Ovarian tissue cryopreservation in young cancer patients for fertility preservation

Abstract

Several options are currently available to preserve fertility and give female cancer survivors a chance to have children at a later date, including the cryopreservation of embryos, oocytes, and ovarian tissue. Selection of the most suitable strategy to preserve fertility depends on the type and timing of anticancer therapy, the cancer, the patient's age, and the presence of the patient's partner. Several studies have shown that the ovarian tissue can be successfully frozen and later grafted in the human womb. To date, approximately 30 live births have been achieved after the transplantation of frozen‐thawed ovarian tissue. At present, the standard procedure for cryopreservation of ovarian tissue is the slow‐cooling method. The slow‐cooling method uses an optimal cooling rate for the target cells, and relies on extracellular ice crystals to gradually dehydrate and equilibrate the tissue. Several groups reported that slow cooling is more efficient than vitrification for the cryopreservation of human ovarian tissue. However, vitrification can be performed under a variety of conditions, and therefore, the choice of methods is important. In addition, vitrification traps aqueous solutions in an amorphous, “vitreous” solid phase that prevents ice crystal formation in tissues. Vitrification methods that were developed using mice and monkey have recently been shown to improve the viability of vitrified ovarian tissues. In this review article, recent topics of ovarian tissue cryopreservation are described.

Introduction

In recent years, advances in diagnostic modalities and multidisciplinary treatment have improved the outcomes for patients with various types of cancer, and the number of cancer survivors has thus been increasing. However, anticancer chemotherapy and radiotherapy often cause premature ovarian failure in female patients. Therefore, it is important to improve the quality of life of female cancer survivors by preventing the loss of fertility. The standard methods of fertility preservation for young female patients with cancer have been the cryopreservation of gametes (oocytes) or zygotes (fertilized ova) and/or the transposition or shielding of the ovaries during radiotherapy. In 2004, the first report was published of a live birth following the cryopreservation and transplantation of ovarian tissue [1]. This technique has since been applied clinically as a new approach to fertility preservation. In Europe and North America, a new field named oncofertility (integrating oncology and reproductive medicine) has been established, and the paradigm of fertility preservation for young female cancer patients is undergoing fundamental changes. It is now 16 years since the first report of ovarian tissue cryopreservation by Donnez [1], and 10 years since he reported the first live birth after transplantation of cryopreserved ovarian tissue [2]. During that period, this technique has become an important option that should be offered to every young female cancer patient. This technique has another advantage in that it is also applicable for girls with cancer who have not yet established an ovulatory cycle, as well as that it avoids transvaginal procedures. In Japan, a group associated with Okayama University and several other institutions first performed ovarian tissue cryopreservation clinically in 2006, and this fertility preservation technique has currently been approved by the ethical committees of nine medical institutions in Japan (as of April 2014).

Ovarian tissue cryopreservation in Europe and North America (slow cooling)

Ovarian tissue cryopreservation has several advantages compared to the cryopreservation of oocytes or zygotes (fertilized ova): (1) A larger number of primordial follicles can be preserved; (2) it is possible to harvest ovarian tissue (the ovary) without considering the ovulatory cycle; (3) ovarian tissue cryopreservation is also applicable to pediatric cancer patients, in whom transvaginal procedures can rarely be performed, as well as adult female patients without a partner; (4) the transplanted ovarian tissue secretes estrogen, and thus serves as hormone replacement therapy.

According to the reports of three groups led by Donnez, Andersen, and Pellicer, respectively, autotransplantation of cryopreserved ovarian tissue has been performed in a total of 60 patients, and the ovarian tissue (or ovary) was harvested before chemotherapy in 73 % [3]. The indication for ovarian tissue cryopreservation was hematological malignancy in 35 % of these patients, other malignancies in 45 %, and nonmalignant disorders such as Turner's syndrome or a family history of premature ovarian failure in 20 %. Ovarian function was restored (with development of follicles) after autotransplantation in 93 % of the patients, while histological examination did not identify follicles in most of the patients without restoration of ovarian function [3]. The transplanted ovarian cortical slices were not thick enough to contain primordial follicles in some cases of failure. Yamada et al. [4] reported that primordial follicles were chiefly localized in the ovarian cortex zone from 0.4 to 1.0 mm beneath the tunica albuginea in females aged from 10 to 40 years. The localization of primordial follicles may vary somewhat with age, and follicles may also be present in the cortical zone deeper than 1.0 mm beneath the tunica albuginea. However, fertility can probably be preserved if the harvested ovarian cortical tissue includes the zone reaching a depth of at least 1.0 mm beneath the tunica albuginea [3, 5].

The slow‐cooling method is the current gold standard for ovarian tissue cryopreservation, since it was employed in all of the cases where live birth has been achieved after cryopreservation and transplantation, and live birth has resulted from natural pregnancy in more than half of these patients [6]. Most of the patients who became pregnant as a result of ovarian tissue cryopreservation and transplantation were under 30 years old, so the patient's age at the time of cryopreservation may be a prognostic factor in a successful pregnancy. One patient who underwent ovarian tissue cryopreservation at the age of 17 years and autotransplantation at 25 years subsequently gave birth to three children over three years. It has been reported that a period of 3.5–6.5 months (a median of 4.5 months) is required after transplantation until the 17 beta‐estradiol level increases and the follicle‐stimulating hormone level declines. This period was 3.5–4.5 months when ovarian tissue was harvested before chemotherapy and 5.5–6.5 months when ovarian tissue was harvested after chemotherapy [7]. Thus, the delay until recovery of hormone levels is suggested to depend on the number of viable follicles at the time of harvesting the ovarian tissue (ovary). Based on the above findings, it is important to perform histological examination of the harvested ovarian tissue (resected ovary) in order to determine the amount of ovarian tissue that should be thawed and the appropriateness of autotransplantation.

The slow‐cooling method of cryopreservation has not been well‐validated with regard to restoration and maintenance of hormone levels after transplantation. There have been reports of an increase in the detection of empty follicles during harvesting of ova after ovarian stimulation, as well as reduced development of the ova that are obtained [7, 8]. During the freezing process, water in the cryoprotection medium freezes and the salt concentration of the medium consequently increases, which is thought to cause damage to ovarian tissue. Formation of ice crystals may also cause physical injury to the ovarian cells and tissues, so there is a possibility that the balance between the development of oocytes and maturation of the granulosa cells may be lost in the follicular stage after cryopreserved tissue is thawed and transplanted [9]. It has recently been recognized that sodium levels influence cell membrane permeability and play a role in apoptosis. In fact, replacing sodium in the cryopreservation medium with a substance that does not cross the cell membrane has been tried in an attempt to reduce damage to the ova when ovarian tissue cryopreservation and transplantation are performed, resulting in the improvement of follicular survival [10].

Ovarian tissue cryopreservation by vitrification

Since 2009, various experiments on vitrification as a technique for ovarian tissue cryopreservation have been reported. Keros et al. [10] froze human ovarian tissue by the slow‐cooling method or vitrification and observed tissue specimens by electron microscopy, reporting that vitrified tissue showed significantly less stromal damage. In Japan, Kagawa et al. vitrified both bovine and human ovarian tissues, and then evaluated the viability of ova in the thawed tissue specimens, reporting that the ova in thawed vitrified tissues were comparable to those in fresh ovarian tissues. They then developed a vitrification device for ovarian tissue cryopreservation (Cryotissue^® Kit; Kitazato BioPharma Co., Ltd., Shizuoka, Japan) [5].

We separately developed a new device for ultra‐rapid vitrification of ovarian tissue and assessed the effect on oocytes in the preserved tissue by electron microscopy. The optimal vitrification conditions were explored by testing two different cryoprotectants [VSEGP consisting of 35 % (v/v) ethylene glycol (EG) + 5 % (w/v) polyvinylpyrrolidone (PVP) + 0.5 M sucrose and VSED consisting of 20 % (v/v) EG + 20 % (v/v) dimethyl sulfoxide (DMSO) + 0.5 M sucrose] and three freezing times (5, 10, or 20 min) [11]. We found that the histological morphology of cortical follicles and organelles were closest to normal when ovarian tissue was subjected to ultra‐rapid vitrification using a freezing time of 5 min with VSEGP as the cryoprotectant [11]. We then assessed this vitrification technique in cynomolgus monkeys and found that ovarian function was restored at a mean of 126 days after transplantation of thawed vitrified ovarian tissue [12]. In January 2010, based on these preclinical results [12], we initiated a clinical study (“Ovarian Tissue Cryopreservation and Autotransplantation for Young Female Patients with Cancer or Immunological Disease Intended for Improvement of QOL”) that was approved by the Ethical Committee of St. Marianna University School of Medicine. In December 2012, transplantation of thawed vitrified ovarian tissue in a patient with premature ovarian insufficiency successfully resulted in a live birth at our institution, which was the first live birth reported after the preservation of ovarian tissue by vitrification [13].

Amorim (from the group of Donnez) reported that the slow‐cooling and vitrification methods were comparable with respect to their effects on preantral follicles and primordial follicles, whereas vitrification was superior to slow cooling in terms of maintaining the morphology of secondary follicles and stromal cells [14]. They also performed cryopreservation of ovarian tissues from women aged 30 to 41 years by the slow‐cooling method and two vitrification methods. Then, the thawed ovarian tissues were transplanted into nude mice and the effects of cryopreservation were assessed after 1 week by investigating follicular morphology and the occurrence of apoptosis. Amorim reported that the optimum results were obtained by vitrification using an EG‐DMSO‐PVP‐sucrose cryoprotectant (Table1) [15]. They subsequently conducted an experiment in baboons using this optimum vitrification protocol. After ovarian tissue was preserved by vitrification, the residual ovarian cortex was resected and autotransplantation of the thawed ovarian tissue was performed at the anatomical site in the ovary. At 5 months after transplantation, histological examination revealed the development of follicles and corpora lutea [16]. Similarly, Ting et al. [17] assessed oocyte morphology after the vitrification of macaque ovarian tissue and concluded that it was a useful cryopreservation method. The reported compositions of the vitrification fluids used by the above‐mentioned groups for cryopreservation are shown in Table 1.

Table 1.

Composition contents of ovarian tissue vitrifaction solution: 2009–2013

Keros, Hovatta et al. [10]

0.38 M EG + 0.35 M DMSO + 0.38 M PrOH + PVP

0.75 M EG + 0.7 M DMSO + 0.75 M PrOH + PVP

1.5 M EG + 1.4 M DMSO + 1.5 M PrOH + PVP

Equilibrium time: 5 or 10 min

Kagawa, Silber et al. [5]

20 % EG + 20 % DMSO + 0.5 M sucrose

Equilibrium time: 15 min

Hashimoto, Suzuki et al. [11]

35 % EG + 5 % PVP + 0.5 M sucrose

Equilibrium time: 5 min

Amorium, Donnez et al. [15]

26 % EG + 10 % DMSO + 2.5 % PVP + 1 M sucrose

Equilibrium time: 11 min

Ting, Zelinski et al. [17]

26 % EG + 25 % glycerol + 0.2 % PVP + 0.2 % PVA + 0.4 % PG

Equilibrium time: 5 min

EG ethylene glycol, PVP polyvinylpyrrolidone, PrOH propanediol,

PVA polyvinyl acetate, PG polyglycerol

To perform cryopreservation by slow cooling, expensive equipment is required. The greatest merit of ovarian tissue vitrification is that it can be performed by using a kit in any place at any time (Cryotissue^® Kit; Kitazato BioPharma Co., Ltd., Shizuoka, Japan, Ova Cryo Device TypeM^® Kit; Kitazato BioPharma Co., Ltd., Shizuoka, Japan). However, further investigation is still required to determine whether slow cooling or vitrification is superior for ovarian tissue cryopreservation. In the USA, a randomized, controlled clinical study comparing these two methods of ovarian tissue cryopreservation has been planned and the protocol is currently being prepared (private communication from Prof. T. Woodruf of the Oncofertility Consortium).

Conclusions

The updated Adolescent and Young Adult Oncology Guidelines (for individuals aged from 15 to 39 years) of the National Comprehensive Cancer Network (http://www.nccn.org/patients/patient_guidelines/aya/index.html) state that ovarian tissue cryopreservation is still at the experimental stage clinically, as is the case with respect to oocyte cryopreservation and the use of gonadotropin‐releasing hormone for ovarian protection. Nevertheless, ovarian tissue cryopreservation can protect a large number of oocytes, and transplantation of cryopreserved ovarian tissue also has a hormone replacement effect because the transplanted ovarian tissue secretes estrogen. In addition to fertility preservation, this technique has the potential to improve estrogen deficiency symptoms, prevent cardiovascular damage associated with low estrogen levels, and mitigate the loss of bone density. Moreover, ovarian tissue cryopreservation provides mental support for young cancer patients who undergo chemotherapy and radiotherapy with various levels of fear and anxiety. It is hoped that ovarian tissue cryopreservation methods will be further optimized in the future and adopted clinically for strong ethical reasons, and that these new fertility preservation techniques become well‐known among doctors and also among patients facing early loss of fertility. Oncologists and reproductive medicine specialists should discuss the indications for and issues related to ovarian tissue cryopreservation, and it may be necessary to develop a registry and establish a system for outcome evaluation.

Conflict of interest

Dr. Suzuki declares that there is no conflict of interest.

Human Rights

All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2005. Informed consent was obtained from all patients for inclusion in the study.