Cryopreservation- medical considerations and techniques

Cryopreservation: An innovation in reproductive medicine

Rapid advancement in the field of assisted reproductive technologies (ART) has boosted a parallel increase in the use of cryopreservation techniques for infertile individuals.¹   Cryopreservation, in reproductive medicine, is defined as the preservation of structurally intact sperms, oocytes or embryos at ultra-low temperatures.^2,3   Although embryo cryopreservation is a widely accepted practice, oocyte cryopreservation is also emerging as an attractive option for fertility preservation.⁴   The technique is considered an innovation in the arena of ART, particularly as an adjunct to in-vitro fertilization (IVF) owing to its multiple advantages. The possibility to cryopreserve oocytes to be used in IVF treatment has not only expanded the reproductive options for patients undergoing gonadotoxic treatments for cancer, but also holds the promise of preserving the reproductive potential of healthy women whose personal circumstances (most often the absence of a partner) disallow them to conceive in their most fertile years. Further, cryopreservation permits women to voluntarily postpone pregnancy until a later age (e.g., after establishing career, etc.) as their fertility remains preserved.^1,5

Oocyte cryopreservation: An overview

Preservation of female genetics can be done through the cryopreservation of oocytes which involves their stimulation, retrieval and storage. However, prior to these steps, the candidate (patient or egg donor) is thoroughly screened for infectious diseases tests like HIV I-II, Hepatitis C, Hepatitis B surface antigen and VDRL. Once the possibility of infection is ruled out, the ovaries are stimulated to achieve multiple follicle growth and egg production. This is followed by the process of follicular development which entails oocyte maturation via human chorionic gonadotropin (hCG) administration. The mature oocytes are finally retrieved under ultrasound guidance and cryo-stored.⁶

Several attempts have been made in the past to develop feasible and efficient methods for effective cryostorage of oocytes. At present, two basic techniques rule the field of oocyte cryopreservation—slow freezing and vitrification.⁷

Cryopreservation techniques: Slow freezing versus vitrification

The renaissance of oocyte cryopreservation began in the middle of the 20^th century when reasonable rates of oocyte survival were achieved with slow or controlled-rate freezing, the foremost technique to be developed for preserving oocytes.^7,8   The procedure involves exposing the oocytes to low concentrations of permeating cryoprotectants such as glycerol or ethylene glycol, followed by loading into appropriate containers (freezing vials or straws). The oocytes are then cooled to a subzero temperature of about -7°C after which seeding is initiated to induce extracellular freezing. Seeding of the extracellular matrix together with a very slow cooling rate give rise continuous oocyte dehydration until a temperature at which the intracellular matrix vitrifies is reached. The vials or straws are then cooled slowly to a temperature of about -30°C to -40°C and plunged into liquid nitrogen for long-term storage.⁷

While slow-freezing has been extensively used for oocyte cryopreservation, success rates obtained have been highly variable across clinical settings, casting doubts on the reliability of the method. Studies have demonstrated that human oocytes are sensitive to ice damage during freeze-thaw, due to their large surface area/volume ratio and plasma membrane permeability properties.⁸   Both experimental and clinical research have revealed much to suggest a high probability of damage to meiotic spindle integrity and chromosome alignment leading to low oocyte survival rates following slow-freezing.⁹

Vitrification, on the contrary, has emerged as a more efficient technique and is gradually replacing slow freezing in clinical settings. In this method, both intra- and extracellular matrices apparently vitrify after cellular dehydration has already occurred. Compared to slow-freezing, vitrification involves the use of greater concentrations of cryoprotectants. Thus, ice cannot form in any part of the sample. As no ice forms, cooling does not have to be slow. Rapid cooling has been shown to enhance oocyte survival rates by up to 37%. Vitrification is additionally associated with a lesser probability of causing damage to meiotic spindle and chromosomal integrity. Furthermore, unlike slow-freezing method, which demands extensive equipments to regulate the cooling rate, vitrification is relatively inexpensive and does not require special equipment, making it a more desirable option.⁷

The above findings were corroborated by a very recent study conducted to compare the clinical success rates with slow freezing and vitrification of mature human oocytes in infertile patients. The results unveiled significantly higher oocyte survival, fertilization, implantation and pregnancy rates with vitrification compared to slow-freezing.¹⁰

Medical considerations

Although, the history of successful oocyte cryopreservation resulting in live birth stretches back to more than 3 decades ago, robust research on enhancing the clinical outcomes has occurred only in the past decade.⁴   The American Society of Reproductive Medicine Practice Guideline Committee¹¹   has asserted that oocyte cryopreservation must no longer be considered experimental for medical indications, outlying elective oocyte cryopreservation. In addition, novel evidences have shown the implantation and pregnancy rates obtained from cryopreserved oocytes to be comparable to those accomplished after a fresh egg donation.⁴   This has widened the scope of application of oocyte cryopreservation and paved the way for better regulation of the factors affecting clinical outcomes.

Factors affecting cryopreservation: Focus on the role of cryoprotectants

The success of human oocyte cryopreservation is influenced by an array of morphological and biophysical factors. Morphological factors such as the quality, size and maturity of the oocyte, the presence of the cumulus oophorus (cumulus cells play a putative role in sheltering the oocytes against unfavorable effects of cooling) apparently affect oocyte survival after thawing. The principle biophysical factor is the intracellular ice formation that may be responsible for cell lysis during cryopreservation. Researchers suggest that intracellular freezing can be prevented by sufficient cell dehydration mediated by cryoprotectants. The protective action of cryoprotectants is intriguing and primarily attributable to their unique property of salt-buffering, that safeguards the oocytes from the deleterious effects of high salt levels during cryopreservation. The exposure time of cryoprotectants is also critical to ensure oocyte survival. It must be sufficiently long to allow adequate dehydration, but must not exceed the optimum duration as that can lead to cryoinjury. Evidence-based research suggests an exposure time of 10 minutes to be optimum for oocyte survival.

When all the above-mentioned medical considerations are taken into account, high clinical success rates can be obtained with oocyte cryopreservation.^12,13      Pooled data from published trials shows oocyte survival rates after thawing in the range of 70-95% and fertilization rates between 70-90%.¹⁴

Concluding remarks

Oocyte cryopreservation, an attractive strategy to preserve female reproductive potential, has enjoyed a renaissance in the past decade. Slow-freezing and vitrification techniques rule the field of oocyte cryopreservation. Published data provides compelling evidence in favor of the superiority of vitrification in obtaining higher clinical success rates than slow-freezing. However, a variety of biophysical and morphological factors, regardless of the technique, dictate the feasibility and success outcomes of cryopreservation.

References

1. Konc J, Kanyo K, Kriston R, Somoskoi B, Cseh S. Cryopreservation of embryos and oocytes in human assisted reproduction. Biomed Res Int. 2014;2014:307268.
2. Pegg DE. Principles of cryopreservation. Methods Mol Biol. 2007;368:39-57.
3. Bredkjaer HE, Grudzinskas JG. Cryobiology in human assisted reproductive technology. Would Hippocrates approve? Early Pregnancy. 2001 Jul;5(3):211-3.
4. Cil AP, Seli E. Current trends and progress in clinical applications of oocyte cryopreservation. Curr Opin Obstet Gynecol. 2013 Jun;25(3):247-54.
5. Mertes H. The portrayal of healthy women requesting oocyte cryo-preservation. Facts Views Vis Obgyn. 2013;5(2):141-6.
6. ART: Step-by-Step Guide. Available from http://www.sart.org/detail.aspx?id=1903. Accessed on 2.12.2014.
7. Saragusty J, Arav A. Current progress in oocyte and embryo cryopreservation by slow freezing and vitrification. Reproduction. 2011 Jan;141(1):1-19.
8. Boldt J. Current results with slow freezing and vitrification of the human oocyte. Biomed Online. 2011 Sep;23(3):314-22.
9. Huang JY, Chen HY, Tan SL, Chian RC. meiotic spindles and chromosome abnormalities.Effect of choline-supplemented sodium-depleted slow freezing versus vitrification on mouse oocyte. Fertil Steril. 2007 Oct;88(4 Suppl):1093-100. Epub 2007 Jun 4.
10. Levi Setti PE, Porcu E, Patrizio P, Vigiliano V, de Luca R, d’Aloja P, Spoletini R, Scaravelli G. Human oocyte cryopreservation with slow freezing versus vitrification. Results from the National Italian Registry data, 2007-2011. Fertil Steril. 2014 Jul;102(1):90-95.e2.
11. Practice Committee of American Society for Reproductive Medicine; Practice Committee of Society for Assisted Reproductive Technology. Mature Oocyte Cryopreservation: a guideline. Fertil Steril. 2013;99:37-43.
12. Fabbri R. Cryopreservation of human oocytes and ovarian tissue. Cell Tissue Bank. 2006;7(2):113-22.
13. Fabbri R, Porcu E, Marsella T, Rocchetta G, Venturoli S, Flamigni C. Human oocyte cryopreservation: new perspectives regarding oocyte survival. Hum Reprod. 2001 Mar;16(3):411-6.
14. Manipalviratn S, Decherney A. Clinical application of human oocyte cryopreservation. Rev Recent Clin Trials. 2008 May;3(2):104-10.