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Submitted by Dr. Hesham Al-Inany, M.D. Lecturer, Gynaecology
& Obstetrics dept. Kasr El-Aini hospital, Cairo University, Egypt.
During Gamete Micromanipulation sperms are injected into an ovum to assist in
union of the gametes.
Evaluation of the embryos
The embryo quality depends on two basic factors : the gamete quality
and availability of optimal culture conditions. There is a need to improve
culture conditions and media to increase the rate of embryo cleavage and
development in vitro.
A clear correlation between the morphological appearance of the embryo,
its growth rate and the chances to implant have been noticed. Therefore,
a grading system of morphology is in use to evaluate the embryo, to choose
the good ones for replacement and freezing .
Grade I : Equal sized blastomeres and no cytoplasmatic fragments.
Grade II : Unequal sized blastomeres with or without slight fragmentation
aside the polar bodies.
Grade III : Equal sized blastomeres with some anucleated fragments (>20%).
Grade IV : Unequal sized blastomeres , highly fragmented (> 50% ) and
No replacement of embryo of grade IV morphology and no cryopreservation
of embryos of grade III or IV. Inspite of this grading system, there is
no absolute selection criterion available for determining egg and embryo
viability ( Ron-EL et al, 1993 ) .
Embryo cryopreservation is now firmly established in cases with excessive
fertilized embryos avoiding the necessity to replace large numbers of
embryos with the risk of multiple pregnancies. To avoid the formation
of intracellular ice , cells must be sufficiently dehydrated by the time
they reach the temperature at which intracellular ice nucleation can occur.
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Embryo freezing protocols
- Slow cooling and slow thawing protocol: using dimethylsulfoxide
(DMSO). The embryo is then cooled at a rate of 2C/min to - 6C. Thawing
is performed at a rate of 8C/min to room temperature (Trounson and Mohr,
- 1,2-propanediol 1.5M and sucrose are used as cryopretectant. The
embryo remains for 15min in the medium with the cryoprotectants cooling
as above but the thawing is very rapidly performed, reaching room temperature
at <1min. (Renaud and Barbinet ,1984).
- (III) The ultrarapid method first described by Trounson and Sjoblam
(1988), is a modification of the vertification method in which glass
rather than ice is formed. A high concentration of 2.5M DMOS and 0.25M
sucrose in buffered saline phosphate is mixed with 10% heat-inactivated
Embryos are suitable for replacement after thawing if the zona is intact
or if at least half of the prefreezing numbers of blastomeres are intact.
The eight-cell stage embryo is usually preferred for freezing because
of its ease in handling and the probability of enough intact blastomeres
to be replaced (Ron-El et al,1993).
Mature unfertilized oocytes could be fertilized, specially in countries
in which freezing of human embryos is considered unacceptable, however
rare pregnancies after freeze-thawing oocyte with later fertilization
were described . Lastly, intensive research in this field will certainly
open new horizons in ART.
Two out of three embryo replacement fail to implant, and the proportion
of the successfully implanting embryos is even much lower. The successful
nidation of an embryo after its replacement is influenced by various factors
including the age of the patient, the number and the quality of the embryos
and the receptivity of the uterus (Ron- EL et al, 1993 ).
According to Behrman S.J. et al, 1994, the reduced implanting capacity
of micromanipulated embryos can be explained by:
- The micromanipulation procedure may be performed in a relatively
harsh manner, resulting in some form of permanent cellular damage. Damage
may not immediately be manifest in the embryo.
- Enviromental factors such as pH and temperature may be important
to avert indirect damage during micromanipulation.
- Procedures for rising and pipetting micromanipulated embryos should
be carefully performed to avoid leakage of cytoplasm and blastomeres
through the artificial gaps.
- Routine clinical procedures may need adjustment as well. The prophylactic
use of antibiotics and low dose corticosteroids in female partners should
The common practice is replacement of embryos into the uterine cavity
through the cervical canal. This transcervical replacement must be carried
out as quickly and atraumatically as possible. It is believed that the
chance of successful implantation increases with the ease and neatness
of the replacement. The appearance of blood on the tip of the catheter
is considered as a bad prognostic sign.
Patient lies in the lithotomy position, the bladder is emptied just
prior to the embryo replacement and the procedure is carried out without
any anesthesia or analgesia.
Gentle catheterisation of the cervical canal with a sterilized catheter
is performed to feel the ease and direction. Once a free passage is demonstrated
the embryologist loads the embryos into a new catheter and hands it over
to the surgeon.
After the replacement, the catheter flushing is promptly examined under
the microscope to ensure that none of the embryos have been left behind.
Patient lies on her back for one hour and does not get up for additional
two hours. Bed rest at home is advised for 48 hours.
Finally, it is important to recognize that micromanipulated embryos are
susceptible to physical damage and should be replaced with more care than
zona intact embryos (Cohen et al, 1992).