OOCYTE FREEZING

OOCYTE FREEZING:

Human oocyte cryopreservation is a procedure to preserve a woman’s eggs also known as mature oocyte cryopreservation which is used to preserve reproductive potential in women. The eggs are extracted, frozen and stored. Intention of the procedure is that, in the future, the woman may choose to have the eggs thawed, fertilized, and transferred to the uterus as embryos to facilitate a pregnancy.

When the egg is frozen, the ice crystals that form can destroy the integrity of the cell. To prevent this, the egg must be dehydrated prior to freezing. This is done using cryoprotectants which replace most of the water within the cell and inhibit the formation of ice crystals.

Births resulting from previously frozen oocytes with those from fresh oocytes have not shown an increased risk of congenital anomalies.

Egg Freezing Success rates have been estimated between 4-12% per oocyte.

SUCCESS RATE:

31.5% – at age of 25

25.9% – at age of 30

19.35 – at age of 35

14.8% – at age of 40

INDICATIONS:

Women with cancer requiring chemotherapy and/or pelvic radiation therapy that may affect fertility.

Surgery that may cause damage to the ovaries.

Risk of premature ovarian failure because of chromosomal abnormalities (e.g. Turner syndrome, fragile X syndrome), or family history of early menopause.

Ovarian disease with risk of damage to the ovaries.

Genetic mutations requiring removing the ovaries (e.g. BRCA mutation).

Fertility preservation for social or personal reasons to delay childbearing.

How Will the Eggs be used in the Future?

When the woman is ready to use the frozen eggs to achieve pregnancy, these cryopreserved eggs are placed in warming solution and assessed. Those eggs that survived the freezing process are fertilized with intracytoplasmic sperm injection (ICSI), where a single sperm is injected directly into the egg, and the fertilized eggs will grow in culture until the embryo(s) are ready to be transferred into the uterus to achieve pregnancy, typically 3-5 days after fertilization.

How Long Can the Eggs be Stored?

Storing the eggs for longer durations does not appear to have negative effects. As elderly maternal age when carrying a pregnancy is associated with higher risks of pregnancy complications, such as high blood pressure, diabetes.

 CRYO: -vitrification, no ice crystals are formed inside cells as freezing occurs ultra-rapidly.

-slow freezing can lead to ice formation due to a very slow freezing process (approx. 2 hours).

1) slow-cooling method has not been used for oocyte freezing.

2) Flash-freezing process- vitrification : Vitrification is much faster but requires higher concentrations of cryoprotectants to be added. Vitrification eliminates ice formation inside and outside of oocytes on cooling, during cryostorage and on warming. Vitrification is associated with higher survival rates and better development compared to slow-cooling when applied to oocytes in metaphase II (MII)

During the freezing process, the zona pellucida, or shell of the egg can be modified preventing fertilization. Thus when eggs are thawed sperm is injected directly into the egg with a needle rather than allowing sperm to penetrate naturally by placing it around the egg in a dish. This injection technique is called ICSI Intracytoplasmic Sperm Injection.

SEMEN FREEZING:

Cryopreservation of human spermatozoa is introduced in the 1960’s. It as an efficient procedure for management of male fertility before therapy for malignant diseases, vasectomy or surgical infertility treatments. spermatozoa seem to be less sensitive to cryopreservation damage because of the high fluidity of the membrane and the low water content.

SUCCESS RATES:

35, the success rate is around 19%

35–39, the success rate is around 15%

40–42, the success rate is around 7%

 

1) Assisted reproduction technologies in cases of preservation of male fertility before radiotherapy or chemotherapy which may lead to testicular failure or ejaculatory dysfunction. semen cryostorage seems to be the only proven method that may offer these couples a chance of having children in the future as cancer therapy may lead to damage, resulting in subfertility or sterility due to gonad removal or permanent damage to germ cells caused by adjuvant therapy

2) Diabetes and autoimmune disorders may lead to testicular damage.

3) Donor insemination programmes cryopreservation is necessary to have enough time to screen donors for infectious agents, such as the human immunodeficiency (HIV) and hepatitis B viruses

4) Azoospermic patients, who have undergone testicular sperm extraction or percutaneous epididymal sperm aspiration, sperm cryostorage is also used to avoid repeated biopsies or aspirations

5) Assisted reproduction treatment to preemptively freeze the semen sample to avoid inconveniences due to failed ejaculation often associated with “semen collection stress”

Slow Freezing

The slow freezing technique proposed by Behrman and Sawada consists of progressive sperm cooling over a period of 2–4 h in two or three steps, either manually or automatically using a semi programmable freezer.

The manual method is performed by simultaneously decreasing the temperature of the semen while adding a cryoprotectant in a stepwise manner and after plunging the samples into liquid nitrogen. It has been shown that the optimal initial cooling rate of the specimen from room temperature to 5°C is 0.5–1°C/min. The sample is then frozen from 5°C to −80°C at a rate of 1–10°C/min. The specimen is then plunged into liquid nitrogen at −196°C

Rapid Freezing

Rapid freezing was first proposed by Sherman. This technique requires direct contact between the straws and the nitrogen vapours for 8–10 min and immersion in liquid nitrogen at −196°C. Inside nitrogen vapours there is a thermal gradient, as a function of the distance and the volume of the liquid below. The sample is initially mixed in dropwise manner with equal volume of cold cryoprotectant; the mixture is loaded into the straws and left to incubate at 4°C for 10 minutes. The straws are then placed at a distance of 15–20 cm above the level of liquid nitrogen (−80°C) for 15 min; after this stage, the straws are immersed in liquid nitrogen. During cooling it is preferable to place the straws in horizontal position to minimize the heat difference between the two ends. This technique has some drawbacks among which; for example, low reproducibility, indeed, the temperature drop curve cannot be controlled, and the freezing temperatures may vary from −70, −80, and −99°C.

REFREEZING

In terms of the level of sperm DNA fragmentation, up to three cycles of freezing and thawing can be performed without causing a level of risk significantly higher than following a single cycle of freezing and thawing. This is provided that samples are refrozen in their original cryoprotectant and are not going through sperm washing and provided that they are separated by density gradient centrifugation or swim-up before use in assisted reproduction technology

Cryopreservation of Small Numbers of Spermatozoa

The conventional methods of sperm cryopreservation are not ideal to cryopreserve small numbers of cells, such as epididymal and testicular spermatozoa. Efficient cryopreservation of surgically retrieved spermatozoa.

Cryoprotectants: glycerol, ethylene glycol, dimethyl sulphoxide, and 1,2-propanediol.

Cryoprotectants are low-molecular-weight and highly permeable chemicals used to protect spermatozoa from freeze damage by ice crystallization. Cryoprotectants act by decreasing the freezing point of a substance, reducing the amount of salts and solutes present in the liquid phase of the sample and by decreasing ice formation within the spermatozoa. The cryoprotectants are added in an equal volume of semen in a dropwise manner, gently mixed at room temperature, and then placed at 37°C for 10–15 minutes to allow for proper equilibration between the cells and the medium. It is necessary that the medium interacts with the cells. The effectiveness of cryoprotecting substances is also a function of the time of interaction between the cryoprotectants and the cells. Glycerol is the permeating cryoprotectant most widely used for human sperm acting on: the membrane structure, permeability and stability of the lipid bilayer, the association of surface proteins and the cellular metabolism. Its employment gives an unfavorable outcome on membrane and acrosome structure, although allowing the freezing of poor quality Glycerol may cause few alterations such as: presence of an undulating membrane, alteration in acrosomal internal membrane, nucleus inhomogeneity and disorganization in mitochondrial crests. Following this observations other protective substances were proposed such as the dimethyl sulfoxide which has deleterious effects on human sperm when used at 4°C, and the 1,2-propanediol slightly used in sperm cryopreservation.

 

–The primary cause of cellular damage during cryopreservation is the formation of intracellular or extracellular ice crystals. During the freezing process, the cooling rate plays an important role in determining the extent of cryoinjury to the spermatozoa

–A rapid cooling rate causes severe intracellular ice formation, since the efflux of water across the membrane is impaired, thus, inducing supercooling.

 

Cryo preservative

Slow freezing

For a long time, slow freezing has been the method of choice for freezing sperm, oocytes, embryos, or blastocysts. Most typically, embryos are frozen 1, 3 or 5 days after the sperm and egg were put together. Freezing is a stressful process for an embryo, and only embryos that are growing well in the laboratory will tolerate the freezing procedure.

How does slow freezing work?

Step 1. Before an embryo can be frozen, all the water that it contains must be removed, otherwise ice crystals will form inside the cells, which is deadly for the cells. To prevent the embryo from shriveling as the water is extracted, we replace the water with “antifreeze,” or a solution of cryoprotective agents such as glycerol, ethylene glycol etc.

Step 2. When most of the water has been removed the embryo, it is inserted into a carefully labeled vial and placed in the cooling chamber of a controlled rate freezer.

Step 3. The embryo is then cooled very slowly at -0.30C per minute, hence the freezing process is termed slow freezing. This allows precise control over the freezing process to maximize water extraction from the embryo and to prevent formation of large ice shards that could pierce the embryo.

Step 4. The cooled vial is placed into carefully labeled metal canes and lowered into the tank with other frozen embryos. The entire process takes several hours and the embryos are stored frozen at – 196 degree C in liquid nitrogen.

Sperm freezing is much easier and more reliable. It needs glycerol as CPA with the addition of sucrose. Egg freezing has for a long time been considered to be more difficult. It is now becoming more reliable and successful with the advent of vitrification. The CPAs most commonly used for blastocyst and egg freezing are EG and DMSO.

Vitrification

Vitrification is a newer technique that incorporates a higher concentration of cryoprotective agents in combination with ultra-rapid cooling or flash freezing. This method requires addition of cryoprotective agents prior to cooling.

Again, the cryoprotective agents act as antifreeze, which lowers the freezing temperature and increase viscosity. The solution turns into amorphous solid or vitrifies (meaning turning it into a glass-like substance) when submerged into liquid nitrogen for flash freezing.

What are some of the advantages of vitrification over slow freezing?

This ultra-rapid process is so fast that it literally allows no time for intracellular ice to form. As a result, vitrification avoids trauma to the embryo. It takes approximately 30 minutes to vitrify 10 embryos.

Vitrified embryos have a better than 95% freeze-thaw survival rate, as compared to 50% survival with slow freezing.

Due to high survival rates with vitrification, oocyte freezing is possible compared to slow freezing which has uncertain survival rate.

Why is it more difficult to freeze oocytes?

An egg is a single free-living cell. They have always been more difficult to freeze than one-cell (zygote) or early 2 to 8-cell embryos indicating the problem was more than the fact of being a single cell.

To survive freezing, a cell needs to be dehydrated. Since water expands as it turns to ice, it could burst the cell. Therefore, the water in the cell is replaced with an “antifreeze” or cryoprotectant.

Since an egg is a single free-living cell, it can be dehydrated quickly. However, when the egg is released from the ovary, it is in a very critical phase of development that is very vulnerable to the freezing process.

Since the egg is preparing for a sperm, the DNA within the egg is in a very delicate phase of reorganization. The egg is in the process of getting rid of half of its DNA, a process that is not completed until after the sperm has entered the egg. Freezing the egg may fatally disrupt the DNA reduction process.

 

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