1.1
- Preparing Tyrode’s solution - adjust pH to 2.1 with
N/5 HCl – filter – ( 0.45 mm)and store at 20oC
- Adjust pH for 2.1 with N/5 HCl
-
Filter – ( 0.45mm) and store at 20 oC
1.1.1
- Using Tyrode’s solution (Irvine Scientific)
Solution
ready for use – Tyrode’s Solutin Acidified (pH 2.1 –
2.5) – Ref. 99252.
1.
2 - Main indications
-
Implantation failure
- Patient’s age
-
Frozen and Thawed embryos
1.3
- Equipment
The
equipment used consists of na inverted Nikon Microscope (Eclipse
or Diaphot) with phase Hoffman’s phase contrast equipped with
two motor driven coarse control manipulators and two hydraulic micromanipulators
(MM-18 and MO – 109 Narishige Co Ltd, Tokyo, Japan). The holding
and hatching micropipettes are placed in the holder and controlled
by two microinjectors IM-6 (Narishige Co Ltd).
1.
4 - Technique
After
72 hours (Day 3) from the oocyte retrieval, when the embryos are
in a stage of development corresponding to 8 to 10 cells, we proceed
to do the hatching with Tyrode’s solution.
The
micromanipulator is adjusted. The dish is prepared with one central
droplet containing Tyrode’s solution and circled by 8 droplets
containing injection medium, one embryo is placed in each one of
those droplets. Cover with mineral oil. Procedure perfofmed over
heated platinum at 37oC.
Load
the hatching pipette with Tyrode’s solution and fixate the
embryo with the holding pipette. Position the hatching pipette at
3 o’clock, choosing the space between two blastomeres or na
embryo fragmentation area. Spray the solution over the zona pellucida
moving the hatching pipette up and down, thus making a hole in the
zona. Remove excess Tyrode’s solution. Repeat procedure in
all embryos. Wash the embryos in culture medium and proceed to transference
2 hours after this procedure.
HATCHING
SCHEMES
2
- Laser Hatching
Some
criteria used in embryology for choosing laser hatching are:
-
Thermal efect canot damage embryo.
-
Prevent genetic damage
- Minimal absorbance by DNA
- Avoids mechanical vibration and facilitates technical handling
having bigger efficiency.
-
Ability to pierce the zona pellucida, precisely dimensioning the
hole size and form .
There
are two types of Laser: CONTACT and NON CONTACT. The contact laser
needs sterilized material, micromanipulators and better technical
ability.
The
non-contact laser doesn’t need pipettes or micromanipulators,
the technique is simpler, there’s much more control over the
laser effect, like form, dimension and orientation, results are
instantaneous and procedure is relatively simple.
2.1
- Equipment
FERTILASE
– MTMT Medical Tehnologies Montreux AS, coupled to the Inverted
Microscope.
2.
2 - Main indications
The
main indications are the same as conventional hatching. Laser hatching
can be used for Pre-Implantation Genetic Diagnosis, Polar Body Biopsy
and Fragment Remotion.
2.
3 - Non contact laser technique
The
embryo may remain in the same culture dish, lid closed. Choose area
having bigger space between blastomeres or area of embryonary fragmentation,
and position the target area. Fire laser pressing a button. Opening
must be made between 12 and 15 mm. The embryo will be ready for
tranference.
