Safe Fertility Restoration After Cancer: New Approaches Using Frozen Ovarian Tissue

Table of Contents

• Introduction: The Promise and Challenges of Ovarian Tissue Freezing
• How This Research Was Conducted
• Five Experimental Safety Strategies
• In-Vitro Maturation of Oocytes
• Constructing an Artificial Ovary
• Purging Strategies to Eliminate Cancer Cells
• Oocyte Maturation Through Xenotransplantation
• Stem Cell-Based Oogenesis
• What This Means for Cancer Patients
• Current Limitations and Challenges
• Recommendations for Patients and Doctors
• Source Information

Introduction: The Promise and Challenges of Ovarian Tissue Freezing

Ovarian tissue cryopreservation (freezing and storing ovarian tissue) has become an established fertility preservation technique, especially valuable for young girls before puberty and patients who need to start cancer treatment immediately. This method involves removing and freezing ovarian tissue before chemotherapy or radiation, which can damage fertility. The technique has proven remarkably successful, with over 200 reported births worldwide after transplanting frozen-thawed ovarian tissue back into patients.

Major studies show encouraging success rates. A well-documented series from five European centers reported a 26% chance of having one or more live births after transplantation. Another multicenter study found an even higher success rate of 41.6% for at least one delivery after ovarian tissue transplantation. These results have led professional organizations like the American Society for Reproductive Medicine and European Society of Human Reproduction and Embryology to classify the technique as innovative and usual care rather than experimental.

However, significant safety concerns remain. The frozen ovarian tissue, typically stored before cancer treatment begins or after remission, may contain metastasized cancer cells. After thawing and transplantation, these microscopic cancer cells could potentially develop into tumors and reintroduce the cancer. While this risk appears relatively small for most solid tumors, ovarian tissue from patients with blood-borne malignancies like leukemia has up to a 50% chance of containing malignant cells.

Even the slightest risk of reintroducing cancer remains alarming, which has spurred the development of innovative techniques to prevent this dangerous possibility. This systematic review examines five different experimental strategies that could provide safer fertility restoration options for cancer patients in the future.

How This Research Was Conducted

This systematic review followed rigorous scientific standards according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. The research protocol was registered on the International Prospective Register for Systematic Reviews (PROSPERO, Registration ID CRD42020197284) before the study began.

Researchers conducted comprehensive searches across three major medical databases—MEDLINE (using PubMed), EMBASE, and the Cochrane Library—on July 8, 2021. They developed a detailed search strategy in consultation with an information specialist from Radboud University Nijmegen Library, using combinations of Medical Subject Headings terms and free text words related to ovarian tissue freezing and fertility preservation.

The team established strict inclusion criteria for studies. Only original research aiming for safe fertility restoration in cancer patients using cryopreserved ovarian tissue qualified for inclusion. Studies had to involve experiments with human ovarian tissue, be published in English between January 1, 2000, and July 8, 2021, and focus on preventing cancer reintroduction. The year 2000 was chosen as the starting point because the first human transplantation of frozen-thawed ovarian tissue was reported that year.

Two authors independently reviewed all identified studies using a systematic review web application called Rayyan QCRI. They screened titles, abstracts, and keywords for relevance, then retrieved full texts of potentially eligible articles. Any disagreements were resolved through discussion or by a third reviewer. The team also examined reference lists of selected publications to identify additional studies missed in electronic searches.

From an initial 12,722 records identified through database searches plus 18 additional studies from references, researchers removed duplicates and screened 8,914 records based on titles and abstracts. Of these, 166 full-text articles were assessed for eligibility, and ultimately 31 studies met all inclusion criteria for qualitative synthesis. These studies dated from 2004 to 2021 and were grouped according to the five different safety strategies they investigated.

Five Experimental Safety Strategies

The systematic review identified five distinct experimental approaches being developed to safely use cryopreserved ovarian tissue for fertility restoration after cancer treatment. Each strategy aims to prevent the reintroduction of malignant cells during fertility restoration while utilizing the patient's frozen ovarian tissue.

The five strategies include:

• In-vitro maturation (IVM) of oocytes: Isolating and maturing eggs from ovarian tissue in the laboratory to perform IVF without transplanting tissue back into the patient
• Artificial ovary construction: Creating a biological scaffold to reseed pre-antral follicles while eliminating cancer cells
• Purging strategies: Techniques aimed at eradicating contaminating malignant cells from ovarian cortex tissue
• Xenotransplantation: Maturing oocytes by transplanting ovarian tissue into immunodeficient animals
• Stem cell-based oogenesis: Using stem cells to generate new eggs for fertility restoration

All these strategies remain in the experimental stage and have not yet reached clinical trials. They represent promising but preliminary approaches that require significantly more research to establish their safety, effectiveness, and potential risks. The ethical aspects associated with these techniques, particularly xenotransplantation and stem cell approaches, also need thorough discussion before clinical application.

Despite their experimental status, these innovative approaches might eventually provide safe fertility restoration options for cancer patients, especially those considered to have a high risk of ovarian metastases. The following sections examine each strategy in detail, including the current state of research, technical challenges, and potential for future clinical application.

In-Vitro Maturation of Oocytes

In-vitro maturation (IVM) involves collecting immature eggs from ovarian tissue and maturing them in the laboratory setting. The resulting mature oocytes can then be fertilized through in-vitro fertilization (IVF), and the embryos transferred to the patient without needing to transplant potentially contaminated ovarian tissue back into the body. This approach completely avoids the risk of cancer reintroduction since no tissue transplantation occurs.

Researchers have developed IVM techniques using oocytes collected through three different procedures. This review focuses specifically on IVM of oocytes obtained from ovarian tissue collected during ovariectomy (ovary removal), as this is the technique applicable to already cryopreserved ovarian tissue stored by cancer patients.

Several sophisticated culture systems have been developed for IVM of oocytes from isolated follicles or intact ovarian tissue fragments. These systems sometimes involve multiple steps, including follicle isolation and in-vitro growth before final oocyte maturation. Different developmental stages of oocytes require different maturation techniques, necessitating complex multi-step approaches.

Telfer and colleagues pioneered a two-step culture system in 2008. Their research showed that human unilaminar follicles could be activated in fragmented fresh cortical tissue. During the second step, secondary follicles were isolated from the tissue. Additional culture in the presence of activin A (a protein that stimulates follicle development) led to further growth of these secondary follicles. This represented a significant advancement in IVM technology.

More recently, McLaughlin and colleagues developed an even more complex multistep culture system in 2018. In this system, fresh ovarian cortex tissue was fragmented, and primordial and primary follicles were allowed to grow to secondary follicles while still embedded in the tissue. The secondary follicles were then manually dissected and cultured with activin A and FSH (follicle-stimulating hormone). This final culture step produced cumulus-oocyte complexes containing metaphase II (MII) oocytes—eggs mature enough for fertilization.

However, researchers noted that these in-vitro matured oocytes showed some abnormalities compared to naturally matured eggs. They produced atypical large polar bodies (a structure normally extruded during egg maturation) with a ratio of 4:1 to 3:1 of oocyte size to polar body size, and the degree of cumulus expansion (a normal process during maturation) was less pronounced than in in-vivo matured oocytes.

An alternative approach involves culturing isolated pre-antral follicles in three-dimensional alginate matrices. These matrices provide physical support to help maintain follicle structure, resulting in better follicle growth and oocyte maturation to the MII stage. Studies using this approach have also demonstrated increased production of 17β-oestradiol (a form of estrogen), indicating improved follicular function during the maturation process.

Constructing an Artificial Ovary

The artificial ovary approach involves creating a biological scaffold that can support follicle survival and development while eliminating any cancer cells that might be present in the ovarian tissue. This strategy aims to provide a safe environment for follicular development that can be transplanted back into the patient without risk of cancer recurrence.

Researchers have experimented with various natural and synthetic materials to create optimal scaffolds for an artificial ovary. These materials include fibrin (a protein involved in blood clotting), agarose (a substance derived from seaweed), Matrigel (a gelatinous protein mixture), and fibrinogen/thrombin combinations. Each material offers different advantages for supporting follicle survival and growth.

Studies have shown that the mechanical properties of the scaffold material significantly impact follicle survival and development. The ideal matrix should provide adequate physical support while allowing necessary nutrient exchange and follicle growth. Research indicates that fibrin matrices, in particular, show promise because their ultrastructure and rigidity most closely resemble that of human ovarian cortex tissue.

In addition to completely artificial matrices, scientists are exploring the possibility of using decellularized human ovarian cortex. This process involves removing all cellular material from donated ovarian tissue, leaving behind only the extracellular matrix framework. This natural scaffold could then be reseeded with the patient's own follicles after they have been purified of any cancer cells.

The artificial ovary approach offers several potential advantages. It could allow doctors to screen the scaffold for cancer cells before transplantation, ensuring complete safety. It also provides the opportunity to optimize the environment for follicle development potentially improving success rates compared to conventional transplantation. However, significant technical challenges remain in creating a fully functional artificial ovary that can support complete follicular development and restoration of normal hormonal function.

Current research focuses on identifying the optimal scaffold materials, developing effective reseeding techniques, and ensuring long-term survival and function of transplanted follicles. While promising, the artificial ovary approach remains in early experimental stages and will require extensive further research before clinical application.

Purging Strategies to Eliminate Cancer Cells

Purging strategies aim to eliminate malignant cells from ovarian tissue before transplantation while preserving the viability of healthy follicles. This approach could allow patients to use their own cryopreserved tissue safely, maintaining the natural ovarian environment that supports optimal follicular development.

Several techniques are being explored for purging ovarian tissue of cancer cells. These include physical separation methods, chemical treatments, immunological approaches, and photodynamic therapy. Each method aims to selectively target and destroy cancer cells while sparing the precious ovarian follicles.

Physical separation techniques exploit differences in size, density, or other physical properties between cancer cells and follicles. For example, some researchers have used density gradient centrifugation to separate smaller cancer cells from larger follicular structures. These methods show promise but may not eliminate all cancer cells, particularly if they're similar in size to follicular cells.

Chemical purging approaches involve treating ovarian tissue with anti-cancer agents that selectively target malignant cells. The challenge lies in finding agents that effectively kill cancer cells without damaging the sensitive ovarian follicles or compromising future fertility. Researchers are investigating various chemotherapeutic agents at different concentrations and exposure times to identify optimal purging protocols.

Immunological methods use antibodies that specifically target cancer cell markers. These antibodies can be conjugated with toxins (creating immunotoxins) or used to flag cancer cells for destruction by the immune system. This approach offers high specificity but requires identifying reliable cancer-specific markers that aren't present on follicular cells.

Photodynamic therapy involves using light-sensitive compounds that are taken up preferentially by cancer cells. When activated by specific light wavelengths, these compounds produce toxic oxygen species that kill the cancer cells. This method shows promise for surface decontamination but may be less effective for cancer cells deep within tissue fragments.

The major challenge with all purging strategies is ensuring complete elimination of cancer cells while maintaining follicle viability and function. Even a few remaining cancer cells could potentially cause disease recurrence. Researchers are developing sensitive detection methods to verify complete cancer cell eradication before transplantation, but this remains technically challenging.

Oocyte Maturation Through Xenotransplantation

Xenotransplantation involves transplanting human ovarian tissue into immunodeficient animals to support follicle development and oocyte maturation. The mature oocytes can then be retrieved from the animal host and used for IVF, avoiding the need to transplant tissue back into the human patient and thus eliminating cancer recurrence risk.

This approach takes advantage of the natural ovarian environment provided by the animal host to support complete follicular development. The animal's circulatory system provides necessary hormones and nutrients, potentially leading to better quality oocytes compared to entirely in-vitro systems.

Researchers typically use immunodeficient mice as hosts for xenotransplantation studies. These animals lack a functional immune system, preventing rejection of the human tissue. The ovarian tissue is usually transplanted to sites that allow easy monitoring and retrieval, such as under the kidney capsule or into the ovarian bursa.

Studies have demonstrated that human ovarian tissue can survive and function in mouse hosts, with follicles progressing through various developmental stages. However, the efficiency of complete follicular development to produce mature, fertilizable oocytes remains relatively low. Researchers are working to optimize transplantation techniques and host conditions to improve outcomes.

Ethical considerations represent a significant concern with xenotransplantation approaches. The use of animals as hosts for human tissue raises various ethical questions that must be carefully addressed. Additionally, regulatory hurdles for techniques involving animal-human combinations are substantial and vary between countries.

Safety concerns include the potential transmission of animal pathogens to humans through the retrieved oocytes or the remote possibility of human cells contaminating the animal host. Strict protocols are needed to ensure complete separation between human and animal biological systems and to prevent any cross-species contamination.

Despite these challenges, xenotransplantation provides a valuable research tool for studying human follicular development and testing safety strategies. It may also offer a viable pathway to fertility restoration for some patients if ethical and safety concerns can be adequately addressed through further research and regulatory development.

Stem Cell-Based Oogenesis

Stem cell-based approaches aim to generate new oocytes from various types of stem cells, potentially providing an unlimited source of eggs for fertility restoration without needing ovarian tissue transplantation. This revolutionary approach could completely avoid the risk of cancer recurrence since it wouldn't use potentially contaminated ovarian tissue.

Researchers are exploring several types of stem cells for oogenesis (egg formation). These include embryonic stem cells, induced pluripotent stem cells (created by reprogramming adult cells), and ovarian stem cells. Each cell type offers different advantages and challenges for generating functional human oocytes.

Embryonic stem cells have the potential to differentiate into any cell type, including oocytes. However, their use involves significant ethical considerations, and the resulting eggs would have genetic material from the embryo donor rather than the patient, unless created through therapeutic cloning techniques.

Induced pluripotent stem cells (iPSCs) offer a potentially more acceptable alternative. These are created by reprogramming a patient's own adult cells (such as skin cells) back to an embryonic-like state. The iPSCs could then be differentiated into oocytes with the patient's genetic material. This approach avoids ethical concerns associated with embryonic stem cells and ensures genetic compatibility.

Some research suggests that the ovary itself may contain stem cells capable of generating new oocytes throughout life, challenging the long-held belief that women are born with all the eggs they'll ever have. If confirmed, these ovarian stem cells could potentially be harvested, expanded in culture, and differentiated into mature oocytes.

The process of generating functional oocytes from stem cells is extremely complex and not yet fully understood. Researchers must replicate the intricate process of oogenesis, which normally occurs during fetal development and involves complex genetic and epigenetic programming. Current techniques have succeeded in producing oocyte-like cells in mice, but generating fully functional human oocytes capable of normal fertilization and development remains a significant challenge.

Safety concerns with stem cell-based approaches include the potential for abnormal genetic or epigenetic programming that could lead to developmental abnormalities in resulting embryos. Extensive research is needed to ensure that stem cell-derived oocytes undergo normal meiosis (cell division) and have correct chromosomal composition and epigenetic markings.

While stem cell-based oogenesis represents perhaps the most revolutionary approach to safe fertility restoration, it remains in the earliest stages of research. Significant basic science advances are needed before this approach can be considered for clinical application, but it holds tremendous promise for the future of fertility preservation.

What This Means for Cancer Patients

This research represents crucial progress toward addressing the most significant safety concern in ovarian tissue transplantation—the potential reintroduction of cancer. For cancer survivors, particularly those with blood cancers like leukemia where ovarian involvement risk reaches 50%, these experimental approaches could eventually provide safe pathways to biological parenthood.

Currently, patients considering ovarian tissue transplantation must undergo rigorous safety screening. Doctors use various methods to detect cancer cells in stored tissue, including immunohistochemistry (staining for cancer-specific markers), molecular analysis of tumor-specific transcripts, and sometimes xenotransplantation into immunodeficient mice to confirm absence of metastatic cells. However, all these tests are destructive to the tissue being analyzed and cannot be applied to the fragments that will actually be transplanted.

Even when tested fragments show no cancer cells, remaining tissue could still harbor micrometastases due to sampling limitations. This uncertainty creates significant anxiety for patients and doctors considering transplantation. The experimental strategies reviewed here aim to eliminate this uncertainty by either avoiding tissue transplantation altogether (IVM, stem cell approaches) or ensuring complete cancer cell removal before transplantation (purging, artificial ovary).

For now, ovarian tissue transplantation should only be performed in fertility clinics with extensive experience, following thorough case review by a multidisciplinary team. The procedure is generally considered safe for most solid tumors where ovarian involvement is limited, but remains controversial for blood cancers with high metastasis risk.

Patients who have stored ovarian tissue should discuss these emerging options with their fertility specialists. While none are clinically available yet, understanding the research landscape can help in making informed decisions about future fertility restoration possibilities. Patients might also consider participating in clinical trials once these approaches advance to human testing stages.

The development of these safety strategies particularly benefits young cancer patients who undergo fertility preservation before puberty, when egg freezing isn't possible. These patients have few options beyond ovarian tissue freezing, making safe transplantation techniques especially valuable for their future fertility restoration.

Current Limitations and Challenges

All five strategies remain in experimental stages with significant limitations that must be addressed before clinical application. The in-vitro maturation approach faces efficiency challenges—current techniques produce relatively few mature oocytes compared to the number of follicles initially present in ovarian tissue. The quality of in-vitro matured oocytes also raises concerns, as they often show abnormalities in polar body size and cumulus expansion compared to naturally matured eggs.

Artificial ovary development struggles with creating scaffolds that perfectly mimic the natural ovarian environment. While fibrin matrices show promise in resembling human ovarian cortex ultrastructure, maintaining long-term follicle survival and function in artificial environments remains challenging. Researchers also need to develop reliable techniques for reseeding scaffolds with purified follicles without damaging these delicate structures.

Purging strategies face the fundamental challenge of completely eliminating cancer cells while preserving follicle viability. Current detection methods may not identify minimal residual disease, creating the risk of false negative results. The aggressive treatments needed to kill cancer cells often damage healthy follicles as well, reducing the pool of available follicles for transplantation.

Xenotransplantation approaches involve significant ethical considerations and regulatory hurdles. The use of animal hosts raises concerns about animal welfare, potential cross-species disease transmission, and ethical acceptance of human-animal biological combinations. These concerns have limited research progress and will likely delay clinical translation even if technical success is achieved.

Stem cell-based oogenesis faces perhaps the most fundamental biological challenges. Scientists still don't fully understand the complex process of human egg development, making it difficult to replicate in the laboratory. The risk of epigenetic abnormalities and chromosomal errors in stem cell-derived oocytes presents serious safety concerns that must be thoroughly addressed before clinical consideration.

All these approaches share common limitations including small sample sizes in current studies, variability between patients, and lack of long-term follow-up data. Most research has used tissue from women with benign conditions rather than cancer patients, potentially limiting applicability to the intended population. The freeze-thaw process itself may damage tissue and reduce the effectiveness of these techniques when applied to cryopreserved samples.

Funding and regulatory challenges also slow progress. These innovative approaches require substantial investment in basic research before clinical trials can begin, and regulatory pathways for such novel techniques remain uncertain in many countries. Ethical review processes for techniques involving stem cells or xenotransplantation are particularly complex and time-consuming.

Recommendations for Patients and Doctors

Based on this comprehensive review, several recommendations emerge for patients considering fertility preservation and doctors providing oncological care. First, ovarian tissue cryopreservation remains a valuable option for fertility preservation, especially for prepubertal patients and those requiring immediate cancer treatment. Patients should discuss this option with their oncology team before treatment begins.

Patients with blood cancers like leukemia should understand the higher risks associated with ovarian tissue transplantation due to the up to 50% chance of ovarian involvement. These patients should consider additional fertility preservation options if possible, such as egg or embryo freezing after puberty induction, and should participate in detailed counseling about the risks and benefits of tissue transplantation.

Doctors should ensure that ovarian tissue transplantation is only performed in specialized centers with extensive experience and following multidisciplinary review. Current safety assessment protocols, including immunohistochemistry, molecular analysis, and sometimes xenotransplantation testing, should be rigorously followed despite their limitations.

Patients with cryopreserved ovarian tissue should maintain realistic expectations about these experimental safety strategies. While promising, none are clinically available yet, and translation to human applications will likely take several years. Patients should stay informed about research developments through their fertility specialists.

The research community should prioritize studies that directly address the limitations identified in this review. Particular focus should be placed on improving the efficiency of in-vitro maturation techniques, developing more reliable purging methods, and addressing the ethical concerns surrounding xenotransplantation and stem cell approaches.

Collaboration between fertility specialists, oncologists, researchers, and ethical review boards is essential to advance these technologies responsibly. Patients should be encouraged to participate in ethical discussions about these emerging technologies, ensuring that development aligns with patient values and concerns.

Finally, funding agencies and regulatory bodies should recognize the importance of developing safe fertility restoration options for cancer survivors. Increased investment in basic and translational research could accelerate progress toward clinically viable solutions that eliminate cancer recurrence risk while restoring fertility.

Source Information

Original Article Title: Strategies to safely use cryopreserved ovarian tissue to restore fertility after cancer: a systematic review

Authors: Lotte Eijkenboom, Emma Saedt, Carlijn Zietse, Didi Braat, Catharina Beerendonk, Ronald Peek

Publication: RBMO Volume 45 Issue 4 2022

Note: This patient-friendly article is based on peer-reviewed research originally published in a scientific journal. It preserves all key findings, data, and conclusions from the original systematic review while making the information accessible to patients and caregivers.