Cryopreservation of embryos

    Last updated date: 03-Mar-2023

    Originally Written in English

    Cryopreservation of embryos

    Cryopreservation of embryos

    Embryo cryopreservation, also known as embryo freezing, is the freezing and storage of embryos for future use. An embryo is a fertilized egg that has been fertilized by sperm. This procedure is intended to assist patients with fertility and reproduction. A fertilized egg is frozen during embryo cryopreservation. Fertility programs may also provide egg freezing, which is the freezing of unfertilized eggs.


    What Embryo Cryopreservation?

    Embryo cryopreservation entails in vitro fertilization, a method in which eggs are extracted from a woman's ovary and joined with sperm in a laboratory to generate embryos. The embryos are frozen and can subsequently be thawed and implanted in a woman's uterus. Embryo cryopreservation is a kind of fertility preservation. It may be beneficial for cancer patients who wish to have children after undergoing radiation therapy, chemotherapy, or certain forms of surgery, all of which can induce infertility. Also known as embryo banking and embryo freezing.

    The choice to freeze embryos is a personal one. Costs vary greatly, and medical insurance may not cover fertility procedures. You must examine your objectives, the expenses, ethical difficulties, your partner's preferences, and other factors.

    The most successful method for preserving fertility in women is embryo cryopreservation. Because of the relatively high cost and high success rate of sperm cryopreservation options for men, embryo cryopreservation is reserved for use in maintaining fertility in women before or after cancer.


    Why people need Embryo Cryopreservation?


    Embryo freezing is frequently performed after people have undergone fertility treatments. In vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) are examples.

    These treatments fertilize eggs with sperm and can result in the formation of additional embryos. You can freeze additional embryos to use later if you:

    • Cancel or postpone implantation into your uterus after an egg has already been fertilized.
    • Desire to postpone IVF till a later date.
    • Desire a backup plan in case first fertility treatment methods fail.
    • Rather than destroying unused embryos, an individual can choose to give them to other people attempting to conceive or to researchers.


    Delaying Pregnancy:

    It sometimes comes down to timing. Nowadays, it's typical for women to prioritize other elements of their lives (for example, their careers) before having a kid. In this circumstance, women may choose to freeze their eggs in order to use them later.

    Women's egg quality and quantity decline as they age, especially beyond the age of 35. Because frozen embryos include younger eggs, there is a lower chance of pregnancy problems if a woman decides to have a child later in life.


    Upcoming Medical Treatments:

    Other times, embryo cryopreservation allows women who are receiving medical procedures that may compromise fertility to freeze embryos and use them later. Women who have genetic abnormalities that may impair fertility are also more likely to desire to preserve their embryos.

    A woman or person designated female at birth (DFAB) with cancer, for example, may desire to store fertilized eggs before beginning chemotherapy or radiation therapy if the treatment may harm their capacity to get pregnant. A transgender man (transitioning to male) may also preserve eggs or embryos before beginning hormone therapy or undergoing gender affirmation surgery.


    Additional Embryos:

    Multiple embryos are frequently generated when a patient undergoes IVF. Couples may choose to freeze additional embryos rather than kill them. The couple can utilize the frozen embryos to conceive another child later, or they can be stored and donated to someone else through a donor program.

    Many same-sex couples and LGBTQ+ persons who want to have children may choose in vitro fertilization using a sperm or egg donor. Embryo freezing also allows them to save their embryos for later use.


    What happens before Embryo Cryopreservation process?

    Preparing for embryo

    Preparing for embryo cryopreservation:

    You must provide your permission to freeze embryos. Your healthcare professional will provide you consent papers to read and sign. The paperwork should provide details such as:

    • The number of embryos that will be frozen.
    • How long will they be kept (often 10 years)?
    • What happens when the storage time runs out.
    • What happens if you die or become ill before the storage time expires?
    • What uses are permitted for embryos (for example, only your fertility treatments or if they can be donated to research or to another infertile couple)?

    Your healthcare practitioner can also advise you on the ideal embryonic stage to freeze one or more embryos. The following phases are susceptible to freezing:

    • Cleavage stage: After 72 hours, when the solitary cell has proliferated to between four and eight cells.
    • Blastocyst stage: After five to seven days, when the solitary cell has increased to 200 to 300 cells.


    Egg Retrieval:

    A woman typically produces one mature egg every ovulation cycle. To increase the odds of recovering a healthy egg for cryopreservation, the woman must first undergo 8-10 days of hormone injections, which allow several eggs to grow and develop in the ovaries.

    The egg retrieval method is a straightforward one that needs conscious anesthesia. It has only minor side effects, such as slight cramps.



    The next stage in embryo cryopreservation is insemination. An egg must be fertilized by sperm before it can develop into an embryo. The egg is inseminated in a petri dish, and once fertilized, it must develop for 5-7 days.

    More than one egg can be recovered and inseminated. The number of viable embryos to freeze frequently varies on the woman's age (more embryos are viable when the woman is under 35 years of age).


    Genetic testing: 

    This step is not required for all couples, but it is an option for those who wish to screen their embryos for genetic problems.

    When one parent is a carrier of a known genetic disorder, such as Huntington's Disease, preimplantation genetic diagnosis (PGD) can protect you from unintentionally passing the problem on to your future child.


    Embryo freezing procedure

    Embryo freezing procedure

    Cryopreservation is required before an embryo may be preserved. This requires draining the cell of water and replacing it with a cryoprotectant chemical (CPA). This is done to keep ice crystals from developing on the cells.

    Fertility doctors can use two methods for freezing embryos: slow freezing and vitrification.


    Traditional Slow freezing of Embryos:

    The most difficult issue during cryopreservation of embryos and oocytes is to avoid the production of ice crystals and toxic concentrations of solutes, which are the two primary causes of cell death associated with cryopreservation, while maintaining intracellular organelle functionality and embryo viability.

    To do this, the freezing fluid in which the cells are suspended must be treated with cryoprotective additives (CPA). CPA promotes cell dryness and minimizes intracellular ice production.

    CPA is classified into two types: intracellular/membrane-permeating (propylene glycol, DMSO, glycerol, and ethylene glycol) and extracellular/membrane-nonpermeating substances (i.e., sucrose, trehalose, glucose, amid, ficoll, proteins, and lipoproteins).

    The permeable CPA displaces water via an osmotic gradient and partially replaces intracellular water, whereas the extracellular CPA raises extracellular osmolarity, establishing an osmotic gradient across the cell membrane and assisting in cell dehydration prior to cryopreservation. At the same time, it inhibits water from entering the cell quickly after thawing during rehydration/dilution from the penetrating CPA.

    Cell dehydration is primarily determined by the permeability qualities of the cell membrane. The permeability of embryos from various species to water and penetrating CPA varies. Embryos are often less permeable to Glycerol (G) than they are to Propylene glycol (PG) or Ethylene glycol (EG). Furthermore, the embryos are less permeable during early stages of development.

    This variation in membrane permeability may have a significant influence on the result of slow freezing of embryos, but it is regulated by increasing the concentration of the nonpermeable CPA and the ambient temperature. Higher survival rates were found when the concentration of nonpermeating CPA was raised, and the overall pregnancy rates of frozen-thawed embryos appeared to be comparable to those of fresh embryos.

    Prior to slow freezing, the embryos are dehydrated by being exposed to a combination of permeable and nonpermeable CPA (duration: 10 minutes). With few exceptions, low concentrations of PG and sucrose are employed for early cleavage stage embryos and G for blastocyst stage embryos in the case of human embryos. Mouse and cow embryos were frozen to very low temperatures of minus 80°C-120°C using a slow cooling rate (between minus 0.3°C-0.5°C/min) in the initial successful CP treatment.

    As a result, the process took a very long time (several hours). A variation of this approach was documented in some research, in which sheep and bovine embryos were slowly chilled at a rate of 0.3°C/min, but only to minus 30-35°C before being put into liquid nitrogen (LN2). The CP process was significantly shortened as a result of this change (1.0–1.5 hours).

    This short approach has now become the treatment of choice for freezing domestic animal embryos. Despite the great outcomes obtained with animal embryos, human embryos are generally frozen at a low rate of 0.3°C/min to approximately minus 30°C to 40°C, followed by an increasing pace of minus 50°C/min to a temperature of minus 80°C-150°C before being put into LN2.

    Slow cooling is considered to prolong the dehydration process until minus 30°C, after which any leftover water is super cooled. Ice nucleation (seeding) is manually induced between 5 and 8°C (close to the true freezing point of the solution) during the slow cooling phase.


    Vitrification (Ultrarapid Cryopreservation) of Embryos:

    Vitrification (i.e., a glass-like condition) is an alternate method of embryo cryopreservation that has lately been regarded as a revolutionary technology; however, the first successful embryo vitrification was published in the mid-1980s.

    Vitrification differs from slow freezing in that it does not allow ice crystals to form in the intracellular and extracellular regions. Vitrification is the solidification of a solution by a dramatic increase in viscosity at low temperatures without the development of ice crystals, which is accomplished by combining a high concentration of CPA with an extremely high (ultrarapid) cooling rate.

    Unlike slow freezing, where dehydration of the embryos begins during equilibration in the freezing solution prior to slow cooling and continues during slow cooling to minus 30-35°C, during vitrification, cells are dehydrated primarily before the start of the ultrarapid cooling by exposure to high concentrations of CPA, which is required to achieve a vitrified intracellular and extracellular state afterwards.

    The high concentration of CPA, which might be harmful to cells, is a possible concern linked with the vitrification method. However, CPA toxicity can be reduced by combining multiple CPA, lowering the relative concentration of each CPA, and limiting embryo exposure time to the solution to a minimum.

    Permeating (e.g., EG, G, DMSO, PG, acetamide) and nonpermeating (e.g., sucrose, trehalose) agents are included in the freezing solutions usually used for vitrification. In certain methods, macromolecules such as polyethylene glycol, ficoll, or polyvinylpyrrolidone are added to the vitrification solution.

    The macromolecules help vitrification with lower CPA concentrations by increasing viscosity. The amount of the solution in which the embryos are vitrified has lately been reduced significantly (0.1-2 L) in order to further improve the cooling rate (>10.000°C/min) required for effective vitrification. Special carrier systems (open versus closed) have been created to do this.

    For safety considerations, closed systems have been designed. When comparing the open and closed systems, employing closed carriers resulted in good survival rates, but with numerous vesicles throughout the cytoplasm of the embryo, which might be a result of not reducing the temperature quickly enough in the closed system. However, due to improved outcomes, the use of vitrification—particularly for cryopreservation of human blastocysts—has lately expanded significantly.

    Technically, vitrification is quite difficult to conduct since the vitrification solutions are very concentrated, viscous, and tiny in volume, and the embryos must be handled for just a short period of time (1 minute) before to and during vitrification. As a result, in order to attain the best/highest survival rate, the embryologist doing vitrification must be well qualified. This is not the case with slow freezing, in which embryos are slowly frozen (using a specific cell freezer), because slow freezing is a more flexible procedure.


    What happens after Cryopreservation of Embryos?

    Woman Conceive

    When a woman or couple decides they want to conceive, the embryos are thawed. To eliminate the CPAs and restore the cells' normal water content, the embryos are progressively thawed and immersed in specific solutions.

    The procedure is known as frozen embryo transfer or FET. To prepare for embryo implantation, the prospective mother may be given estrogen tablets or injections to strengthen the uterine lining, followed by progesterone therapy to make the uterus receptive to the embryo. In a "natural" FET cycle, a patient is observed until ovulation occurs, and the embryo is placed into the uterus around five days later.

    When the uterus is ready, the doctor inserts a catheter into the vagina, across the cervix (the entrance of the uterus), and into the uterus, and uses a syringe to delicately inject one or more frozen embryos, along with a little quantity of fluid, into the uterus. Following that, the embryologist checks the catheter under a microscope to confirm that the embryo is no longer within the device. A blood pregnancy test about 10 days after the embryo transfer can establish if the operation was effective.


    What are the advantages of embryo cryopreservation?

    storage fertility

    Embryo cryopreservation has reduced the number of fresh embryo transfers while increasing the efficiency of the IVF cycle. Similarly, embryo cryopreservation is an important tool in situations of embryo transfer (ET) cancellation due to ovarian hyperstimulation risk, uterine hemorrhage, increased blood progesterone levels on the day of triggering, or any other unforeseen events.

    If you are having difficulty becoming pregnant later in life, embryo freezing can assist in these cases:

    • Advancing age.
    • Gender transition.
    • Infertility issues.
    • Social/personal factors, such as seeking a higher level of education or having career commitments that need you to postpone childbearing for several years.
    • Treatment that may damage fertility (for example, chemotherapy or pelvic radiation therapy for cancer).
    • Upcoming military deployment.
    • Women who are single may be concerned about their age and want to freeze eggs or embryos created with donor sperm.


    What is the success rate of Embryo Cryopreservation?

    Illustration Egg Freezing

    Approximately 95% of frozen embryos survive the treatment. Following the freezing process, the embryos are maintained in liquid nitrogen and can be kept frozen for many years.

    One advantage of embryo cryopreservation is that it allows fertility specialists to transfer embryos in the future without having to extract eggs again.

    Because embryo cryopreservation has a high success rate, it is a popular treatment. According to research, newborns born following cryopreservation had no more developmental defects than babies born from fresh embryos; however, longer-term studies are needed to corroborate these findings.

    According to some studies, transferring frozen embryos rather than fresh embryos may boost the odds of a healthy pregnancy and result in better results for both the mother and the infant.

    Frozen embryo transfer is the process of thawing and implanting an embryo into a woman's uterus. The procedure is frequently successful. However, rates vary greatly depending on a variety of circumstances, including:

    • Both parents’ overall health.
    • Mother’s age at the time of the egg retrieval.
    • Presence of fertility issues, such as endometriosis, fibroids, and uterine polyps.
    • Previous success or failure of fertility treatments and pregnancies.

    Your healthcare practitioner will assist you in comprehending the aspects that may influence your chances of success.


    What are the risks or complications of this procedure?


    Women who have their embryos cryopreserved may encounter minor negative effects. Typically, any difficulties or adverse effects of embryo freezing occur during the extraction of the eggs by the doctor.

    • Some of the common side effects include:
    • Cramping or bloating
    • Feeling full
    • Light bleeding
    • Changes in vaginal discharge
    • Infection
    • Overstimulation of the ovaries

    While embryo freezing is a very safe and simple technique, one of the biggest dangers linked with embryo extraction for preservation is the possibility of multiple pregnancies. Multiple pregnancies may increase the chance of difficulties for both the mother and the fetus, which is why many fertility clinics advise women under the age of 38 to transfer just one embryo at a time.

    Embryo freezing does not increase the likelihood of congenital defects or health issues in subsequent pregnancies. In reality, frozen-thawed embryo outcomes study showed decreased rates of preterm delivery, low birth weight, growth limitation, and perinatal death.

    The main risks associated with embryo cryopreservation are:

    • Embryos are damaged during the freezing procedure.
    • Embryos that are unfit for freezing.
    • Inability to conceive after embryos have been thawed and implanted.
    • An increase in the prevalence of pregnancy-related medical disorders such as preeclampsia and placenta accrete spectrum.
    • Multiple births are possible if more than one embryo is implanted (twins or triplets).



    IVF cycle

    Embryo cryopreservation is the process of preserving embryos at subzero temperatures in order to keep them viable for future pregnancies.

    During an in vitro fertilization (IVF) cycle, 10 or more eggs are extracted from a woman's ovaries and mixed with sperm from a male. Up to four eggs can be implanted into a woman's uterus. The surviving embryos are then cryopreserved (frozen) in liquid nitrogen canisters at temperatures of 320 degrees Fahrenheit below zero.

    Embryo cryopreservation necessitates the presence of an available and consenting spouse or the use of donor sperm. The woman's oocytes are extracted, followed by in vitro fertilization with her partner's or donor sperm. The embryos are subsequently treated by cryoprotectants and frozen at subzero temperatures until they are needed. When the choice is taken to utilize the embryo, it is thawed and arrangements are made for implantation. Survival rates per thawed embryo are adequate, however they vary greatly depending on the circumstances and treatments used.

    Cryopreservation of embryos is a type of fertility preservation. It might help cancer patients who want to have children after having radiation therapy, chemotherapy, or certain types of surgery, all of which can cause infertility.

    Advantages of embryo cryopreservation include its relatively high success rate in the general population and the existence of well-tested protocols. Disadvantages include the need for a partner or donor gametes, as well as the potential exposure risk of ovarian stimulants needed for oocyte retrieval.