Cryopreservation is the process of preserving living cells, tissues, and other biological samples by placing them in a deep freezer at extremely low temperatures. The samples are typically stored at −196°C or below.
All of a cell’s biological processes come to an end at such low temperatures, and the cell dies. The process of cryopreservation enables cells to withstand freezing and thawing.
The cell membrane may rupture as a result of ice build-up inside the cells. By controlling the freezing rate and selecting the freezing media properly, this may avoided.
Cryopreservation Process
This procedure involves the preservation of biological materials such as cells, oocytes, spermatozoa, tissues, ovarian tissues, pre-implantation embryos, organs, etc. at extremely low temperatures without compromising the viability of the cells. Typically, this approach makes use of dry ice and liquid nitrogen.
Cryopreservation Steps
The following are all the steps in the technique for preserving the biological samples that were obtained:
1. Harvesting or Selection of material
When choosing biological materials, it’s vital to consider a few factors such volume, density, pH, shape, and damage-freeness.
2. Addition of cryo-protectant
In order to decrease the freezing point of the medium and enable a slower cooling rate, which lowers the danger of crystallization, cryoprotective chemicals such as glycerol, FBS, salts, sugars, and glycols are added to the samples.
3. Freezing
In this method of cryopreservation, several freezing techniques generally, shield cells from harm and cell death caused by their contact with warm solutions of cryoprotective chemicals.
4. Storage in liquid nitrogen
before transferring them to the storage containers. The cryopreserved samples are stored in a freezer at a temperature of -80 °C for a minimum of 5 to 24 hours.
5. Thawing
The procedure of warming biological samples to regulate the pace of cooling and avoid cell harm brought on by crystallization.
Best Age for cryopreservation
According to research, women who have frozen their own eggs before turning 30 had the greatest live birth rates. These eggs are probably of higher quality, increasing the likelihood that you will utilise them.
According to HFEA data, most women freeze their eggs around the age of 37. This raises a red flag since around this age, fertility begins to decline. It is preferable to freeze your eggs before you turn 36 if you want to guarantee better quality eggs and a higher likelihood of having a live birth.
This is the general suggestion that you freeze sperm before you turn 36 since sperm quality also decreases with age.
Cryopreservation of Embryos
preserving one or more embryos by freezing in preparation for use in the future. In vitro fertilization, in which eggs from a woman’s ovary are taken and sperm is added to create embryos in a lab. This is a step in the process of embryo cryopreservation. The embryos can also store, thaw, and then implant in a woman’s uterus. One method of preserving fertility is embryo cryopreservation. It could be helpful for cancer patients who wish to have a family after undergoing infertility-causing procedures like radiation treatment, chemotherapy, or some types of surgery. also known as “freezing” and “embryo banking.”
Is embryo cryopreservation worth it?
The choice to freeze embryos is a private one. The cost of fertility treatments varies greatly, and medical insurance may not cover them. You’ll need to take into account your objectives, the associated expenses, moral dilemmas, your partner’s preferences, and other factors.
Does freezing damage embryos?
Freezing harms embryos. If they are thawed, they may suffer harm also. A few or perhaps all of the frozen embryos might not survive.
Whether or not your embryos are acceptable for freezing, thawing, and implantation in this a healthcare professional helps more clearly.
Before embryo cryopreservation
Consent is mandatory. to freeze embryos. First, get proper consent signed by your healthcare professional. The documentation should include information like:
1. Number of frozen embryos.
2. How long is this stored (often 10 years).
3. What happens when the storage time expires.
4. What happens if you die or become too ill to make decisions before the storage period is over.
5. What are the required embryos that allow using for (for example, only your fertility treatments or if they can be donated to research or to another infertile couple).
Additionally, your health professional can assist you in determining the ideal time to freeze one or more embryos. Freezing is possible at the following stages:
1. Cleavage stage: When the single cell has multiplied to between four and eight cells after about 72 hours.
2. Blastocyst stage: When the single cell has multiplied to between 200 to 300 cells after five to seven days.
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During embryo cryopreservation
Embryos can freeze using either vitrification or slow freezing.
1. Vitrification
1. The embryos should give a cryoprotective agent (CPA). CPA is a liquid that shields cells from ice crystals and behaves like antifreeze.
2. Place the embryos right away in liquid nitrogen tanks that are set at a temperature of 321° Fahrenheit (-196.1° Celsius).
2. Slow freezing
Although slow freezing is no longer popular, certain fertility doctors could still practice it. During slow freezing, fertility experts:
1. Give the embryos less of a cryoprotective agent (CPA) than apply it during vitrification.
2. Place the embryos in a device that steadily lowers their temperature over the course of around two hours.
3. Take the embryos out of the cooler and place them in liquid nitrogen tanks at a temperature of -321°F (196.1°C).
It’s interesting to note that the embryos maintain their biological age after freezing. So the embryo hasn’t aged if you freeze them at age 35 and utilize them again at age 50.
After embryo cryopreservation
A fertility doctor will do the following if subsequent use of frozen embryos is necessary:
1. From the liquid nitrogen, remove the embryos.
2. Let their body temperatures gradually rise to normal. To remove the CPA, soak them.
3. Make use of the embryos as directed (for example, transfer them into your uterus).
Oocyte Cryopreservation
Egg freezing is popular with mature oocyte cryopreservation. It is a technique that mostly preserves a woman’s future capacity to get pregnant. Unfertilized eggs are taken from your ovaries freeze and keep it for later use. In vitro fertilization is the process of combining a frozen egg with sperm in a lab and implanting it in your uterus (in vitro fertilization).
The eggs freeze quickly during the vitrification process, which leaves less time for ice crystal formation. New cryoprotectants are applied with a high concentration of anti-freeze chemicals.
An anti-freeze-like cryoprotectant bath with a low concentration is first used to soak the oocyte. To assist remove some water from the egg, some sucrose is added. The egg is next moved to a high concentration anti-freeze cryoprotectant for a very brief period of time before being moved right away to liquid nitrogen. When the egg is defrosted and utilized to transplant the lady with the egg.
Risks of Oocyte Cryopreservation
Risks associated with egg freezing include:
1. Conditions brought on by the usage of reproductive medications. Rarely, the use of injectable fertility medicines, such as a synthetic follicle-stimulating hormone or luteinizing hormone to stimulate ovulation, might cause your ovaries to swell and hurt shortly after ovulation or egg retrieval (ovarian hyperstimulation syndrome). Indicators and symptoms include bloating, nausea, vomiting, and diarrhea. The likelihood of experiencing a more severe, even fatal, variant of the condition is even more remote.
2. Problems with egg retrieval. The colon, bladder, or blood artery can occasionally be infected, hemorrhaged, or be damage when an aspirating needle uses to remove eggs.
3. Emotional dangers although there is no guarantee of success, egg preservation might give women hope for a future pregnancy.
The likelihood of miscarriage will largely depend on your age at the time your eggs will get freeze. Due largely to aged eggs, elderly women experience increased miscarriage rates.
Cryopreservation of Sperm
A solution is to add the semen sample to preserve it from freezing and thawing. After that, the sample is transferred to polypropylene vials and maintained in liquid nitrogen to freeze.
This process secures the chances of conception in the future. Also possible is the deposit, freezing, and short-term storage of the sperm in cryobanks. Later, this sperm may apply to certain infertility therapies. In the future, the sperm may be defrosted and used for treatments like intrauterine insemination (IUI) or in vitro fertilization (IVF).
Sperm frozen and thawed
A specific freezing solution is used by andrologists to transfer prepared sperm into tiny vials. The vials are placed in liquid nitrogen-filled storage freezers. The sperm must maintain its freezing point at minus 196 degrees Celsius at all times.
The sperm from each patient is normally divided into a number of vials and stored in a number of storage tanks. The remaining sperm samples are safe even if one freezer breaks down. Sperm may be kept forever frozen.
The sperm is gently defrosted in the lab until it reaches room temperature before being used. It is examined after thawing to make sure there are still active, motile sperm present. Unfortunately, between 50 and 70 percent of sperm may not survive the freeze-thaw cycle in some instances. The ones who do survive, though, might have an edge in terms of fertility. According to studies, using these sperm in IVF may increase the likelihood that a pregnancy will result.
How many sperm and eggs should Freeze
Each ejaculation a man has contains a large number of sperm. About 20 million to 100 million sperm cells per milliliter of ejaculate are present. Every time a healthy man ejaculates, 1.5 to 5 ml of semen is produced. Therefore, freezing will only require one or two healthy sperm samples.
Eggs present a slightly more difficult situation. According to research, in order to increase her chances of becoming pregnant by 75%, a 34-year-old woman has to freeze 10 eggs. For the same chance, a woman who is 37 has to preserve 20 eggs. Additionally, in order to increase her chances of becoming pregnant by 75% by the time she is 42, 61 eggs must be frozen.
The fact that not all retrieved eggs will survive the process of being frozen, thawed, fertilized, developing into an embryo, and implanted in the uterus, as well as the fact that egg quality declines as women age are both accounted for by this rise.
Every stimulation cycle’s egg collection yields a different amount of eggs. The woman’s age, ovarian reserve, and how her body reacts to stimulation all have an impact on this. While some women may be able to generate so many eggs in a single cycle, others might require several.
Chances of success
Not every egg, sperm, or embryo that is harvested makes it through the freezing and thawing process. The freezing techniques, like gradual freezing or flash freezing, are one of the causes.
It takes a few hours for the slow freezing to finish and the final storage temperature to be reached. However, the newest flash-freezing technique, called vitrification, is significantly faster, doesn’t produce ice crystals, and doesn’t harm the egg, embryo, or blastocyst.
Once the eggs, sperm, and embryos are thawed and ready to be used, flash freezing appears to have higher success rates. The survival percentage of embryos during freezing and thawing is 95%. (Compared to 50 percent survival with slow freezing). This is a more effective method for freezing eggs, which have a survival rate from freeze-thaw of over 90%. (Compared to the approximately 66 percent survival rate of eggs during slow freezing).
