, 1993 ).
This capability raises the prospect that embryos may be biopsied and subjected to genetic analysis during cleavage. If the result of such analysis can be obtained before embryo transfer, then the physician may have the ability to screen for genetic diseases even before implantation. This advance would eliminate the unwanted consequence of chorionic villous biopsy or amniocentesis ( Tarin & Handyside, 1993 ).
Prenatal diagnosis by embryo biopsy have limitations. Cleaving embryos must be removed from the female for testing. Embryos those are fertilized in vivo can be flushed from the reproductive tract. However, the efficiency of this procedure must be improved before it is routinely applied (Tarin & Handyside, 1993 ).
However, in ART this procedure can be applied especially if the number of embryos available for intrauterine transfer is more than that can be transferred in one cycle. This is highly important in cases of severe male factor with high percent of abnormal forms ( oligoasthenozoospermia ) in which incidence of chromosomal abnormalities is high. By this procedure complete genetic testing can be done, and transfer of genetically diseased embryos would be avoided (Tarin & Handyside,1993 )

Methods of embryo biopsy

Cleavage stages

  1. Aspiration method: The blastomeres are removed by suction with a micropipette. The sample micropipette can either be forced through the ZP like a needle or can make direct contact with the blastomere through a hole or slit in the zona made previously using either acid Tyrode's solution or a sharpened dissection pipette, respectively.

  2. Extrusion method: can be performed in three different ways Displacement : an opening is made in the ZP with a beveled pipette, then, the beveled pipette is inserted at a second point. A gentle flow of medium injected through the pipette is used to dislodge, displace and make one or more blastomeres protrude beyond the ZP through the first puncture site.
    Stitch and Pull: The biopsy is carried out by drilling a hole through the zona with acid Tyrode's solution. Afterward, single cells are removed using stitching movements with a microneedle .
    Push: After drilling the zona, the blastomeres are extruded through hole by pushing against the zona with a microneedle at some distance from the hole.
  3. Mechanical division: Removal of the ZP by chemical or mechanical force. Immediately, thereafter, the blastomeres are separated by aspirating the embryos with a Pasteur pipette.

Blastocyst stage

  1. Aspiration : The sample micropipette sucks a number of cells away from the mural trophectoderm cell layer opposite the inner cell mass while a microneedle cuts the cell contacts.
  2. Stitch and Pull: uses two siliconised glass microneedles to manipulate the embryo and to withdraw several mural trophectoderm cells through the ZP.
  3. Herniation : A tear or slit is made in the ZP opposite the inner cell mass. The embryo are left in culture for several hours until a definite herniation of mural trophectoderm cells is visualized. Then , the herniating cells are cut off.

Recently, Tarin and Handyside (1993 ), studied embryo biopsy strategies for preimplantation diagnosis, in order to analyze different biopsy methods embryo stages and cellular masses that can be removed for preimplantation diagnosis of genetic diseases to find optimal biopsy conditions compatible with the subsequent development of the conceptus, the reliability of genetic analysis.
They reported that the displacement and push methods may be more suitable than the stitch and pull and the aspiration approaches at cleavage stages. also the aspiration and stitch and pull procedures may assure higher success rates than the herniation procedure at the blastocyst stage . The mechanical division method and the use of acid Tyrode's solution would not be advisable before the eight-cell stage.
Human embryos at the two-cell stage and blastocyst stage may not be suitable for preimplantation diagnosis because of an excessive reduction of cellular mass at the two-cell stage and low or zero pregnancy rate after transfer at the blastocyst stage.
Biopsy of a quarter of the embryonic cellular mass or day 2 after insemination may increase biochemical pregnancies if the cleavage rate is not preserved. Tarin and Handyside, (1993) concluded that, at the present time, biopsy of a quarter of the embryo on day 3 after insemination may be the most feasible approach for preimplantation diagnosis.

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