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Assisted Hatching (AH)

After fertilization, the zona pellucida (ZP) of mammalian eggs and embryos undergoes transformation into a hard coat that prevents polyspermy, protects the embryo, and maintains its integrity. Once it is in the uterus, the blastocyst must exit the ZP (hatching) to allow interaction between the trophectoderm and the endometrial cells for the process of implantation.

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.

Following fertilization, a process of zona hardening occurs that is evident from the increased resistance to dissolution by different chemicals. This procedure may be a consequence of the in vitro culture.

The process of assisted hatching (AH) in human embryos was first described in 1990 by Cohen et al in Human Reproduction. The process showed an increased implantation rate. The size of the hole seems to be adequate at 30–40 µm.

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?

Several different methods for AH have been introduced over the years, but comparative studies are lacking. In most of these techniques a full thickness gap is created in the ZP.

The most common techniques used today are:
• Mechanical hatching
• Acid Tyrode hatching
• Laser hatching
• Pronase thinning

Embryos at the 6–8-cell stage, at day 3 after insemination, or at the blastocyst stage, at day 5 or 6 after insemination, can be manipulated for AH. It is important that the size of the hole created in the zona is large enough to avoid trapping of the embryo during hatching, but not large enough to permit blastomere loss.

Mechanical Hatching
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

Laser 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

The following system facilitates microscopic objective-delivered accessibility of laser light to the target The device is usually attached to an inverted microscope below the objective turret. The laser AH software is designed for easy positioning, focus and measurement of embryos, and simple alignment of the laser. Usually low power is used for perforating very thin (15 µm) or hard ZP.

The type of laser equipment influences the different methods used, i.e. varying in energy, time, and number of pulses needed to open the ZP.

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.

For this process a solution of pronase is used (10 IU/ml pronase is diluted 10x by the G2 medium). The embryo is transferred to the pronase solution oil for ~60 sec for initial stretching, and softening of the ZP. The aim is to thin the ZP without complete removal. The embryo is then quickly examined at the heated stage of an inverted microscope to observe whether the zona size has expanded, is faint, and if the perivitelline space has increased in size. If these criteria are not met, the embryos are further incubated with pronase for an additional 30–60 s. The embryos are then transferred to fresh G2 medium and gently washed twice and placed into the incubator until transfer.

In an effort to establish some uniformity in AH indications and techniques the American Society for Reproductive Medicine (ASRM) summarized all prospective randomized controlled trials ( from 1996 to 1999) in a special committee report, in which different techniques for AH were compared to conventional ART in matching controls. Some of these studies showed a clinical benefit for AH, and some did not. The committee’s opinion was that “AH may be clinically useful and that individual ART programs should evaluate their own patient populations in order to determine which subgroups may benefit from the procedure”. The ASRM committee’s other conclusion was that “routine or universal performance of AH techniques is unwarranted”.

There are a great deal of controversies and debates about the use and results of treatment.
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.

Adverse effects

Monozygotic twinning has been described as a consequence of AH. This may occur by trapping the hatching blastocyst in the ZP gap. It may also be possible that one of the blastomeres could be damaged during the procedure, although every effort is made to avoid this.

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