Assisted Hatching (AH)
5-day blastocyst hatching from its shell Hatched blastocyst
Failure to hatch due to intrinsic abnormalities of the blastocyst, ZP or endometrium, is considered one of the major factors limiting implantation.
Day-3 embryo with thick zona Embryos with cytoplasmic fragmentation
8-cell embryo (1. zona pellucida, 2. blastomere, 3. fragmentation)
What should be the indication?
• Embryos with thick zona (>15 µm)
• Patients with elevated day-3 follicle-stimulating hormone (FSH)
• Embryos with extensive fragmentation or slow rates of cell division
• Cell death after freezing and thawing
• Advanced maternal age
• Multiple implantation failure
How is assisted hatching (AH) performed?
The most common techniques used today are:
• Mechanical hatching
• Acid Tyrode hatching
• Laser hatching
• Pronase thinning
Embryos denuded of corona cells are micromanipulated in microdrops of HEPES-buffered medium under paraffin oil. As mentioned above, the procedure is performed at 37°C, under an inverted microscope.
The embryo is held in position by gentle suction from the holding pipette and the microneedle is passed through the ZP at the largest perivitelline space and advanced tangentially, from the 1 o’clock to the 11 o’clock position. The embryo is then released from the holding pipette and held by the microneedle. The microneedle is brought to the bottom of the holding pipette and the embryo undergoes gentle friction until a cut is made. Once again the embryo is rotated until the slit is visible at the 12 o'clock position. The embryo is firmly held by the holding pipette and the ZP is cut in a similar manner, creating a cross-shaped slit.
Inserting the pipette through the ZP
Gentle friction against the holding pipette
Acid Tyrode assisted hatching (AH)
An embryo is stabilized with a holding pipette held at the 9 o'clock position and a 10 µm pipette containing acid Tyrode solution is oriented at the 3 o'clock position adjacent to an area of an empty perivitelline space. It is recommended that the embryo be held in such a way that the micropipette containing acid Tyrode faces a large perivitelline space, or an area with cytoplasmic fragments of the embryo. A 30-µm diameter defect in the zona is then created by using a mouth-controlled delivery system to blow the acid Tyrode over the external surface of the zona. The embryo is then rinsed several times to wash off the excess acid Tyrode and return it to the standard culture media until transfer. Limiting embryo exposure to acid Tyrode by adequate and quick manipulation is necessary to avoid harmful effects on embryo development.
8-cell embryo in the process of assisted hatching
The use of laser technology in the reproduction field was first described in 1989 by Tadir et al. A few technologies were developed:
A. The Contact Laser
This procedure is performed on a microscope slide, while the embryo is placed in a drop of medium covered with paraffin oil. The embryo is held with a holding pipette, and the laser is delivered through a microscopic laser glass fiber, fitted to the manipulator by a pipette holder, in direct contact with the ZP. Several pulses are necessary to penetrate the ZP. Because each laser pulse removes only small portions of the ZP, the fiber tip should be continuously readjusted to guarantee close contact with the remaining zona. The main disadvantages of contact mode lasers are the necessity for sterile micropipettes and optical fibers to deliver the laser beam to the target.
Microscope attached to a laser system Zona pellucida after laser treatment
B. Noncontact laser
Day-3 embryo after assisted hatching with laser
C. Assisted hatching (AH) by pronase thinning of the zona pellucida
Zona pellucida thinning is the aim of the following technique, i.e. to narrow the ZP without complete lysis and perforation. By not breaching the zona, the potential risk of blastomere loss and embryonic infection is minimized.
There are several advantages to the use of the the laser system, as it facilitates a rapid, simple and accurate performance of the procedure, with relative safety. There is also less operator variability, therefore results obtained with the same system can be compared with greater reliability.