Effects of malignancies on fertility preservation outcomes and relevant cryobiological advances

A decrease in cancer deaths has resulted in the possibility of child bearing for many young adult cancer survivors. Most antitumor treatment modalities are detrimental to female fertility, and methods for fertility preservation before gonadotoxic treatment, including cryopreservation of oocytes, embryos and ovarian tissue, have therefore been developed. This review focuses on the ovarian function of cancer patients, the safety and efficacy of fertility preservation methods, and the pregnancy outcomes of these patients. Breast cancer and hematological tumors constitute the majority of cancers in reproductive-aged female oncology patients. Ovarian function may not be impacted by breast cancer cells, while in patients with hematological malignancies, decreases in anti-Müllerian hormone and antral follicle counts have been demonstrated. In most cases, patients can undergo ovarian stimulation without delaying treatment, and a new stimulation protocol known as dual stimulation, which may be more efficient, has now been developed. Birth outcomes are also acceptable in cancer patients.     

Global uptake of fertility preservation by women undergoing cancer treatment: An unmet need in low to high-income countries

Although the incidence of cancers is on the rise globally, mortality has continued to decrease due to advances in early detection and treatment. Cancer treatments such as chemotherapy and radiotherapy can impact the reproductive capacity of survivors by inducing premature ovarian failure and subsequent infertility causing significant psychological distress with decreased quality of life. Despite the increasing need for fertility preservation services for the rising number of cancer survivors and the recent advances in assisted reproductive technology, many women with cancers in low, middle, and to a lesser extent, high-income countries have no access to these services. This article, therefore, presents an overview of the effect of cancer treatment on fertility, options of fertility preservation, and factors influencing fertility preservation utilization by women who had a cancer diagnosis. In addition, we discuss the availability, practices, and outcomes of fertility preservation services in low, middle, and high-income countries and highlight pragmatic steps to improving access to oncofertility care for women with cancers globally.     

Radiations and female fertility

Hundreds of thousands of young women are diagnosed with cancer each year, and due to recent advances in screening programs, diagnostic methods and treatment options, survival rates have significantly improved. Radiation therapy plays an important role in cancer treatment and in some cases it constitutes the first therapy proposed to the patient. However, ionizing radiations have a gonadotoxic action with long-term effects that include ovarian insufficiency, pubertal arrest and subsequent infertility. Cranial irradiation may lead to disruption of the hypothalamic-pituitary-gonadal axis, with consequent dysregulation of the normal hormonal secretion. The uterus might be damaged by radiotherapy, as well. In fact, exposure to radiation during childhood leads to altered uterine vascularization, decreased uterine volume and elasticity, myometrial fibrosis and necrosis, endometrial atrophy and insufficiency. As radiations have a relevant impact on reproductive potential, fertility preservation procedures should be carried out before and/or during anticancer treatments. Fertility preservation strategies have been employed for some years now and have recently been diversified thanks to advances in reproductive biology. Aim of this paper is to give an overview of the various effects of radiotherapy on female reproductive function and to describe the current fertility preservation options.     

Intraovarian Transplantation of Female Germline Stem Cells Rescue Ovarian Function in Chemotherapy-Injured Ovaries

Early menopause and infertility often occur in female cancer patients after chemotherapy (CTx). For these patients, oocyte/embryo cryopreservation or ovarian tissue cryopreservation is the current modality for fertility preservation. However, the above methods are limited in the long-term protection of ovarian function, especially for fertility preservation (very few females with cancer have achieved pregnancy with cryopreserved ovarian tissue or eggs until now). In addition, the above methods are subject to their scope (females with no husband or prepubertal females with no mature oocytes). Thus, many females who suffer from cancers would not adopt the above methods pre- and post-CTx due to their uncertainty, safety and cost-effectiveness. Therefore, millions of women have achieved long-term survival after thorough CTx treatment and have desired to rescue their ovarian function and fertility with economic, durable and reliable methods. Recently, some studies showed that mice with infertility caused by CTx can produce normal offspring through intraovarian injection of exogenous female germline stem cells (FGSCs). Though exogenous FGSC can be derived from mice without immune rejection in the same strain, it is difficult to obtain human female germline stem cells (hFGSCs), and immune rejection could occur between different individuals. In this study, infertility in mice was caused by CTx, and the ability of FGSCs to restore ovarian function or even produce offspring was assessed. We had successfully isolated and purified the FGSCs from adult female mice two weeks after CTx. After infection with GFP-carrying virus, the FGSCs were transplanted into ovaries of mice with infertility caused by CTx. Finally, ovarian function was restored and the recipients produced offspring long-term. These findings showed that mice with CTx possessed FGSCs, restoring ovarian function and avoiding immune rejection from exogenous germline stem cells.     

Fertility preservation and preimplantation genetic assessment for women with breast cancer

Breast cancer is the most common cancer diagnosed among reproductive aged women, and its treatment can compromise future fertility. Options for fertility preservation include oocyte or embryo cryopreservation after ovarian stimulation (OS), which are the most established choices and are applicable for adult women with cancer. Ovarian tissue freezing may also be appropriate, as it offers potentially the least delay. The recognisation of the role of BRCA1 and BRCA2 mutations in some women has led to the involvement of preimplantation genetic diagnosis (PGD), recently renamed preimplantation genetic testing for monogenic disorder (PGT-M), whereby embryos are created by IVF and cell(s) are removed and genetically analyzed for specific disease-related mutations. PGT-M offers a valid option for women wishing to avoid transmission of the predisposition for hereditary breast cancer to their offspring. The aim of this paper is to provide an overview of the factors that influence fertility preservation in newly diagnosed breast cancer patients, and to illustrate the option of PGT-M to enable conception of an unaffected child.     

Retrievable hydrogels for ovarian follicle transplantation and oocyte collection

Cancer survivorship rates have drastically increased due to improved efficacy of oncologic treatments. Consequently, clinical concerns have shifted from solely focusing on survival to quality of life, with fertility preservation as an important consideration. Among fertility preservation strategies for female patients, ovarian tissue cryopreservation and subsequent reimplantation has been the only clinical option available to cancer survivors with cryopreserved tissue. However, follicle atresia after transplantation and risk of reintroducing malignant cells have prevented this procedure from becoming widely adopted in clinics. Herein, we investigated the encapsulation of ovarian follicles in alginate hydrogels that isolate the graft from the host, yet allows for maturation after transplantation at a heterotopic (i.e., subcutaneous) site, a process we termed in vivo follicle maturation. Survival of multiple follicle populations was confirmed via histology, with the notable development of the antral follicles. Collected oocytes (63%) exhibited polar body extrusion and were fertilized by intracytoplasmic sperm injection and standard in vitro fertilization procedures. Successfully fertilized oocytes developed to the pronucleus (14%), two‐cell (36%), and four‐cell (7%) stages. Furthermore, ovarian follicles cotransplanted with metastatic breast cancer cells within the hydrogels allowed for retrieval of the follicles, and no mice developed tumors after removal of the implant, confirming that the hydrogel prevented seeding of disease within the host. Collectively, these findings demonstrate a viable option for safe use of potentially cancer‐laden ovarian donor tissue for in vivo follicle maturation within a retrievable hydrogel and subsequent oocyte collection. Ultimately, this technology may provide novel options to preserve fertility for young female patients with cancer.     

Cryopreservation of Human Ovarian Tissue

New and often aggressive treatment schemes allow the successful healing of many young patients with cancer, but the price the young women have to pay is high: many of them lose ovarian function and fertility. Due to the improved long-term survival of adolescents and young women with malignancies undergoing gonadotoxic chemotherapy, preservation of future fertility has been the focus of recent ubiquitarian interest. A feasible solution is the cryopreservation of ovarian tissue. Ovarian tissue, after thawing, can be used in three different ways: 1. grafted into its normal site (orthotopic); 2. grafted into a site other than its normal position (heterotopic), necessitating recourse to in vitro fertilization (IVF); 3. grown and in vitro matured in order to obtain metaphase II oocytes for an IVF program. It is believed that protein supplementation, in cryopreservation solution, is essential for improving ovarian tissue cryopreservation. The aim of this study was to evaluate the ultrastructural appearance of human ovarian tissue cryopreserved in 1.5 M 1,2 propanediol (PROH), 0.2 M sucrose using different protein sources: fetal calf serum (FCS), plasmanate or syntetic serum substitute (SSS). Fresh and frozen/thawed ovarian tissues were compared by transmission electron microscope (TEM), to evaluate the appearance of stromal and follicle cells as affected by different protein sources. Our data indicate that FCS is a better protein support for ovarian tissue cryopreservation when compared to SSS or Plasmanate. In addition the follicles are more resistant to the cryopreservation with respect to stroma.     

Cryopreservation/transplantation of ovarian tissue and in vitro maturation of follicles and oocytes: Challenges for fertility preservation

Cryopreservation of ovarian tissue and in vitro follicle maturation are two emerging techniques for fertility preservation, especially in cancer patients. These treatment regimes are opening up more options and allow for more suitable choices to preserve fertility according to the patient's specific circumstances. If these technologies are to become widely accepted, they need to be safe, easy to perform and must obtain favorable results. The generation of healthy eggs with the normal genetic complement and the ability to develop into viable and healthy embryos requires tight regulation of oocyte development and maturation. Novel freezing techniques such as vitrification, along with whole ovary cryopreservation and three-dimensional follicle cultures, have shown favorable outcomes. The scope of this article is to take a comprehensively look at the challenges still faced in order for these novel technologies to be routinely employed with the aim of successful fertility preservation.     

Variability in the Practice of Fertility Preservation for Patients with Cancer

Fertility is important to women and men with cancer. While options for fertility preservation (FP) are available, knowledge regarding the medical application of FP is lacking. Therefore we examined FP practices for cancer patients among reproductive endocrinologists (REs). A 36 item survey was sent to board-certified REs. 98% of respondents reported counseling women with cancer about FP options. Oocyte and embryo cryopreservation were universally offered by these providers, but variability was noted in reported management of these cases—particularly for women with breast cancer. 86% of the respondents reported using letrozole during controlled ovarian stimulation (COS) in patients with estrogen receptor positive (ER+) breast cancer to minimize patient exposure to estrogen. 49% of respondents who reported using letrozole in COS for patients with ER+ breast cancer reported that they would also use letrozole in COS for women with ER negative breast cancer. Variability was also noted in the management of FP for men with cancer. 83% of participants reported counseling men about sperm banking with 22% recommending against banking for men previously exposed to chemotherapy. Overall, 79% of respondents reported knowledge of American Society for Clinical Oncology FP guidelines—knowledge that was associated with providers offering gonadal tissue cryopreservation (RR 1.82, 95% CI 1.14–2.90). These findings demonstrate that RE management of FP in cancer patients varies. Although some variability may be dictated by local resources, standardization of FP practices and communication with treating oncologists may help ensure consistent recommendations and outcomes for patients seeking FP.     

Fertility Preservation Options for Men and Women With Cancer

Approximately 0.2% of Americans aged 20 to 39 years are childhood cancer survivors. Advances in cancer detection and therapy have greatly improved survival rates for young cancer patients; however, treatment of childhood cancers can adversely impact reproductive function. Many cancer patients report a strong desire to be informed of existing options for fertility preservation and future reproduction prior to initiation of gonadotoxic cancer therapies, including surgery, chemotherapy, and radiotherapy. This article discusses, in detail, the effects of cancer treatment on fertility in men and women, and outlines both current and experimental methods of fertility preservation among cancer patients.Key words: Fertility preservation, Childhood cancer, Sperm cryopreservation, Testicular tissue cryopreservation, Spermatogonial stem cell cryopreservation, Embryo cryopreservation, Oocyte cryopreservation, Ovarian tissue cryopreservationIn 2014, an estimated 15,780 new cancer cases were diagnosed among children and adolescents younger than age 20 years, resulting in 1960 deaths. In addition, 1 in 285 children will be diagnosed with cancer before age 20, and approximately 0.2% of Americans aged 20 to 39 years are childhood cancer survivors.¹ Advances in cancer detection and therapy have greatly improved survival rates for young cancer patients; however, treatment of childhood cancers can adversely impact reproductive function (eg, men who survive childhood cancer are half as likely as their siblings to father a child).² Many cancer patients report a strong desire to be informed of existing options for fertility preservation and future reproduction.³ Therefore, the American Society of Clinical Oncology and the American Society for Reproductive Medicine recommend that consideration of fertility preservation be included prior to initiation of gonadotoxic cancer therapies, including surgery, chemotherapy, and radiotherapy.^4–6Infertility as a result of cancer treatment can be psycho logically upsetting for many patients,^3,7,8 and data suggest that those who pursued fertility preservation usually cope better with their cancer treatment.⁹ Infertile cancer survivors have an option to become parents through adoption or gamete donation, but most declare a preference for having a biological child.^3,10 Schover and colleagues³ found that 51% of newly diagnosed young male cancer patients reported a desire to have children in the future, and this rate increased to 77% for those who did not have children at the time of diagnosis. The desire to become a biological parent persists in male cancer survivors, as 70% reported wanting to father a child after chemotherapy treatment.⁹ A history of cancer treatment may be perceived by some to pose an increased risk to the health of future offspring; however, several studies have shown that male cancer survivors have not demonstrated an increased risk for having a child with birth defects or cancer.^11,12 Recently, a retrospective cohort study conducted in the United States showed no increased risk of malformations or premature birth in the offspring of male cancer survivors.¹³The optimal time for consideration of fertility preservation is before the initiation of any oncologic therapy that can affect gametogenesis; thus, it is critical that fertility preservation is discussed with all patients at the time of diagnosis and before treatment starts. Practitioners who provide care for cancer patients should be aware of the relationship between cancer treatment and infertility. Moreover, they need to be able to appropriately refer patients to a reproductive medicine specialist in a timely fashion for further counseling and fertility preservation. Although fertility concerns are paramount to young adults with cancer, many oncologists still do not routinely address these concerns.^3,14 In a survey of 200 young male cancer survivors who were primarily treated at a comprehensive cancer center, only 51% recalled being offered sperm cryopreservation prior to their cancer treatment.³ Further, it is important to recognize the psychologic stressors associated with a new cancer diagnosis and associated late effects of cancer treatment, such as infertility or early menopause. Findings from several studies support the importance of counseling patients regarding their risk for fertility issues and educating providers regarding the potential fertility preservation options that are available. For example, Babb and colleagues¹⁵ found that, at many institutions, this counseling is already taking place and there is a high rate of discussion with newly diagnosed patients regarding infertility.     

Human oocyte morphometry before and after cryopreservation: A prospective cohort study

The cryopreservation of human oocytes is an important strategy to spare fertility in women submitted to gonadotoxic therapy, ovarian surgery, or even to allow gestation by assisted reproduction technology after natural ovarian senescence. Methods to predict oocyte resistance to cryopreservation are still based on qualitative morphological assessment. In this study we evaluated whether morphometric characteristics of mature oocytes before vitrification and after warming are related to successful fertilization by intracytoplasmic sperm injection (ICSI). This was a prospective cohort study including 28 infertile women and 71 oocytes. Morphometric assessments included oocyte diameter, perivitelline space (PS), zona pellucida (ZP) and first polar body (PB). Out of 49 warmed oocytes, 27 (55%) survived cryopreservation and their pre-vitrification measures were similar to those of the 22 oocytes that perished. However, the oocytes that eventually failed to be fertilized had undergone more enlargement of the total diameter (p = 0.029) and shrinking of the PS (p = 0.033) after cryopreservation, compared to oocytes that were successfully fertilized. These findings suggest that the morphometric characteristics of fresh oocytes do not predict their survival to vitrification, while fertilization failure is associated with oocyte enlargement and PS shrinking after cryopreservation.     

Restoration of fertility after treatment for cancer

The late effects of chemotherapy and radiation treatment on fertility are an important issue for long-term survivors of cancer who may not have started or completed a family at the time of diagnosis. Attempts at protecting reproductive function using hormonal manipulation have proved largely unsuccessful and other strategies have to be considered. For men, semen cryopreservation allows subsequent artificial insemination of a female partner or ivf but cryopreserved semen is a finite resource, does not allow natural conception and is not an option for prepubertal boys. In an effort to overcome this, research is in progress to investigate whether testicular cells harvested and cryopreserved before the start of chemotherapy can be reintroduced to the testis after treatment and resume normal spermatogenesis. This has been achieved in a mouse model and the results of experimental protocols in men are awaited with interest. For women, harvested mature oocytes are only poorly tolerant of the freezing process although immediate in vitro fertilization and cryopreservation of embryos can be successful. An experimental technique of great interest is the harvesting and cryopreservation of ovarian cortex before the start of sterilizing treatment. In ewes, the reimplantation of autologous ovarian cortical tissue into surgically castrated animals has resulted in resumption of oestrus, conceptions after normal matings and the birth of live offspring. Recently, ovarian function has been re-established using a similar technique in a patient following treatment for Hodgkin's lymphoma, but so far pregnancy has not been reported.     

Short‐term rapamycin treatment increases ovarian lifespan in young and middle‐aged female mice

Although age‐related ovarian failure in female mammals cannot be reversed, recent strategies have focused on improving reproductive capacity with age, and rapamycin is one such intervention that has shown a potential for preserving the ovarian follicle pool and preventing premature ovarian failure. However, the application is limited because of its detrimental effects on follicular development and ovulation during long‐term treatment. Herein, we shortened the rapamycin administration to 2 weeks and applied the protocol to both young (8 weeks) and middle‐aged (8 months) mouse models. Results showed disturbances in ovarian function during and shortly after treatment; however, all the treated animals returned to normal fertility 2 months later. Following natural mating, we observed prolongation of ovarian lifespan in both mouse models, with the most prominent effect occurring in mice older than 12 months. The effects of transient rapamycin treatment on ovarian lifespan were reflected in the preservation of primordial follicles, increases in oocyte quality, and improvement in the ovarian microenvironment. These data indicate that short‐term rapamycin treatment exhibits persistent effects on prolonging ovarian lifespan no matter the age at initiation of treatment. In order not to disturb fertility in young adults, investigators should in the future consider applying the protocol later in life so as to delay menopause in women, and at the same time increase ovarian lifespan.     

Orthotopic Transplantation of Cryopreserved Mouse Ovaries and Gonadotrophin Releasing Hormone Analogues in the Restoration of Function following Chemotherapy-Induced Ovarian Damage

Therapy advances are constantly improving survival rates of cancer patients, however the toxic effects of chemotherapy drugs can seriously affect patients’ quality of life. In women, fertility and premature ovarian endocrine dysfunction are of particular concern. It is urgently we find methods to preserve or reconstruct ovarian function for these women. This study compares GnRHa treatment with ovarian tissue cryopreservation and orthotopic transplantation in a chemotherapy-induced ovarian damage murine model. 56 inbred Lewis rats were divided into 4 treatment groups: Saline control (group I); cyclophosphamide only (group II); cyclophosphamide plus GnRHa (group III); cyclophosphamide and grafting of thawed cryopreserved ovaries (group IV). Body weight, estrous cycle recovery time, ovarian weight, morphology and follicle count, as well as breeding and fertility were compared among groups. Only group IV was able to restore to normal body weight by the end of the observation period and resumed normal estrous cycles in a shorter time compared to other treatment groups. There was a decrease in primordial follicles in all treatment groups, but group III had the greatest reduction. Although, there was no difference in pregnancy, only one animal littered normal pups in group II, none littered in group III and four littered in group IV. Thus, cryopreservation and orthotopic transplantation of ovarian tissue can restore the fertility of rats subjected to chemotherapy in a manner that is superior to GnRHa treatment. We also observed increased rates of hepatic, splenic and pulmonary haemorrhage in group III, suggesting there may be synergistic toxicity of GnRHa and cyclophosphamide.     

Optimizing methods for human testicular tissue cryopreservation and spermatogonial stem cell isolation

Cryopreservation of testicular tissue before cancer therapy for fertility preservation in prepubertal boys with cancer is of great interest in reproductive medicine. Isolation of spermatogonial stem cells (SSCs) from cryopreserved tissues would be a suitable cell source to re-establish spermatogenesis after cancer therapy. We herein establish optimized protocols for cryopreservation of human testicular tissue and isolation of SSCs from cryopreserved tissue. We developed a freezing protocol that provided high testicular cell viability and supported structural integrity and tubular epithelium coherence similar to fresh tissue. Then, we established a protocol that allowed efficient isolation of functional SSCs from cryopreserved tissues. Isolated cells were found on the testicular basement membrane after xenotransplantation. Our results demonstrated the preservation of testicular tissue structure and high cell viability with efficient isolation of SSCs after testicular cryopreservation, which is promising for future therapeutic applications in fertility preservation.     

Premature ovarian failure

The diagnosis of premature ovarian failure is based on the finding of amenorrhoea before age 40 associated with follicle-stimulating hormone levels in the menopausal range. Screening for associated autoimmune disorders and karyotyping, particularly in early onset disease, constitute part of the diagnostic work up. There is no role for ovarian biopsy or ultrasound in making the diagnosis. Management essentially involves hormone replacement and infertility treatment, the most successful being assisted conception with donated oocytes. Embryo cryopreservation, ovarian tissue or oocyte cryopreservation and in vitro maturation of oocytes hold promise in cases where ovarian failure is foreseeable as in women undergoing cancer treatments.     

Nano-Encapsulation of Arsenic Trioxide Enhances Efficacy against Murine Lymphoma Model while Minimizing Its Impact on Ovarian Reserve In Vitro and In Vivo

Advances in cancer therapy have increased the rate of survival of young cancer patients; however, female lymphoma patients frequently face a temporary or permanent loss of fertility when treated with traditional cytotoxic agents. The potential loss of fertility is an important concern that can influence treatment decisions for many premenopausal cancer patients. The negative effect of chemotherapeutic agents and treatment protocols to patients’ fertility–referred to as fertotoxicity–are thus an increasingly important cancer survivorship issue. We have developed a novel nanoscale formulation of arsenic trioxide, a potent drug for treatment of hematological malignancies, and demonstrate that it has significantly better activity in a murine lymphoma model than the free drug. In parallel, we have developed a novel in vitro assay of ovarian follicle function that predicts in vivo ovarian toxicity of therapeutic agents. Our results reveal that the nanotherapeutic agent is not only more active against lymphoma, but is fertoprotective, i.e., it is much less deleterious to ovarian function than the parent drug. Thus, our in vitro assay allows rapid evaluation of both established and experimental anticancer drugs on ovarian reserve and can inform the selection of efficacious and fertility-sparing treatment regimens for reproductive-age women diagnosed with cancer.     

Is there a clinical future for spermatogonial stem cells?

Like every other adult stem cell in the human body, spermatogonial stem cells (SSCs) have the capacity to either renew themselves or to start the differentiation process, namely, spermatogenesis. Due to the continuation of the stem cell population in the testis, several possible options for preservation and re-establishment of the reproductive potential exist. Currently, spermatogonial stem cell transplantation (SSCT) is considered the most promising tool for fertility restoration in young cancer patients. This technique involves the injection of a testicular cell suspension from a fertile donor into the testis of an infertile recipient. Although, SSCT could prove important for fertility preservation, this technique is not without any risk. Testicular cell suspensions from cancer patients may be contaminated with cancerous cells. It is obvious that reintroduction of malignant cells into an otherwise cured patient must be omitted. Decontamination strategies to solve this problem are discussed. Another alternative to preserve male fertility could be in-vitro culture of SSCs. This approach may be applied to generate spermatozoa in-vitro from cultured spermatogonial stem cells, which, in turn, could be used for intracytoplasmic sperm injection. Xenogeneic transplantation and xenografting are two other hypothetical methods to preserve fertility. However, because of the ethical and biological concerns inherent to these approaches, xenogeneic transplantation and xenografting should be limited to research. When SSCT or SSC culture becomes available for clinical use, efficient protocols for the cryopreservation of SSCs and testicular tissue will be of great benefit. The search for an optimal freezing protocol is discussed. Apart from fertility preservation, SSC studies are useful for other applications as well, such as transgenerational gene therapy and cell-based organ regeneration therapy.     

Impact of prepubertal bovine ovarian tissue pre-freeze holding duration on follicle quality

Ovarian tissue cryopreservation prior to gonadotoxic treatment is the only recommended option for fertility preservation in prepubertal girls. Due to the technical complexity of this technique, limited number of centres across the world are equipped to offer the facility. Hence, the retrieved ovarian tissue needs to be maintained at hypothermic temperature (4 °C) for long time during shipment. The time taken between tissue retrieval and cryopreservation could influence the functionality of cells during fertility restoration. This study explored the tissue integrity and follicle quality of ovarian cortical slices subjected to pre-freeze holding for various time durations in vitro. Prepubertal bovine ovarian tissue from < 12 months old animals were handled at hypothermic holding (4 °C) for 0, 24, 48 and 72 h. The tissues were assessed for follicle viability through confocal analysis of live-dead labelled samples, and follicle quality and tissue integrity through histology. Results have shown that follicle viability, and overall follicle quality were not significantly affected at the end of 72 h hypothermic holding. Though, the observation reassures extended hypothermic holding prior to freezing, findings need to be validated in human tissue prior to use in clinical fertility preservation programs.