Cryopreservation of Oocytes (includes storage)

Overview

Oocyte cryopreservation is a growing technology used for assisted reproduction that may help a variety of patient groups. As a result, increasing efficiency is critical to realize the enormous promise of this strategy. However, in addition to various research examining factors related with protocol technique, such as type of cryoprotectant (CPA), kind of storage device, or cooling/warming rates, there are more creative ways for improvement. These alternative techniques include using different stages of oocytes, studying changes in basal media and buffer composition, automating CPA exchange processes and device loading, and examining oocyte cellular composition to increase cryotolerance. Finally, developing more accurate or perceptive markers of "success" is critical for ongoing progress.

What is Cryopreservation of Oocytes? 

Oocyte cryopreservation is the procedure of extracting, freezing, and storing a woman's eggs (oocytes) in order to maintain reproductive potential in women of reproductive age. In 1986, the first human birth from a frozen oocyte was recorded. Over the last few years, oocyte cryopreservation has evolved significantly, with increased overall success of eggs surviving the freezing process. The American Society for Reproductive Medicine no longer considers it an experimental technique. The treatments that lead to increased gamete survival, potential fertilization, and live birth rates provide women far more control than was feasible even five years ago. 

Who Needs Oocyte Freezing?

Cryopreservation of oocytes can be considered for a number of reasons, including:

• Women with cancer who require chemotherapy and/or pelvic radiation therapy, which may have an impact on fertility.
• Surgery that may result in ovarian damage.
• Premature ovarian failure is a risk due to chromosomal abnormalities (e.g., Turner syndrome, fragile X syndrome) or a family history of early menopause.
• Ovarian illness with a risk of ovarian damage.
• Genetic mutations necessitate the removal of the ovaries (e.g., BRCA mutation).
• Fertility preservation is used to postpone birth for societal or personal reasons.

How is Oocyte Freezing Done?

Oocyte Freezing Process:

Prior to the ovarian stimulation cycle, your UCLA fertility expert may do an assessment of the ovarian reserve to predict the possible production of oocytes. Blood tests and a pelvic ultrasound would be performed as part of the evaluation. This will also aid in determining the appropriate medicine dose. Ovarian stimulation is performed in the same way that in vitro fertilization (IVF) is, with injectable hormone drugs. Following stimulation, the oocytes and surrounding fluid in the ovarian follicles are aspirated vaginally while the patient is sedated.

Under the microscope, the oocytes' maturity is determined, and those that are mature are cryopreserved. Currently, vitrification is the preferred approach for cryopreserving oocytes, which is accomplished by rapidly chilling them into liquid nitrogen, where they may be preserved.

How will Oocytes be Used in the Future?

When the woman is ready to use the frozen oocytes to achieve pregnancy, they are put in a warming solution and evaluated. Those oocytes that survived freezing are fertilized with intracytoplasmic sperm injection (ICSI), in which a single sperm is injected directly into the oocyte, and the fertilized oocytes will grow in culture until the embryo(s) are ready to be transferred into the uterus to achieve pregnancy, usually 3-5 days after fertilization.

What are the Chances of a Successful Pregnancy Using Frozen-Thawed Oocytes?

Oocyte Freezing Success Rates:

Clinical pregnancy rates have been estimated to be between 4 and 12% per oocyte. However, because egg freezing is still in its early stages, additional data will be required to have a better understanding of these success rates. In general, the two most critical elements in determining the likelihood of a live delivery are the woman's age at the time of egg freezing and the number of accessible eggs.

How Long Can the Oocytes be Stored?

Keeping the eggs for prolonged periods of time does not appear to have any detrimental consequences. However, data can only be stored for up to four years. It should be noted that greater mother age during pregnancy is connected with an increased risk of pregnancy issues such as high blood pressure, diabetes, and cesarean delivery. Most clinics have a maximum age limit for using these gametes to produce conception.

Effectiveness of Oocyte Cryopreservation?

Cryopreservation of oocytes has proven more difficult than sperm cryopreservation, in part because several regimens induce damage to an egg's zona pellucida (i.e., shell). However, there are currently a variety of cryopreservation techniques available for preserving mature or immature oocytes. While immature oocyte survival rates have reached 60%, the freezing method frequently changes the oocyte development process, and fertility and live birth rates for surviving oocytes are typically lower than those attained with fresh oocytes.

 Recent research that gathered the results of all oocytes cryopreserved utilizing a slow freezing methodology between 1997 and 2005 found an overall post-thaw live birth rate of 1.9%, which is much lower than the rate reached with fresh oocytes. Recent advances in the utilization of vitrification procedures (i.e., quick freezing techniques) have increased post-thaw survival rates, with a number of recent studies reporting >90% oocyte survival after vitrification, with implantation happening in up to 11% of thawed oocytes.

Oocytes Cryopreservation Procedure

Freezing protocols

The length of time required to freeze tissues varies based on the cryopreserving employed and the cells being frozen. Typical freeze methods can be either gradual or fast, and commonly entail wrapping tissues in straws for protection. Slow freezing includes gradually lowering the temperature of reproductive tissues and cryoprotectants. Tissues, for example, can be placed in cryoprotectants at ambient temperature and chilled at a rate of -2oC/min until they reach -7oC. The rate of temperature decrease is then lowered to -0.3oC/min until -30oC is reached, then accelerated to -35oC/min to reach -135oC. Finally, the tissues are immersed in liquid nitrogen and stored at -196oC. 17

Rapid freeze treatments, also known as vitrification methods, on the other hand, immerse the reproductive tissues and cryopreserves in liquid nitrogen at -196oC. The concentration of cryopreserving is raised to avoid the development of ice crystals in the cells.

Storing human tissues

Human tissues are frozen and kept in liquid nitrogen at or below -196oC.

Thawing protocols

A common thawing approach involves immersing the straws holding cryopreserved tissues in water baths and gradually raising the temperature until they reach room temperature. To lessen the harmful impact on the reproductive tissues, the concentration of the cryopreservation media in which they are stored is gradually reduced at the same time.

Media for cryopreservation

Tissues can be cryopreserved using a variety of methods.

1. Glycerol

In the 1940s, glycerol was the first cryopreservant used to freeze human gametes, resulting in the cryosurvival of human spermatozoa. Glycerol is still a popular cryopreservant for human sperm, and some births have resulted from thawed spermatozoa cryopreserved in glycerol for lengthy periods of time (e.g., >20 years).

    2. Dimethyl sulphoxide (DMSO)

In the early 1980s, the DMSO procedure was created. For many years, it was utilized effectively as a cryopreservant. This procedure was employed for the first live births from cryopreserved embryos, as well as the first live birth from a cryopreserved oocyte. DMSO is no longer used because of concerns that it may raise the chance of genetic abnormalities, as well as because the protocol's survival and implantation rates were poor.

    3. Propenediol (PROH) in different concentrations with or without sugar

In the 1980s, propenediol was initially utilized as a cryoprotectant. It effectively replaced DMSO and is still the most widely used medium today. Sucrose-containing treatments are more efficient than those without, according to research, albeit success rates vary depending on the sucrose content. Oocyte survival, for example, significantly improved when 0.2M or 0.3M sucrose concentrations were utilized vs 0.1M sucrose (71% vs 25-50%). However, there are concerns that greater sucrose concentrations (i.e., 0.3M) may impair embryo development.

    4. Sodium-deficient medium – Choline

Recently, scientists aiming to improve oocyte cryopreservation success rates have utilized propenediol/sucrose methods in which sodium is substituted with choline. This is because new information suggests that salt has a detrimental influence on the cryopreservation process. To date, no live births have been documented as a result of the use of such cryopreservation, and there is insufficient data to compare the results of this medium with other cryopreservation regimes.

Benefits associated with Oocytes Cryopreservation

Cryopreservation techniques revolutionized IVF therapy by allowing gametes or embryos generated but not used in one IVF treatment cycle to be kept and used in subsequent treatments. There are numerous associated benefits.

• The effectiveness of assisted reproduction

Cryopreservation improves the efficacy of assisted reproductive therapies by allowing all cells retrieved and/or fertilized during one treatment cycle but not utilized to be kept for use in a subsequent treatment cycle. This substantially eliminates the need for practitioners to undertake ovarian stimulation and oocyte retrieval, which are both costly and physically demanding for the women involved. Prior to cryopreservation, oocyte retrieval was required for each treatment cycle, and oocytes that were not fertilized or utilized were discarded

• Ovarian hyperstimulation syndrome risk is reduced.

Ovarian hyperstimulation syndrome (OHSS) is a dangerous illness that, if left untreated, can lead to death. It is the most prevalent complication of artificial insemination. OHSS occurs as a result of ovarian stimulation with pharmaceuticals, and its occurrence can potentially be minimized by limiting the frequency of ovarian stimulation or by giving women a period of rest between ovarian stimulation and embryo implantation. Because embryos are cryopreserved from one cycle to the next, ovarian stimulation is not required each time a treatment cycle begins, and if the woman's ovaries are hyperstimulated, embryo implantation can be postponed without squandering harvested oocytes. However, a recent study concluded that there was insufficient data to justify the widespread use of cryopreservation as a method for avoiding ovarian hyperstimulation syndrome.

• Embryo preservation for scientific purposes

Cryopreservation allows embryos to be maintained for research reasons or for couples who conceive during their first treatment cycle and have unused, frozen embryos to contribute to research.

• Reduces multiple embryo implantation

Multiple embryos implanted each cycle provide health hazards to both the pregnant woman and her fetus/es, including the potential of multiple pregnancy and related problems such as miscarriage and low birth weight. Prior to the introduction of cryopreserved embryo procedures, there was a push to implant all available embryos since any unused embryos could not be saved. However, it is currently common practice to implant only one or two embryos and cryopreserve any leftover embryos for future treatment cycles.

• Preserving fertility

Cryopreservation allows people whose fertility is endangered to preserve their reproductive cells so that they can conceive with assisted reproductive treatments in the future. Fertility preservation is increasingly being given to prepubescent and postpubescent patients enduring radiation and/or chemotherapy. Women who want to postpone childbirth and/or have a family history of early menopause may also use it.

The limitations and hazards of Oocytes Cryopreservation

The current research found certain gaps in the existing evidence on oocyte cryopreservation. Only a few women have followed up to use their frozen oocytes for future conception. As a result, we have less information about the long-term pregnancy outcomes of frozen oocytes. Furthermore, oocyte freezing technology has improved over time, making it impossible to generalize the findings of oocytes preserved by slow freezing many years ago and anticipate the outcomes of freshly vitrified oocytes. There are very few high-quality prospective studies on newborn outcomes after utilizing frozen oocytes. Long-term prospective randomized research to investigate the fertility rate, live birth rate, and long-term effect on children born by oocyte cryopreservation is desperately needed.

While cryopreservation has become a standard part of assisted reproductive techniques, and cryopreservation techniques have greatly improved in recent decades, fertility, implantation, and/or live birth rates from frozen and thawed reproductive cells remain lower than those obtained using fresh products. This is especially true in the case of oocyte cryopreservation. Human tissue storage can also be expensive and is not covered by Medicare. The hazards are comparable to those associated with ovarian stimulation for IVF, including minor risks of ovarian hyperstimulation syndrome (enlargement of the ovaries and fluid accumulation in the pelvis and belly), infection, and bleeding during the egg retrieval operation.

Issues of ethics and legislation with Oocytes Cryopreservation

Cryopreservation raises a number of ethical concerns. As a result, the freezing and storage of human tissues is strictly regulated all over the world. Patients must be given trustworthy information on the results of assisted reproductive therapies after cryopreservation (e.g., live birth rates, outcomes for kids conceived using the procedures) so that they may make educated decisions about the storage of their tissues.

• Frozen oocytes ownership

Human tissue preservation poses questions concerning ownership, particularly with relation to embryos. The couple who saved the embryos retains ownership of the embryos, and embryos can only be utilized or disposed of with the approval of both parties, until the time limit for storage is reached, in which case they must be disposed of.

• Labeling

It is critical that all preserved reproductive tissues be thoroughly labeled, especially to guarantee that the owners of stored tissues can be identified. Clinics must also verify that their labeling are not easily changed

• Storage of a deceased person's tissues

The provider retains ownership of frozen gonadal tissues, and clinics are obligated to dispose of cryopreserved tissues after a physician's death. Only if the tissue source has clearly stated a desire for the tissues to be utilized in the case of death (for example, for research purposes) may tissues be stored or retrieved for use.

• Time spent in storage

Human tissues can be kept for a maximum of five years.

• Consent

Each person preserving gonadal tissues must get a separate consent form.

Conclusion

Oocyte cryopreservation has matured into a well-established technology during the last three decades. Despite rising fertility awareness, the services are still underutilized. The need of the hour is improved interdisciplinary collaboration between oncologists and reproductive experts, as well as wider availability of oocyte freezing services. Over the last decade, vitrification has been the leading technique of oocyte cryopreservation across the world. Although a closed vitrification system provides an aseptic environment, open vitrification is the most often used method. Planned OC appears to be appropriate for people in their early to mid-thirties. It may be advised at a younger age in selected individuals with decreased ovarian reserve who are at risk of developing primary ovarian insufficiency (POI). Patients should be encouraged to save 15-20 mature oocytes in order to have a successful live delivery.  Several investigations have revealed that frozen-thawed oocytes have acceptable fertilization and pregnancy rates.