What Is Assisted Hatching?

Assisted hatching is the use of physical or chemical methods to artificially create a defect or crack in the zona pellucida of the embryo, which is conducive to the "breaking of the shell" from the zona pellucida or to dissolve and disappear the zona pellucida. In order to help embryos hatch and promote embryo implantation. Increase the likelihood of implantation.

Assisted hatching

Assisted hatching is the use of physical or chemical methods to artificially create a defect or crack in the zona pellucida of the embryo, which is conducive to the "breaking of the shell" from the zona pellucida or to dissolve and disappear the zona pellucida. In order to help embryos hatch and promote embryo implantation. Increase the likelihood of implantation.

Chinese name

Assisted hatching

Foreign name

assisted hatching

Method

Physical or chemical

Acceptor

Parent

Assisted hatching ^[1]

I. Origin and basis of assisted hatching

The concept of assisted hatching was first proposed. jacquescohen. He believes that in the ivf-et process, embryos cannot hatch from the zona pellucida, which is one of the reasons for the high failure of embryo implantation. If assisted incubation of in vitro fertilized embryos (ie, artificially opening or thinning the zona pellucida) will help improve embryo implantation rate.

Their basis is:

Whether human or mouse embryos undergo micro-assisted incubation, the time when the embryos begin to hatch and the morphological characteristics of the incubation will change, indicating that micro-assisted micro-operations can indeed affect the embryo's incubation process;

After artificially opening in the zona pellucida, the embryo implantation rate increased. It may be because assisted hatching increases the chance of the embryo anastomosis endometrial implantation window opening;

embryos with thin zona pellucida are easy to implant;

Any factors that affect the incubation will cause delay and loss of the embryo incubation process, resulting in failure of the implantation.

In 1991, Cohen's laboratory successfully assisted incubation of human in vitro cultured embryos using acidification. Although the implantation rate of embryos after assisted hatching has improved, there is no statistical difference compared with the control group. Further observation and analysis show that: assisted hatching does not have the effect of increasing the implantation rate for all patients and all embryos, but only for those embryos that have difficulty in hatching for some reason, such as hyaline Thick embryos, embryos with abnormal zona pellucida, patients with older maternal age, and patients with high fsh levels. As a result, the concept of selective assisted hatching emerged. That is, according to the specific conditions of the mother and each embryo itself, the embryos that have difficulty in hatching themselves are selected and assisted hatching is given. Assisted hatching helps to improve the implantation rate of embryos. It may be related to helping embryos with abnormal thickness and stiffness of the zona pellucida to break through the mechanical barrier, and helping embryos with poor quality without sufficient energy to break through the zona pellucida.

Second, the characteristics of assisted hatching Since assisted hatching does not improve the implantation of every embryo, in order to avoid exposing the embryo to unnecessary procedures and stimuli, (iohen proposed Indications to assist incubation. In the future ivf practice, other ivf centers have continuously expanded and supplemented these indications. Although different ivf centers have different grasps on the indications, but generally speaking In the future, when deciding whether an embryo needs to be assisted with incubation, the factors usually considered include: the age of the mother, the level of fsh, the history of ivf treatment, the quality of the embryo itself, the development speed of the embryo, the thickness of the zona pellucida, uniformity and shape.

1. Cellophane tape

(1) Thickness of zona pellucida: embryos with thickness of zona pellucida greater than 13um should be assisted incubation;

(2) The shape of the zona pellucida: When the shape of the embryo is irregular, such as an oval, it is often suggested that the zona pellucida has certain functional defects. Therefore, the shape of the embryo is irregular, which is usually an indication of the need for assisted hatching;

(3) Color of the zona pellucida: The color of the zona pellucida is darker. If it is brownish or darker, it should be hatched auxiliaryly.

2. Maternal age

The age of the mother is an important factor in considering the need for assisted hatching. As the mother's age increases, the zona pellucida of its embryo usually becomes thicker and harder, losing its elasticity, making it difficult for the embryo to hatch naturally. At the same time, older embryos from the mother's body usually have poorer quality, which will also make it difficult for the embryo to hatch naturally. Some ivf centers have a maternal age of 38 as a boundary. When the maternal age is 38 years or older, all embryos used for et should be assisted with incubation. And some IVF centers are based on the maternal age of 40 years.

3 Embryo quality

For poor quality embryos with more debris, their ability to implant is usually poor. After assisted hatching, its implantation capacity may improve.

4 Embryo development speed

Embryos that are stunted often have diminished natural hatching capacity and the hatching process is often delayed. After assisted incubation, it helps the embryo to maintain consistency with the opening time of the endometrial implantation window. Embryos with fewer than 6 cells on the 3rd day after fertilization had a higher implantation rate after assisted hatching than the control group without assisted hatching.

5. ivf treatment history

For patients with a history of failed IVF treatment, adjuvant incubation should be given. Because, the failure of natural hatching is likely to be one of the reasons that lead to the failure of their IVF treatment.

6. fsh level

If the level of fsh is too high (greater than 15 miu / ml) on the 3rd day of the cycle, it usually indicates a poor ovarian function. The zona pellucida of such patients is likely to be structurally abnormal or poorly functioning due to a poor follicular development environment. Assisted hatching often increases the implantation rate of such embryos.

7. Frozen embryo

After freezing and thawing, the zona pellucida of the embryo hardens. Therefore, freezing embryos is one of the indications of assisted hatching.

Third, the method and operation of assisted hatching There are currently four methods of assisted hatching: mechanical method, acidification method, laser method and enzyme digestion method. Among them, acidification and mechanical methods are in clinical use. It is widely used, especially the acidification method; the laser method is rarely used clinically because it requires special equipment; the enzyme digestion method is a new method that has appeared in recent years. It has been reported that the enzyme digestion method has obvious help for blastocyst implantation, and the effect of this method has yet to be confirmed by more clinical practice.

(1) Mechanical law

Mechanical method is called pairialzonadissection in English, pzd for short.

1. Assisted production of incubation work tray

(1) Use htf or pbs containing 6% plasmanate (commercial human plasma protein preparation) to make small drops in a falcon 1006 dish. The number of droplets is determined by the number of embryos to be assisted to hatch. 10 in the volume of each droplet;

(2) Cover the droplet with about 4ml of processed mineral oil (minel · aloil);

(3) After closing the lid of the tray, put the working tray into a 37 ° C incubator for pre-heating. Generally, pre-heating is required 30 minutes before use.

2. Preparation of the micromanipulator

The fixed needle is mounted on the left-hand microinjection needle holder, and the pzi) needle is mounted on the right-hand microinjection needle holder.

3 Mechanically assisted hatching operations

(1) Put the embryos to be hatched into the droplets of the working tray, put one in each drop, it is better to put 2 embryos at a time, so as to avoid long operation time and expose the embryos to the non-culture environment for too long.

(2) Lower the fixing needle into small droplets, so that the area with a large yolk gap is between 11:00 and 1 o'clock, and fix the embryo with a fixing needle at 9 o'clock.

(3) The pz [) needle is lowered into the droplet, and the needle is inserted from the part around 1 o'clock of the embryo, and the needle is punctured from the part around 1 l. Avoid damaging the cells of the embryo during operation.

(4) Loosen the fixed needle that fixes the embryo, move it from 9 o'clock to: pzi) needle, and rub the pz [] needle repeatedly with it. Blind until the zona pellucida is penetrated, the pzd needle comes out of the zona pellucida, and a crack is formed.

(5) Rotate the embryo by 90 degrees' and repeat the above operation twice. Eventually, two criss-crossed cracks were formed in the zona pellucida.

4 Wash the assisted incubated embryos 3 times in clean culture medium, and then put them back into the droplets of the original culture dish or the et dish, and wait for et

(Two) acidification method

The acidification method refers to the use of Acid Tyde's Solution (referred to as AT Solution) to dissolve the zona pellucida and form a small hole with a diameter greater than 20 gim in the zona pellucida.

1. Assisted production of incubation work tray

(1) Use the AT solution to make a 10-nine drop above the falcon 1006 plate, and circle it at the bottom of the plate with a red marker to show the difference.

(2) Use 6% plasmanate. HTF or pbs make a volume of 10 drops in the lower part of the dish. The number of droplets is equal to the number of embryos to be assisted to hatch.

(3) Cover the droplets with about 4 ml of treated mineral oil.

(4) After closing the lid of the disc, place the working disc in a 37 ° C incubator for pre-heating. Generally, pre-heating is required 30 minutes before use.

2. Preparation of the micromanipulator

(1) Install the fixed needle on the left-hand microinjection needle holder.

(2) The fixed needle is mounted on the microinjection needle holder on the right hand side, and is connected with the mouth suction control device (moul: h-eontrolledsuc! Ion).

3 Acidification assisted incubation operation

(1) Punch holes in the transparent zone

1) Lower the fixed needle into the AT droplet, use the mouth suction control device to suck the AT solution into the holdingpipette-, and then lift the fixed needle out of the liquid surface.

2) Fix the embryo at 9 o'clock with a fixing pin, and place the area with large yolk gaps or areas with more debris at 3 o'clock.

3) Place the fixed needle at 3 o'clock on the embryo, and use the mouth suction control device to carefully blow the at solution on the transparent band to dissolve it. While blowing the at solution, the fixed needle should be moved up and down slightly The range of the zona pellucida contacting the at solution is about 200um.

4) Once the zona pellucida is dissolved and penetrated, immediately stop blowing the AT solution and quickly and carefully begin to suck back to remove the AT solution in the area.

5) Continue to fix the embryo with a fixed needle while moving the stage of the microscope to move the embryo away from the working area just before, reducing the contact between the acid and the embryo.

(2) Remove the fragments. For embryos containing fragments, the small holes just dissolved in the zona pellucida can be used to suck out the fragments using a fixed needle. When doing this, be careful not to injure embryo cells or suck them out by mistake.

4 Washing of embryos after acidification After the embryos assisted by the acidification method are washed 3 times in clean culture medium, they are returned to the droplets of the original culture dish or the et dish, and wait for et.

(Three) enzyme digestion

In recent years, some IVF centers have used pronast instead of the AT solution to locally dissolve the zona pellucida in order to avoid the possibility of damage to the embryo caused by the acid solution, so as to achieve the purpose of assisting incubation. As blastocyst implantation has received more and more attention, some centers have used blastocysts to be incubated in a certain concentration of pr) nas solution for 24 hours before implantation, so that the zona pellucida is completely digested by enzymes and disappeared to promote Implantation of blastocysts.

(Four) laser method

The laser method refers to the use of a laser beam to punch holes in the transparent zone to assist in hatching ^[2] .