This manuscript reports our experience with OTC at the University of Pennsylvania and CHOP. To date, a total of twenty-one patients have cryopreserved ovarian tissue. Of these, 9 were less than 18 years of age. Patient characteristics, diagnoses and treatment exposures varied widely. The majority of cases were performed in patients who did not have other viable options for fertility preservation, or who did not respond to ovarian stimulation. Notably, 38 % pursued another method of fertility preservation before or at the time of OTC. To date, no transplants have been performed at our institution.
We found that ovarian tissue cryopreservation was a feasible option for children and adults facing gonadotoxic therapies. Multi-disciplinary collaboration (with a pediatric oncologist, reproductive endocrinologist, pediatric surgeon, pediatric and adult clinical/research nurse and a psychosocial counselor) was required to establish and carry out this experimental protocol at the adult and pediatric university affiliated hospitals. Not only must the investigative team identify and obtain informed consent/assent, the team must urgently coordinate preoperative testing, serologic testing for tissue storage and surgical scheduling in relation to planned cancer therapies. While scheduling surgery in the morning is ideal so that laboratory staff can be available during normal working hours to process and preserve tissue, it is often not possible due to the complex nature of OR scheduling, allocation of OR resources or unavoidable delays on the day of surgery. In our experience, tissue was received after noon in one half of the cases. This highlights the importance for surgeons and laboratory staff to be flexible. When a clinical practice makes a commitment to take care of oncofertility patients, it is important that the lab be open and available to accommodate cases outside of normal working hours.
Of particular interest is the case of an 11 year old pre-menarchal female with Stage IV neuroblastoma, status post recent chemotherapy, who elected to pursue OTC. Her remarkably high FSH level was concerning for follicular depletion; however, a complete absence of preantral follicles was not observed on histological examination of the excised ovarian tissue. This suggests that traditional measures of ovarian reserve may not accurately reflect ovarian follicle counts in pediatric patients shortly after chemotherapy. Hormones such as FSH are unreliable in prepubertal patients and those using hormonal contraception. More investigation is needed on the utility of endocrine values, particularly more novel markers such as anti-mullerian hormone levels, in the setting of pediatric cancer therapy to predict ovarian follicle reserve and ultimately determine whether OTC is of benefit to such patients. Nonetheless, at this time, OTC represents an important direction in fertility preservation for individuals who are unable to pursue other fertility preservation options such as embryo or oocyte cryopreservation.
Improvements in cancer treatments have increased survival rates; however, many therapies increase the risk of infertility and premature ovarian failure [5
]. Interest in fertility preservation has increased and more efforts have been made to offer fertility preservation options to cancer patients as part of treatment. Fertility preservation strategies for females include embryo cryopreservation, oocyte cryopreservation and OTC. While embryo banking is currently considered the only standard (non-experimental) procedure, improved success with experimental techniques such as oocyte banking and ovarian tissue banking offer single females the possibility of having biological children with a future partner [10
]. Selection of an appropriate technology depends on a variety of factors including the patient’s age and health at diagnosis, the overall gonadoxicity of the intended therapy, the urgency of treatment, cost, and the availability of a sexual partner. Many cancer patients do not have enough time to complete ovarian stimulation before starting cancer treatment, or have other concerns about this process. OTC is an attractive technique for fertility preservation because it avoids ovarian stimulation and remains the only option for pre-pubertal females. Rather than freezing individual oocytes, biopsy of the ovarian cortex theoretically represents an efficient way of preserving thousands of primordial follicles at one time. As described in this report, ovarian cortical tissue (whole ovary or biopsy) is generally obtained laparoscopically and dissected into small fragments and cryopreserved. The only method that has resulted in live human births is autotransplantation of cryopreserved tissue at the site of the native ovary that still included ovarian cortex. Sixteen human live births have been reported from previously frozen thawed tissue and are described in Table [10
]. Pregnancies have occurred with and without assistance after transplantation. It is important to note that all of the patients were adults at the time of tissue acquisition, most had a diagnosis of cancer, and all had remaining portions of ovarian cortex at the ovarian site at the time of transplantation of the thawed tissue.
Live births from orthotopic transplantation of frozen-thawed ovarian tissue
Potential limitations of OTC must be acknowledged. Most importantly, there is a significant concern regarding the potential for reseeding tumor cells following ovarian transplantation procedures in cancer survivors with tumors that might involve the ovaries. Although many types of cancer virtually never metastasize to the ovaries, leukemias are systemic in nature and therefore pose a significant risk. A recent study of 18 patients with leukemia (CML or ALL) showed that leukemic tumors occurred (4/18 cases) after thawed human ovarian cortical tissue was xenografted to mice [15
]. Therefore, patients with leukemia and other tumors which involve the ovaries are counseled that cryopreserved ovarian tissue should not be used in the future for transplantation. In order to achieve pregnancy without transplantation, it would be ideal if oocytes could be matured and fertilized in vitro and embryos transferred to a woman in order for her to achieve pregnancy after cancer treatment. This technology has been successful in the mouse model and advancements have been made in the primate [16
]. Ongoing studies are being conducted to move this technology forward. While auto transplantation of cryopreserved ovarian tissue also has the potential benefit of restoring temporary endocrine function to cancer survivors who develop premature ovarian failure, the duration of endocrine function is very limited [17
Fertility preservation technologies present a variety of ethical challenges and uncertainty, particularly in pediatric patients [18
]. Issues surrounding consent are particularly important to consider, since decisions being made involve a child’s future reproductive desires [19
]. Parents must consider whether to pursue fertility preservation techniques at all, and if so, also determine the disposition of the reproductive tissues in case of death. When minors are sufficiently mature to understand the risks and benefits of the procedure, they must be involved in the process of assent [20
]. Moreover, because fertility preservation techniques such as OTC and oocyte cryopreservation are still considered experimental [7
], enrolling minors in clinical studies presents additional challenges. Protecting vulnerable populations is paramount, and IRB’s at most institutions will only allow studies in which the expected direct benefits of the experimental treatment outweigh its risks [21
]. Because of concerns regarding the protection of minors, our experimental protocol in children was limited to ovarian biopsy only (rather than oophorectomy) in patients planning to receive treatments at the highest risk for permanent gonadotoxicity. In addition, it was initially stipulated that subjects between the ages of 10 and 12 were eligible for the procedure only if they were undergoing a concomitant surgical procedure under anesthesia. Recently, after a series of reports of human births from OTC and transplantation were reviewed, the eligible age range was expanded and the procedure allowed as a stand-alone surgery in at any age (although combining with clinical care is always the preference). It is expected that the inclusion criteria and study procedures for experimental protocols in children will vary somewhat by institution depending on the human subjects concerns at the site. The role of the child advocate should be highlighted here as an effective mechanism for ensuring that children, both those who assent and those who are not capable of assent, are protected.
Cost is a major barrier to accessing many fertility preserving technologies and will likely be an impediment to widespread application of OTC. Although OTC was performed at no cost to patients at our institutions because it was being done within the context of a research protocol, charges and costs of OTC vary. Coordinating OTC at the time of other surgical procedures can not only reduce the risk of the procedure, but also decrease the overall cost in terms of charges for general supplies, OR time and anesthesia costs. Nonetheless, combined procedures are not always possible and there must be a mechanism in place to cover the costs associated with OTC. Advocates of insurance coverage for fertility preservation argue that oncofertility services are different from services for infertile couples because the subsequent infertility is directly caused by medical treatments. For example, fertility preservation services can be likened to reconstructive breast surgery, which is covered for individuals who require a mastectomy for the treatment of breast cancer but not for individuals who desire the procedure for cosmetic purposes [22
]. Still, many insurance companies do not cover fertility preservation services. Research is now beginning to explore the willingness of individuals to pay for ovarian tissue cryopreservation and evidence suggests that OTC is significantly valued [23
]. As evidence of the utility of the use of ovarian tissue to restore fertility continues to build in terms of the numbers of live births, insurers may begin to appreciate this technique and include it within their cadre of covered services.
There are still many unanswered questions about the safety, efficacy, and application of ovarian tissue cryopreservation and subsequent transplantation. For example, it is not yet clear whom we should target for this technology, how much cortical tissue to remove, how to best cryopreserve the tissue, whether the technology is effective in prepubertal females, and how to minimize the risk of transplanting occult malignant cells. It is also important to develop a better understanding of patient and parental beliefs about fertility at the time of diagnosis and the major decision making influences when choosing whether or not to pursue fertility preservation options like OTC. While more research is clearly needed to answer these questions, reports of success from OTC provide hope for cancer survivors without other options to have biological children after treatment. Expanding post-treatment reproductive options will no doubt improve the overall long term quality of life of cancer survivors.