Cryopreservation is the frozen state of biological samples for preserving the viability over an extended period of time. Cryopreservation technology comes under cryobiology, means biology that studies the effects of low temperatures. The path-breaking advancement in the fertility sector was achieved through the cryopreservation of gonadal tissue (ovarian and testicular tissues), gametes (sperm and oocytes) and embryos. This accomplishment by the clinical researchers through cryopreservation technology helped to build the road of success in maintaining the animal kingdom.
Cryopreservation techniques are applicable to both male and female to preserve their fertility and are a useful tool for ART (assisted reproduction techniques).
- Fundamentals of Cryopreservation technology.
- Application of Cryopreservation Technology in animal fertility sector.
- Application of Cryopreservation Technology in human fertility sector.
- Disease conditions require Cryopreservation Technology for fertility preservation.
- Preliminary Classification of cryopreservation techniques.
- Slow-cooling/ Slow-freezing process.
- Application of cryopreservation techniques in fertility preservation.
- Autografting of gonadal tissues.
- Xenografting of gonadal tissues.
- In‐vitro culture of Gonadal tissues.
- Germplasm cryopreservation.
- Advancement of cryopreservation techniques.
- Testis tissue transplantation.
- Ovarian tissue transplantation.
Fundamentals of Cryopreservation technology
The cryopreservation of oocytes or spermatozoa is termed as cryopreservation of reproductive cells and it involves three stages – freezing, storage, and thawing. At the initial stage, isolated reproductive cells expose to cryoprotectants and then cool down to subzero temperature. After freezing, the product is stored in optimum condition to maintain the viability. Thawing, dilution, and removal of the cryoprotectants are followed prior to the implantation. Intracellular ice formation can alter the osmotic pressure and that can damage cell membrane and destroy the viability of the frozen cells. Therefore, proper osmotic pressure management during freezing and thawing procedure is crucial for successful cryopreservation technique. The novel advancement in Cryopreservation technique limits the scope of frozen cellular damage as it maintains the cellular physical characteristics, which is essential for viability.
Cryopreservation technique maintains the viability of the cryopreserved cells and tissues without altering functionality or genetic sequence for centuries, which highly increase the significance of this technique in the fertility sector.
Application of Cryopreservation Technology in animal fertility sector
Cryopreservation Technology is applied to improve the number of endangered animals, rare and/ or valuable animals. It has been observed that the existence of many animals become extinct and some become endangered due to a large number of juvenile mortality, or inbreeding depression. Poor animal management and endemic attack of fatal diseases are often responsible for such outcomes. The initiative to save animals made the researchers keen to work on animal fertility sector. One of the major objectives is germplasm conservation in animals and gonadal cryopreservation in conjunction with transplantation is a feasible option for it.
Application of Cryopreservation Technology in human fertility sector
Cancer affected pre-pubertal boys can cryopreserve testicular tissues before starting gonadotoxic treatments or castration surgery. Similarly, cryopreservation of ovarian tissue is the option for pre-pubertal girls to preserve fertility before cancer therapy or other reproductive ill health causing ailments. Ovarian tissue preservation is the only option to preserve the fertility of women who have a higher risk of diminished gonadal function due to premature menopause or administration of chemotherapy. Cryopreservation of testicular tissue and ovarian cortex are promising techniques applicable to preserve fertility in cancer patients who require chemotherapy in urgent basis.
Researchers showed their keen interest to explore cryopreservation technology and its role in fertility sector. Advanced fertility treatment procedures such as ICSI (intracytoplasmic sperm injection), gametes isolation and preservation, in-vitro and in-vivo fertilization, embryo culture and maturation are all based on sophisticated cryopreservation technology.
Disease conditions require Cryopreservation Technology for fertility preservation
Cryopreservation is not only useful for cancer patients who require radiation therapy or chemotherapy, but also for other disease conditions like pelvic diseases, and inflammatory disorders. The abnormal chromosomal conditions like Mosaic Turners syndrome also require cryopreservation technology for fertility preservation.
Preliminary Classification of cryopreservation techniques
The advancement of cryobiology helps to develop more efficient cryopreservation techniques. At the preliminary phase, the only slow-cooling process was available, but advancement in innovation and evolution in this field has invented the vitrification technique. Vitrification technique is simpler and efficient than slow-cooling process.
Conventional testicular tissue cryopreservation is conducted under a slow-cooling process. In this process, the cooling rate is maintained at the optimum level to avoid intracellular ice crystal formation. Uniform cooling of the specimen by maintaining optimize conductivity is a challenge in the slow cooling process. Controlled slow freezing and uncontrolled slow freezing are two types of slow freezing process. Controlled slow freezing and vitrification technique can only be used for ovarian tissue cryopreservation but, the uncontrolled slow freezing protocol can cryopreserve human testicular tissues. Comparison of controlled slow freezing and vitrification technique provides a conflicting result. However, there are sufficient study findings to prove that controlled slow freezing provides promising result by developing pregnancies and live births.
In this process, the liquid is solidified due to the quick enhancement of viscosity with the rapid cooling process to avoid crystallization. Therefore, intracellular and extracellular tissues get protection from the ice formation. Vitrification process is a cost-effective technique. High osmolality solution is used with quick immersion in liquid nitrogen and resultant of this leads to shortening the freezing time. At the initial developmental phase, ovarian tissues can be preserved by using vitrification. But the chance of necrosis of the ovarian tissue is high. Later improvement of vitrification technique become a consistent approach for the oocyte cryopreservation. Novel advancement of the technique launched needle immersion vitrification, which gives better survival of human follicle tissues and ovarian tissues.
Application of cryopreservation techniques in fertility preservation
Autografting of gonadal tissues
Female cancer survivors with premature ovarian failure can get potential benefit from autografting of cryopreserved ovarian tissue. This process helps to restore the reproductive hormonal functionality. Orthtopical and heterotopical are two autografting cryopreservation techniques applicable to transplant ovarian tissue. The acceptance of ovarian tissue cryopreservation and autografting procedures are increasing worldwide. The two principal goals of ovarian tissue functioning restoration are an improvement of the quality of life and reinstallation of reproductive function. There are many success stories that provide evidence of restoring human fertility by opting ovarian tissue autografting process. Alternatively, testicular tissue transplantation can also be used in case of animals, however, there is no clinical study report obtained on autografting of testicular tissues in humans.
Xenografting of gonadal tissues
Xenografting has a dual purpose for cancer affected patients. It allows follicle maturation and also acts as an assisted tool for detection of cancer transmission and relapsing. Antral follicle developed after inducing xenografting of frozen ovarian tissue. A human research study has also found that xenografting of cryopreserved immature testicular tissue can proceed spermatogenesis and proliferation of spermatogonia, which may help in sustaining the male fertility.
In‐vitro culture of Gonadal tissues
An animal study has reported that the in-vitro culture of cryopreserved ovarian cortex increases the follicular survival and growth. Autografting of testicular tissues has the possibility to reintroduce malignancy. In-vitro culture spermatogenesis in testicular tissues has been developed to avoid this adverse effect. An animal research study showed that in-vitro spermatogenesis improves functional sperm number in the test animal testis.
Utilization of a cryopreservation technique is a promising field in the fertility sector. Variety of germplasm cryopreservation increases the range of fertility treatment. Currently, cryopreservation of sperm is well-established technique, frequently applied to protect male fertility in case of threatened damage of male reproduction possibilities. Sperm cryopreservation technique, sperm-cryo-banking, and semen cryopreservation are the emerging field in cryobiology. They support the male fertility for cancer survivors and other biotechnological applications. Both, semen preservation and DNA cryopreservation techniques help to maintain biodiversity by protecting endangered animal species, in addition to saving human fertility.
Oocytes and embryo cryopreservation are two approved female germplasm preservation techniques that can preserve female genetics. But at the initial developmental phase of female gametes, the cryopreservation techniques were much difficult than male gametes. Same male gametes cryopreservation techniques cannot give successful result due to composition, size, and associated structural differences.
The major concern for female gametes cryopreservation is intracellular ice formation. Even, slow cooling rates can damage the intracellular structures of female gametes due to intracellular ice formation. The clinical researchers had given tremendous efforts to solve these problems by modifying the cooling rate. Rapid cooling during the evolution phase gives significant improvements. In some cases, plasma membrane stabilizers are used during cryopreservation to overcome the cooling effect of oocytes. Linoleic acid–albumin (proteins), Butylated Hydroxytoluene (antioxidant) or trehalose or sucrose (sugar) are usually added as plasma membrane stabilizers. Chromosomal segregation is a common problem associated with freshly collected oocytes. But, the addition of choline supplementation and a higher concentration of sucrose in oocytes cryopreservation technique protect from chromosomal segregation. However, it is necessary to mention that improper oocytes cryopreservation can increase the risk of chromosome abnormalities.
Oocyte cryopreservation can be possible within 2 to 3 weeks, and therefore it is a very wide application in the fertility sector to uphold female fertility in medical emergency cases, such as cancer affected young female patients.
Cryobanking of embryos is not only applied in human but this can also be used as an assisted tool to increase the number of an endangered animal. Prolog preservation of both oocytes and spermatozoa comes under cryopreservation of gametes and play a significant role in assisted reproduction techniques. Cryopreservation of gametes has broken the geographical distance barrier between donors and receivers. The in-vitro fertilization or artificial insemination through cryopreservation can be possible for the individuals who have located far away with a good chance of getting potential availability of viable gametes.
Germplasm cryopreservation offers single parenting options for both male and female. The genetic material of dead or alive endangered male and female population can be retrieved and preserved through spermatozoa, oocytes, and embryo cryopreservation.
Advancement of cryopreservation techniques
Continuous improvement in technology also allows enduring preservation of gametes and embryos. Modernization of research strategies now initiating the development of novel technologies for most favorable isolation process and conserving technology of male and female gametes at the earliest phase and their consequential maturation. Gonadal tissue transplantation supports early-phase haploid gamete maturation and development.
Testis tissue transplantation
Testis tissue transplantation can be possible due to the development of cryopreservation techniques. Testis tissue transplantation provides the scope to preserve fertility for young males and children, who have undergone cancer treatment or suffering from a disease that can cause sterility. Gonadal tissues are retrieved before commencing cancer treatment and preserve the fertility by using cryopreservation techniques. The cryopreserved gonadal tissues are stored in cryo-banking for future use.
Testicular tissue cryopreservation is the only technique available to preserve pre-pubertal male fertility. Testicular tissue cryopreservation also allows preserving different testicular cells for acquiring spermatogonial stem cells and permits the cell to cell contacts between somatic and germ cells. Cryopreserved immature testicular tissue can also be used for fertility treatment in different ART.
The tissue texture of immature testis is different from the adult testis. The proper cryopreservation can only allow further development of the immature testis. Therefore both, the accurate cryopreservation protocol and the adherence of the protocol are very crucial for the successful development of the cryopreserved immature testis. Adult cryopreserved testicular tissue or cryopreserved testicular tissues after completion of the full developmental stage are used as a source for sperm. The extracted sperm from these tissues is used for fertility treatment. Moreover, cryopreserved spermatozoa and semen are well-established techniques commonly used in fertility sector.
Cryopreservation techniques can also be applied in fetal testicular tissue transplantation and these tissues can enhance serum testosterone level and thus improve sperm quality. The in-vitro research report also showed that cryopreserved pre-pubertal testicular tissues can able to produce the hormone.
Ovarian tissue transplantation
Ovarian functionality and follicular developmental mechanism in the female reproductive system can be assessed by ovarian tissue transplantation. But, it is quite impossible to transplant fresh ovarian tissue immediately due to lack of synchronization between donor and recipient. Cryopreservation techniques give the opportunity to preserve ovarian tissue. Cryopreserved ovarian tissue is stored in the cryo-banking facility for future use. Research evidence has established that cryopreserved-thawed ovarian tissue can able to survive under the favorable organ culture system for a long period of time and can be used for transplantation purpose. The first human baby was born in 2004by using autologous transplantation of frozen-thawed ovarian tissue. Since then, 37 children have been born through cryopreserved ovarian tissue and that makes clear the successful utilization of this technique in the fertility sector.
Advancement of the fertility sector is only possible through the utilization and further development of cryopreservation technique. Cryopreservation becomes essential adjunction to current fertility treatments. Use of cryopreservation techniques, including gametes cryopreservation, embryo cryopreservation at their different maturation stage increases the success of fertility treatment. The individualized fertility treatment is only possible through such different types of cryopreservation. Cryopreservation techniques not only preserve human fertility but also helps in balancing the biodiversity of the animal kingdom by maintaining the endangered animal population.