Assisted Hatching in Artificial Reproductive Technology - Everybody has consumed sprouts at some time in their lives. Have you ever had a bad grain or pulse that is so difficult to chew that it causes dental pain? If you look, you'll see that grain must not have sprouted. Certain recalcitrant grains don't seem to sprout at all, or hatch, in other words, even though your mother had maintained the identical circumstances for each grain. In a similar vein, certain obstinate embryos in an IVF lab are difficult to hatch, even when given all the necessary circumstances. An embryo must hatch out of its zona pellucida in order to deposit itself onto the endometrium and develop into a baby, much like a seed must sprout in order to attach itself onto the soil and grow into a plant. In this case, the embryo serves as the seed, and the endometrium as its soil. When embryos that resemble the hard grain refuse to sprout, what should we do? In this instance, we breach the zone pellucida wall. If you are considering for a Fellowship in Embryology, then this is a crucial topic for your career as an embryologist.
What is Assisted Hatching?
An embryo's zona pellucida is purposely weakened in a lab setting by making a hole on the surface that may simplify implantation. After hatching, either naturally or most likely with assistance, a herniated embryo or blastocyst emerges from the zona pellucida.
Why do we perform assisted hatching in IVF?
According to research, between 25 and 30 percent of embryos do not hatch under ideal circumstances in vitro, such as in a lab. It must completely hatch before it is implanted into the uterine endometrium. Therefore, it cannot go through the reactions or fully hatch before the endometrium is prepared. We can use hatching to ensure that the embryo is hatched inside the implantation window, as this could be the cause of implantation failures.
So, let's look into what zona pellucida actually is like the thing that we are trying to fight with or the thing that we are trying to break. It is an acellular matrix and it is composed of sulfated glycoproteins called Zp1, Zp2 and Zp3. It basically protects the embryo and it maintains the integrity of the embryo. It is a protective structure that helps the embryo stay intact.
How does a natural hatching happen?
Either no IVF technology was utilized, or the baby is growing naturally. The zona thins due to the blastocyst's early growth. The zona will then breach due to growth and zona thinning, and lysins from the uterus and embryo help in hatching and zona thinning. By breaking down the zona, these lysins are enzymes that facilitate hatching. The blastocyst finally emerges from the zona pellucida due to the expansion and function of lysin. Interactions between the endometrium and trophectoderm cells lead to implantation.
The interaction between the embryo and the endometrium is as follows: first, the embryo hatches, followed by opposition, adhesion, and invasion.
What are the causes for impaired hatching and what makes the embryo to not hatch?
· The first reason we are looking at here is zona hardening.
· The next cause for impaired hatching is increased zona thickness.
· The final reason is reduction in zona lysins.
What are the benefits of early hatching? Why are we doing the hatching?
It could be of great trauma to the embryo like we are manipulating so much of the natural process. It could like hinder from the implantation happening and it could result in a failed embryo transfer.
Cellular energy requirements for hatching might be insufficient in poor prognosis patients. Many IVF cases come with high age, PCOS and many other poor prognosis patients. Here the cellular energy requirement for hatching might be insufficient. So, this hatching we are externally mechanically breaking the zona making it less difficult for the embryo to break it. Assisted hatching decreases energy requirement for hatching. It optimizes implantation window. So, in normal hatching five days after fertilization embryo has 48 hours to implant after you know the fifth day. The implantation window is narrow in stimulated cycle 72 to 120 hours post fertilization. If it crosses the 120th hour the implantation window is crossed and the embryo transfer will fail. We hatch it before that 120th hour so that we help or aid implantation to occur.
What are the techniques we use to artificially or assist the embryo in hatching?
· First thing is mechanical. You can mechanically tear the zona open. Partial zona dissection on day two is made in this technique. So, we perform this technique on the day two.
How many cells are there in the day two?
It could be eight cells. The first day it divides into two cells and then the second day it could divide into four cells and sometimes in second day it could be as many as eight cells too. So, embryo held by holding pipette.
· The second one is chemical like how we saw the lysins naturally thin the zona. We can use chemicals to break the zona and then enzymatic again enzymes can be used to completely dissolve the zona pellucida and then laser assisted technique where you use lasers to break small parts of zona pellucida so that the blastocyst can herniate out and hatching occurs.
· Piezo assisted technique this is a new technique where you use a needle and you pierce a little bit of zona and then piezo is a mechanism where it needle vibrates vigorously so that the hole is formed in the embryo and the herniation and hatching is aided.
The enzymatic method is done on day 5 embryos. Why is it that we do it on day 5 embryos? The cell-to-cell anchorage or the interaction or adherence or the junctional complexes between the adjacent blastomeres is very less before this. So, at day 5, it has formed like proper blastocyst, very good blastocyst where the blastomeres are very well adhered to each other.
The laser-assisted drilling is atraumatic and most popular method. In enzymatic method, you are basically completely dissolving an entire part of the embryo. In chemical methods, you are spraying extremely acidic chemicals onto the embryo. So, these are very big traumas to the embryo. So, laser-assisted drilling is the least traumatic technique that we have come up with.
The precautions we should take while using laser-assisted drilling is:
· We should avoid thermal effects.
· Any heat that is causing the embryos or that is being formed around the embryos, so that could be eliminated. So, the wavelength should be used above absorption maximum of DNA, which is more than 265 nanometres and low ablation threshold to minimize mechanical vibration.
So, there must be absolutely no vibration when we are performing the laser-assisted drilling. Why is that? Because we are focusing the laser on the embryo and if the embryo moves, the laser could damage another part of the embryo and one good thing about laser-assisted drilling is that the handling of it is very easy and you need not be very highly skilled to perform laser-assisted drilling.
What are the types of laser systems?
· Contact laser system
How does the contact laser systems to hatch the embryo out?
The laser beam is inserted and then directed by a glass or optical fiber. The fiber is fitted to micromanipulator by a pipette holder and is brought in direct contact with the zona, but the tip is not forced further into it before application of the laser beam. This prevents any contact of the fiber with the surface of the ooplasm or blastomere once it penetrates the zona. So, the laser should either be disposable or sterilizable before being used on other patients’ embryos.
· Non-contact laser system
These are the most popular assisted hatching techniques. So, this is very easy to use and laser beam guided through an optic lens is focused on the embryo and complete sterility is maintained. Why? Because none of the parts of the equipment is touching the embryo.
It is completely sterile. So, there is high precision to since it's focused on the embryo at a point it is very precise and very less trauma is given to the embryo.
Piezo-assisted drilling
This is a novel method that creates a hole in the zona pellucida using electromagnetic pulses. Zona has no bending and is cocked off. For almost two seconds, a continuous piezoelectric pulse is therefore delivered. In order to create a hole—or more accurately, a slit—in the zona pellucida, we puncture it with a piezoelectric pulse and stop using electromagnetic pulses for two seconds. Therefore, the tip of the injection needle vibrates strongly in the same plane that is vertical to the needle's long axis due to the piezoelectric pulse generated under these conditions. The vibratory needle is transferred to a neighbouring area while the embryo stays in the same pipette position, and the same process is repeated once approximately 75% of the zona pellucida thickness has been removed.
Disadvantages of Assisted Hatching
· The zona pellucida, the protective covering, may be lost, making our embryo more vulnerable to infection. Our embryo might be infected by microbes. It could therefore be immunocompromised. Conversely, suppose the female body perceives the embryo as a microbe, an opponent, or a foe. In that case, it may fight it under the mistaken impression that it is an enemy, perhaps killing our embryo.
· Increased loss of blastomeres or the whole embryo and natural expansion of blastocysts may fail to occur just like how the stories have been told in our childhood where you know a butterfly was helped to hatch out of its cocoon and the butterfly couldn't survive for longer and why was that because it couldn't bring in all the strength to push itself out of the cocoon so it could not further survive. So, similarly natural expansion of blastocysts may fail to occur.
Monozygotic Twinning
So, this is seen very often in assisted hatching cases where these embryos they split themselves into two and they form identical twins where both of them form into two different offsprings.
Patient selection
When do we select patients to go for assist hatching of embryos?
The indications are woman's age more than 38 years then elevated basal FSH levels, zona thickness more than 18 micrometres, repeated failed IVF or ICSI cycles and embryos generated in in vitro maturation technique and cryopreserved embryos. All these indications could be taken for applying assisted hatching for embryos.
· Another reason why we should not go for assisted reproductive technology in cases where blastomeres not appear to be in interface.
· Medical management: Antibiotics because microorganisms around the embryo must be killed so that it doesn't get infected because there is no zona present. So, antibiotics will help remove all the microbial environment around the embryo and immunosuppressive agents why? Because the body shouldn't see the embryo to be an enemy to us and all our WBC cells and all our fighting mechanisms should be suppressed. Why? Because we want our embryo to be implanted. So, methylprednisolone of 16 mg four times a day is given before the day and tetracycline 16 mg per day starting from the day of opiules or opium pickup is given.
Summary:
Sulfated glycoproteins make up the acellular matrix known as zona pellucida. Reduced zona lysis, increased zona thickness, and zona hardness may be the causes of poor hatching. The implantation window can be increased with assisted hatching. A mechanical, chemical, enzymatic, laser-assisted, or piezo-assisted method may be employed. The downsides to assisted hatching could be due to infection, immunological reasons and monozygotic twinning. So, we can conclude that assisted hatching of human embryos increases the chances of implantation. The most often used aided hatching technique is laser assisted hatching. Why is that? Since laser aided hatchings are non-contact, the embryos are completely sterile because none of the equipment comes into touch with them. Why is it so crucial that we prevent the equipment from touching the embryo in this situation? because the embryo's zona pellucida, which shields it from infections, is now absent. Therefore, it is crucial that no equipment comes into contact with it. We can state unequivocally that assisted hatching procedures, which are often employed in many IVF centers, can increase implantation rates.
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