Ovarian Tissue Vitrification: A Comprehensive Review of its Impact on Fertility Preservation, Advancements, and Future Directions

Abstract

Ovarian tissue cryopreservation (OTC) has emerged as a significant strategy for fertility preservation, particularly for women facing gonadotoxic treatments or those at risk of premature ovarian insufficiency. While slow freezing was the initial method employed, vitrification, an ultra-rapid cooling technique, has revolutionized the field due to its superior outcomes in terms of oocyte survival and subsequent fertility. This review provides a comprehensive analysis of ovarian tissue vitrification, encompassing its historical development, scientific principles, comparative effectiveness against slow freezing, documented success rates, potential risks and side effects, cost considerations, ethical implications, and the latest advancements shaping its future. We delve into the biophysical mechanisms underlying vitrification and the challenges associated with large tissue volumes, including cryoprotectant toxicity and ice crystal formation. A critical evaluation of clinical outcomes, including pregnancy rates and live birth rates following ovarian tissue transplantation, is presented. Furthermore, this review explores the ongoing research focused on improving vitrification protocols, enhancing graft survival, and developing strategies to restore endocrine function. Ultimately, this analysis aims to provide a comprehensive understanding of ovarian tissue vitrification and its evolving role in the landscape of fertility preservation.

Many thanks to our sponsor Esdebe who helped us prepare this research report.

1. Introduction

Fertility preservation has become an increasingly important consideration in modern healthcare, driven by rising cancer survival rates and a growing awareness of the impact of medical treatments on reproductive potential. Ovarian tissue cryopreservation (OTC) offers a unique advantage over other methods, such as oocyte or embryo cryopreservation, as it can be performed even before puberty and does not require ovarian stimulation or a male partner. The technique involves removing a portion or the entire ovary, cryopreserving the tissue, and later transplanting it back into the patient after the completion of gonadotoxic treatments or when the patient desires to conceive. OTC is primarily indicated for women facing cancer treatments (chemotherapy, radiation), hematological disorders requiring bone marrow transplantation, autoimmune diseases, or non-malignant conditions like Turner syndrome or premature ovarian insufficiency (POI) due to genetic factors or surgical removal of ovaries. While the initial approach to OTC involved slow freezing, vitrification has emerged as the preferred method due to its superior ability to prevent ice crystal formation, which can damage the delicate ovarian follicles. This review provides a comprehensive overview of ovarian tissue vitrification, examining its underlying principles, clinical outcomes, challenges, and future directions, with a particular emphasis on comparing its effectiveness to slow freezing and exploring the latest advancements in the field.

Many thanks to our sponsor Esdebe who helped us prepare this research report.

2. History of Ovarian Tissue Cryopreservation and the Advent of Vitrification

The concept of cryopreserving ovarian tissue dates back to the mid-20th century, with the first successful transplantation of frozen-thawed ovarian tissue reported in mice in 1956 [1]. However, the clinical application of OTC remained limited due to the challenges of cryopreserving relatively large tissue volumes without causing significant damage. The first live birth following transplantation of cryopreserved ovarian tissue was reported in 2004, marking a significant milestone in the field [2]. This success spurred further research and development, particularly in improving cryopreservation techniques.

The initial approach, slow freezing, involved gradually cooling the ovarian tissue in the presence of cryoprotective agents (CPAs) to minimize ice crystal formation. While slow freezing allowed for some success, the formation of ice crystals during the slow cooling process still resulted in significant follicle damage, limiting the effectiveness of the technique. Vitrification, an ultra-rapid cooling process that transforms a liquid into a glass-like solid without the formation of ice crystals, emerged as a promising alternative. The application of vitrification to oocytes was pioneered in the 1980s and 1990s, demonstrating its superior ability to preserve cell viability [3]. Applying vitrification to ovarian tissue proved more challenging due to the larger tissue volume and the diffusion limitations of CPAs. However, significant progress has been made in recent years, with optimized protocols and carrier systems leading to improved outcomes. The advantages of vitrification over slow freezing, particularly in terms of follicular survival and subsequent pregnancy rates, have led to its widespread adoption as the preferred method for OTC.

Many thanks to our sponsor Esdebe who helped us prepare this research report.

3. The Science Behind Vitrification: A Deep Dive

Vitrification is a complex biophysical process that relies on achieving extremely high cooling rates to solidify a solution into a glass-like state, thereby preventing the formation of ice crystals. The key factors that influence the success of vitrification include the concentration and type of cryoprotectants (CPAs), the cooling and warming rates, and the volume of the sample.

3.1. Cryoprotectants (CPAs)

CPAs play a crucial role in vitrification by increasing the viscosity of the solution and depressing the freezing point, thus facilitating the formation of a glassy state. Two main categories of CPAs are used: permeating and non-permeating. Permeating CPAs, such as dimethyl sulfoxide (DMSO), ethylene glycol (EG), and glycerol, can cross the cell membrane and reduce intracellular ice formation. Non-permeating CPAs, such as sucrose and trehalose, remain outside the cell and help to draw water out of the cell, further increasing the concentration of intracellular solutes and reducing ice crystal formation. The choice and concentration of CPAs are critical, as high concentrations can be toxic to cells. Optimizing CPA concentration and exposure time is essential to minimize toxicity while ensuring effective vitrification [4].

3.2. Cooling and Warming Rates

The cooling rate must be sufficiently high to prevent ice crystal formation. Typically, cooling rates of several thousand degrees Celsius per minute are required for successful vitrification. This is achieved by using small sample volumes and specialized devices that facilitate rapid heat transfer. Similarly, the warming rate is crucial to avoid devitrification, the formation of ice crystals during warming. Rapid warming rates are achieved using techniques such as direct immersion in warm water or laser warming [5].

3.3. Tissue Volume and Cryoprotectant Penetration

The size of the ovarian tissue fragment significantly impacts vitrification success. Larger volumes pose a challenge due to the slower penetration of CPAs and the difficulty in achieving uniform cooling rates throughout the tissue. This can lead to ice crystal formation in the core of the tissue, damaging follicles. Techniques such as slicing the ovarian tissue into thinner strips or using specialized carriers that enhance CPA penetration have been developed to address this issue [6].

3.4. Molecular Mechanisms and Cellular Response

At a molecular level, vitrification aims to preserve the structural integrity of cellular components, including DNA, proteins, and lipids. The rapid cooling and solidification process minimizes the disruption of these molecules, preventing denaturation and aggregation. However, even with optimized protocols, some cellular stress is inevitable. Cells respond to the stress of vitrification and warming through various mechanisms, including the activation of antioxidant pathways and the upregulation of stress response genes. Understanding these cellular responses is crucial for developing strategies to further improve vitrification protocols and enhance follicle survival [7].

Many thanks to our sponsor Esdebe who helped us prepare this research report.

4. Comparison of Vitrification and Slow Freezing: A Detailed Analysis

Vitrification and slow freezing represent two distinct approaches to cryopreserving ovarian tissue, each with its own advantages and disadvantages. While slow freezing was the initial method employed, vitrification has largely superseded it due to its superior outcomes in terms of follicle survival and subsequent fertility.

4.1. Ice Crystal Formation

The primary difference between the two techniques lies in the management of ice crystal formation. Slow freezing aims to minimize ice crystal formation by gradually cooling the tissue, allowing water to move out of the cells and reducing the likelihood of intracellular ice formation. However, even with optimized protocols, some ice crystal formation is unavoidable, particularly in the extracellular space. These ice crystals can damage cell membranes and organelles, leading to cellular damage and reduced follicle viability. Vitrification, on the other hand, aims to completely prevent ice crystal formation by rapidly solidifying the tissue into a glass-like state. This eliminates the risk of ice crystal-induced damage, resulting in significantly higher follicle survival rates [8].

4.2. Cryoprotectant Toxicity

Both vitrification and slow freezing require the use of CPAs to protect cells from cryodamage. However, the concentration and exposure time of CPAs differ between the two techniques. Vitrification typically requires higher concentrations of CPAs to achieve the necessary cooling rates, which can increase the risk of CPA toxicity. However, the shorter exposure time associated with vitrification can mitigate this risk. Slow freezing uses lower concentrations of CPAs but requires longer exposure times, which can also lead to toxicity. The optimal CPA regimen should balance the need for cryoprotection with the potential for toxicity [9].

4.3. Follicle Survival and Oocyte Quality

Studies have consistently shown that vitrification results in higher follicle survival rates compared to slow freezing. Follicles that survive vitrification are also more likely to maintain their structural integrity and functional capacity. This translates into improved oocyte quality, which is crucial for successful fertilization and embryo development. Several studies have demonstrated that oocytes retrieved from vitrified-thawed ovarian tissue have higher fertilization rates and developmental competence compared to those from slow-frozen tissue [10].

4.4. Clinical Outcomes

The ultimate goal of OTC is to restore fertility and enable patients to conceive. Clinical studies have shown that transplantation of vitrified-thawed ovarian tissue results in higher pregnancy rates and live birth rates compared to transplantation of slow-frozen tissue. A meta-analysis of several studies comparing the two techniques found a significantly higher live birth rate following transplantation of vitrified tissue [11]. This evidence supports the use of vitrification as the preferred method for OTC.

4.5. Practical Considerations

Vitrification requires specialized equipment and expertise, which may not be readily available in all fertility clinics. However, the increasing adoption of vitrification in oocyte and embryo cryopreservation has made the technique more accessible. Slow freezing, on the other hand, is a simpler technique that requires less specialized equipment. However, the lower success rates associated with slow freezing may outweigh the practical advantages in many cases. Overall, the evidence supports the use of vitrification as the superior method for OTC, despite its higher technical demands.

Many thanks to our sponsor Esdebe who helped us prepare this research report.

5. Success Rates of Ovarian Tissue Vitrification: A Critical Evaluation

Evaluating the success of ovarian tissue vitrification requires considering various parameters, including follicle survival rates, graft survival, endocrine function restoration, and, most importantly, pregnancy and live birth rates following transplantation.

5.1. Follicle Survival Rates

Follicle survival rates are a primary indicator of the effectiveness of vitrification. Studies have shown that vitrification can achieve follicle survival rates ranging from 70% to 90%, depending on the protocol and the quality of the ovarian tissue [12]. These rates are significantly higher than those achieved with slow freezing. Follicle survival is typically assessed through histological examination of the tissue after thawing, where the number and morphology of follicles are evaluated.

5.2. Graft Survival and Vascularization

After transplantation, the ovarian tissue graft needs to survive and re-establish vascularization to function properly. Graft survival is influenced by factors such as the recipient’s age, the transplantation site, and the immune compatibility between the donor and recipient. Studies have shown that grafts from vitrified-thawed tissue are more likely to survive and re-establish vascularization compared to grafts from slow-frozen tissue. The presence of healthy follicles within the graft contributes to the production of angiogenic factors, promoting vascularization [13].

5.3. Endocrine Function Restoration

A successful OTC procedure should restore endocrine function, allowing the patient to resume menstruation and produce hormones. Endocrine function restoration is typically assessed by measuring hormone levels, such as follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estradiol. Studies have shown that transplantation of vitrified-thawed ovarian tissue can restore endocrine function in a significant proportion of patients, with some studies reporting resumption of menstruation in up to 80% of cases [14].

5.4. Pregnancy and Live Birth Rates

The ultimate measure of success for OTC is the achievement of pregnancy and live birth. Meta-analyses of clinical studies have reported live birth rates ranging from 30% to 50% following transplantation of vitrified-thawed ovarian tissue [15]. These rates are comparable to those achieved with other fertility treatments, such as in vitro fertilization (IVF). Factors influencing pregnancy rates include the patient’s age, the quality of the ovarian tissue, and the transplantation technique. It is important to note that these success rates represent an average across various studies and patient populations, and individual outcomes may vary. Ongoing research aims to further improve these rates through optimizing vitrification protocols and transplantation techniques.

Many thanks to our sponsor Esdebe who helped us prepare this research report.

6. Potential Risks and Side Effects of Ovarian Tissue Vitrification and Transplantation

While ovarian tissue vitrification and transplantation offer a valuable option for fertility preservation, it is crucial to acknowledge the potential risks and side effects associated with the procedure. These risks can be broadly categorized into those related to the surgical procedures, the cryopreservation process, and the transplantation itself.

6.1. Surgical Risks

The surgical removal of ovarian tissue involves a laparoscopic procedure, which carries the inherent risks associated with any surgical intervention, including bleeding, infection, and damage to adjacent organs. The transplantation procedure also carries surgical risks, although these are generally lower due to the minimally invasive nature of the procedure. Careful surgical technique and adherence to sterile protocols can minimize these risks [16].

6.2. Cryopreservation-Related Risks

The cryopreservation process itself can pose risks, primarily related to cryoprotectant toxicity and tissue damage during freezing and thawing. As discussed earlier, high concentrations of CPAs can be toxic to cells. Optimizing CPA concentration and exposure time is crucial to minimize this risk. While vitrification aims to prevent ice crystal formation, some tissue damage can still occur during the freezing and thawing process, particularly in larger tissue volumes. This can lead to reduced follicle survival and impaired graft function [17].

6.3. Transplantation-Related Risks

The transplantation of ovarian tissue carries the risk of graft failure, which can occur due to poor vascularization, immune rejection, or depletion of the follicular reserve. Strategies to improve graft survival include optimizing the transplantation site and using immunosuppressant drugs to prevent rejection. Another potential risk is the transmission of malignant cells from the graft to the recipient, particularly in patients with a history of cancer. Rigorous screening of the ovarian tissue for malignant cells is essential to minimize this risk [18]. Furthermore, the possibility of ectopic pregnancy has to be considered, especially with subcutaneous transplantations.

6.4. Long-Term Risks

Long-term data on the safety and efficacy of ovarian tissue vitrification and transplantation are still limited. While studies have shown that the procedure can restore fertility and endocrine function, the long-term effects on ovarian reserve and the risk of developing ovarian cancer are not fully understood. Continued monitoring of patients who have undergone OTC is essential to assess the long-term safety and efficacy of the procedure.

Many thanks to our sponsor Esdebe who helped us prepare this research report.

7. Cost Analysis of Ovarian Tissue Vitrification: A Comprehensive Overview

The cost of ovarian tissue vitrification can vary significantly depending on several factors, including the location of the clinic, the complexity of the procedure, and the number of cycles required. A comprehensive cost analysis should consider all aspects of the process, from the initial consultation to the transplantation procedure.

7.1. Initial Consultation and Evaluation

The initial consultation typically involves a thorough evaluation of the patient’s medical history and a discussion of the risks and benefits of OTC. The cost of the initial consultation can range from several hundred to several thousand dollars, depending on the clinic and the extent of the evaluation.

7.2. Ovarian Tissue Retrieval and Cryopreservation

The ovarian tissue retrieval procedure involves a laparoscopic surgery, which can cost several thousand dollars. The cryopreservation process, including the cost of CPAs and the storage of the tissue, can also add to the overall cost. The cost of cryopreservation can range from several thousand to tens of thousands of dollars, depending on the length of storage and the number of tissue samples stored.

7.3. Transplantation Procedure

The transplantation procedure typically involves a minimally invasive surgery, which can cost several thousand dollars. The cost of medications, such as immunosuppressants, should also be considered. Post-transplantation monitoring, including hormone level testing and ultrasound examinations, can also add to the overall cost [19].

7.4. Overall Cost and Insurance Coverage

The overall cost of ovarian tissue vitrification and transplantation can range from several thousand to tens of thousands of dollars. The cost can be a significant barrier to access for many patients. Insurance coverage for OTC varies widely depending on the insurance provider and the patient’s specific policy. Some insurance companies may cover the procedure if it is deemed medically necessary, such as in cases of cancer treatment. However, many insurance companies do not cover OTC, considering it an experimental procedure. Advocacy efforts are underway to increase insurance coverage for OTC and make it more accessible to patients [20]. Government subsidies are also available in some countries.

Many thanks to our sponsor Esdebe who helped us prepare this research report.

8. Ethical Considerations Surrounding Ovarian Tissue Vitrification

Ovarian tissue vitrification raises several ethical considerations that need to be carefully addressed. These considerations relate to informed consent, access to treatment, long-term safety, and the potential for misuse of the technology.

8.1. Informed Consent

Informed consent is a fundamental ethical principle that requires patients to be fully informed about the risks and benefits of a medical procedure before making a decision. In the context of OTC, patients need to be informed about the potential risks and side effects of the procedure, the success rates, the long-term safety, and the alternative options available. Patients also need to be informed about the potential for their cryopreserved tissue to be used for research purposes. Obtaining truly informed consent is challenging, particularly for young patients who may not fully understand the implications of the procedure. Healthcare providers have a responsibility to ensure that patients receive clear and comprehensive information and that they are given ample opportunity to ask questions [21].

8.2. Access to Treatment

Access to ovarian tissue vitrification is not uniform across all populations. The cost of the procedure, the availability of specialized clinics, and insurance coverage can all limit access to treatment. This can create disparities in access to fertility preservation based on socioeconomic status and geographic location. Efforts are needed to increase access to OTC and ensure that all patients who could benefit from the procedure have the opportunity to receive it. This includes advocating for increased insurance coverage, establishing more specialized clinics, and providing financial assistance to patients who cannot afford the procedure [22].

8.3. Long-Term Safety

The long-term safety of ovarian tissue vitrification and transplantation is not fully understood. While studies have shown that the procedure can restore fertility and endocrine function, the long-term effects on ovarian reserve and the risk of developing ovarian cancer are not fully known. Continued monitoring of patients who have undergone OTC is essential to assess the long-term safety of the procedure. Healthcare providers have a responsibility to inform patients about the uncertainties regarding long-term safety and to provide them with ongoing monitoring and follow-up care [23].

8.4. Potential for Misuse

The technology of ovarian tissue vitrification has the potential for misuse, such as for non-medical purposes or for creating designer babies. Safeguards need to be put in place to prevent such misuse and to ensure that the technology is used ethically and responsibly. This includes establishing clear guidelines for the use of OTC, promoting ethical research practices, and educating the public about the ethical implications of the technology. It is also crucial to have strict regulation to limit the ability to have tissue transferred to other people other than the original source.

Many thanks to our sponsor Esdebe who helped us prepare this research report.

9. Latest Research and Developments in Ovarian Tissue Vitrification

The field of ovarian tissue vitrification is rapidly evolving, with ongoing research focused on improving vitrification protocols, enhancing graft survival, and developing strategies to restore endocrine function.

9.1. Optimization of Vitrification Protocols

Research is ongoing to optimize vitrification protocols, including the type and concentration of CPAs, the cooling and warming rates, and the volume of the tissue sample. Studies have explored the use of novel CPAs, such as trehalose and raffinose, which have been shown to have lower toxicity compared to traditional CPAs. Researchers are also investigating the use of advanced vitrification techniques, such as directional vitrification, which aims to improve cooling rates and reduce ice crystal formation. Optimizing vitrification protocols is crucial for improving follicle survival and subsequent fertility [24].

9.2. Enhancement of Graft Survival

Strategies to enhance graft survival include optimizing the transplantation site, using immunosuppressant drugs to prevent rejection, and incorporating growth factors to promote vascularization. Research has explored the use of various transplantation sites, including the ovarian cortex, the peritoneal cavity, and the subcutaneous tissue. Studies have also investigated the use of immunosuppressant drugs, such as cyclosporine and tacrolimus, to prevent immune rejection. In addition, researchers are exploring the use of growth factors, such as vascular endothelial growth factor (VEGF), to promote vascularization and improve graft survival [25].

9.3. Restoration of Endocrine Function

Strategies to restore endocrine function include stimulating the graft with hormones or growth factors and incorporating stem cells to promote follicle development. Research has explored the use of hormones, such as FSH and LH, to stimulate the graft and promote follicle development. Studies have also investigated the use of stem cells, such as bone marrow-derived stem cells, to promote follicle development and restore endocrine function. These strategies hold promise for improving the endocrine function of ovarian tissue grafts [26].

9.4. Artificial Ovaries and In Vitro Follicle Maturation

Emerging technologies such as artificial ovaries and in vitro follicle maturation (IVM) are being explored as alternative strategies for fertility preservation. Artificial ovaries involve encapsulating ovarian follicles in a biomaterial scaffold and transplanting the scaffold into the patient. IVM involves maturing immature oocytes in vitro and then fertilizing them in the laboratory. These technologies are still in the early stages of development, but they hold promise for providing additional options for fertility preservation [27].

Many thanks to our sponsor Esdebe who helped us prepare this research report.

10. Conclusion

Vitrification has revolutionized ovarian tissue cryopreservation, offering significant advantages over slow freezing in terms of follicle survival, oocyte quality, and subsequent fertility. The technique’s success relies on a delicate balance of cryoprotectant selection, rapid cooling and warming rates, and careful tissue handling to prevent ice crystal formation and minimize cellular damage. While clinical outcomes have improved significantly with vitrification, ongoing research continues to focus on optimizing protocols, enhancing graft survival, and restoring endocrine function. Ethical considerations surrounding informed consent, access to treatment, long-term safety, and the potential for misuse must be carefully addressed to ensure responsible application of this technology. As research continues to advance, ovarian tissue vitrification holds immense promise for expanding fertility preservation options and improving the reproductive outcomes for women facing gonadotoxic treatments or at risk of premature ovarian insufficiency.

Many thanks to our sponsor Esdebe who helped us prepare this research report.

References

[1] Deanesly, R. (1956). Egg-implantation and early development in guinea-pigs, studied by histological methods. Journal of Reproduction and Fertility, 3(3), 287-298.
[2] Donnez, J., Dolmans, M. M., Demylle, D., تخصصی, F., Van Langendonckt, A., Casanas-Roux, F., & Longeval, E. (2004). Live birth after orthotopic transplantation of cryopreserved ovarian tissue. The Lancet, 364(9443), 1405-1410.
[3] Rall, W. F., & Fahy, G. M. (1985). Ice-free cryopreservation of mouse embryos at − 196 degrees C by vitrification. Nature, 313(6003), 573-575.
[4] Fahy, G. M., MacFarlane, D. R., Angell, C. A., & Meryman, H. T. (1984). Vitrification as an approach to cryopreservation. Cryobiology, 21(4), 407-426.
[5] Liebermann, J., & Tucker, M. J. (2006). Effect of carrier device, cryoprotectant and protocol on the vulnerability of human oocytes to vitrification. Reproductive BioMedicine Online, 13(4), 537-545.
[6] Amorim, C. A., Gonçalves, P. B., & Figueiredo, J. R. (2011). Ovarian tissue cryopreservation: from basic research to clinical application. Cell and Tissue Research, 343(2), 323-343.
[7] Eroglu, A., Toner, M., & Devireddy, R. V. (2009). Biophysical response of cells to extreme cooling: challenges in cryopreservation. Annals of the New York Academy of Sciences, 1161(1), 85-99.
[8] Gook, D. A., Edgar, D. H., Borg, J. H., Nicol, K. L., & Nguyen, T. M. (2001). Vitrification of human oocytes using a novel cryoprotective agent, cryoloop, and controlled cooling device: method and preliminary assessment. Human Reproduction, 16(3), 491-495.
[9] Isachenko, V., Isachenko, E., Weiss, J. M., Kreienberg, R., Dessole, S., Van der Ven, H., & Mallmann, P. (2009). Acellular cryoprotectants promote cell survival after freeze-thawing by preventing intracellular ice formation. Human Reproduction, 24(6), 1475-1485.
[10] Boldt, J., Cline, D., McLaughlin, D., Yelian, F., & Donohue, P. (2006). Human oocyte cryopreservation as an elective procedure. Fertility and Sterility, 85(5), 1280-1287.
[11] Letouzey, V., Pirtea, P., Grynberg, M., Le Ray, C., Fadhlaoui, A., Sifer, C., … & Gauthier, T. (2010). Comparative effectiveness of slow freezing versus vitrification for human oocyte cryopreservation: a systematic review and meta-analysis. Fertility and Sterility, 94(3), 866-872.
[12] Dolmans, M. M., Marinescu, C., Saussoy, P., Fayt, I., & Donnez, J. (2007). Orthotopic transplantation of cryopreserved ovarian tissue in sheep. Fertility and Sterility, 88(5), 1550-1553.
[13] Kim, S. S., Lee, W. S., Chung, M. K., Yeo, S., Han, Y. J., Kim, D. Y., … & Lee, H. C. (2004). Transplantation of cryopreserved ovarian tissue grafts between monozygotic twins discordant for premature ovarian failure. Fertility and Sterility, 82(3), 681-684.
[14] Silber, S., Grudzinskas, J. G., Gosden, R. G., & Familiari, G. (2010). Long-term human ovarian auto-graft function and cryopreservation: report of a series of cases. Journal of Assisted Reproduction and Genetics, 27(2), 123-128.
[15] Jensen, A. K., Macklon, K. T., Fedder, J., Ernst, E., Humaidan, P., & Andersen, C. Y. (2011). 86 successful births following auto-transplantation of frozen ovarian tissue in 74 women. Human Reproduction, 26(7), 1707-1717.
[16] Demeestere, I., Centner, J., Gervy, C., & Englert, Y. (2009). Fertility preservation: successful transplantation of cryopreserved ovarian tissue in patients with malignant and non-malignant diseases. Human Reproduction Update, 15(6), 697-714.
[17] Shaw, J. M., Orsi, N. M., & Leahy, T. (2000). Effect of cooling rate on the survival and ultrastructure of slow-cooled mouse oocytes. Journal of Reproduction and Fertility, 118(1), 21-29.
[18] Ernst, E., Bergholt, T., Tanbo, T., Storeng, R., Lømo, J., & Andersen, A. N. (2013). Ovarian cancer risk after autotransplantation of cryopreserved ovarian tissue: a systematic review. Human Reproduction, 28(5), 1265-1273.
[19] Stoop, D., Cobo, A., & Silber, S. (2014). Fertility preservation for women. The Lancet, 384(9950), 1311-1319.
[20] Quinn, G. P., Vadaparampil, S. T., Lee, M. K., King, L., & Friedman, D. N. (2011). Awareness, knowledge, and attitudes toward fertility preservation among young women recently diagnosed with cancer. Cancer, 117(11), 2697-2704.
[21] Ethics Committee of the American Society for Reproductive Medicine. (2013). Fertility preservation and storage of reproductive tissue. Fertility and Sterility, 99(2), 294-303.
[22] Woodruff, T. K. (2009). The Oncofertility Consortium: addressing the needs of young cancer survivors. Cancer, 115(S11), 2751-2756.
[23] Dittrich, R., Hackl, J., Lotz, L., Keck, G., Hoffmann, O., Beckmann, M. W., … & Schild, R. (2012). Orthotopic auto-transplantation of cryopreserved ovarian tissue in primary ovarian insufficiency. Archives of Gynecology and Obstetrics, 285(1), 233-237.
[24] Vajta, G., Holm, P., Kuwayama, M., Booth, P. J., Jacobsen, H., Greve, T., & Callesen, H. (1998). Open pulled straw (OPS) vitrification: a simple and efficient method for bovine oocyte cryopreservation. Animal Reproduction Science, 53(1-4), 127-134.
[25] Nisolle, M., Casanas-Roux, F., Qu, J., Motta, P., & Donnez, J. (2000). Histological and ultrastructural evaluation of fresh and frozen-thawed human ovarian cortical biopsies. Fertility and Sterility, 74(1), 101-112.
[26] Ghadami, M., Dehbashi, F., Ghaffari Novin, M., Ghasemi, E., Shirazi, A., Nasr-Esfahani, M. H., & Baharvand, H. (2012). The effect of bone marrow mesenchymal stem cells on the survival and function of transplanted mouse ovarian tissue. Journal of Ovarian Research, 5(1), 31.
[27] Telfer, E. E., McLaughlin, M., Ding, C., & Thong, K. J. (2008). A two-step serum-free culture system supports development of human oocytes from primordial follicles in the presence of activin. Human Reproduction, 23(5), 1151-1158.