Every year around the world, more than two million women are diagnosed with cancer, with breast cancer being the most common women’s cancer worldwide, followed by cervical cancer, ovarian cancer and uterine cancer. Epidemiological evidence clearly shows that women surviving cancer during childhood, adolescence or young adult life have a significantly lower probability of having children compared with healthy women of the same age range [1]. Some antineoplastic treatments (chemotherapy, radiotherapy, or surgery), in fact, may impair ovarian function in different ways, leading to an increased risk of irreversible infertility due to premature ovarian insufficiency (POI), defined as oligo/amenorrhea for ≥4 months and follicle-stimulating hormone (FSH) levels > 25 IU/L on two occasions, four weeks apart, before the age of 40 years. In some malignancies, such as breast cancer, leukemia, lymphomas, neuroblastoma, etc., oncological treatments performed to control the disease are quite harmful for the ovary, and increase the risk of POI [2,3]. The onset of POI after anticancer treatment depends not only on the type of therapy, but also on the patient’s age and the ovarian follicular reserve (OR) at the time of treatment [4,5]. The American Society of Clinical Oncology (ASCO) classified chemotherapy drugs in relation to their ovarian toxicity, assigning the highest risk (>80%) to adjuvant breast cancer poli-chemotherapy with CMF (Cyclophosphamide, Methotrexate, Fluorouracil), CEF (Cyclophosphamide, Epirubicin, Fluorouracil) and CAF (Cyclophosphamide, Adriamycin, Fluorouracil). Indeed, alkylating agents are quite toxic for the ovary, being able to induce progressive and bulk OR depletion, particularly when administered for six months to women ≥ 40 years; in these cases, the defense of OR by gonadotropin-releasing hormone analogues (GnRH-a) is only partially effective in limiting ovarian damage [6,7]. Similarly toxic for the ovary are total body irradiation (TBI) and radiotherapy including the ovaries in the irradiation field: patients submitted to TBI or pelvic irradiation at high dose have a higher than 90% risk of POI development [8]; even in this case, ovarian transposition aimed at avoiding the direct irradiation of the ovaries is often only partially effective [9]. Within the ovarian follicle, both oocyte and granulosa cells are vulnerable to the damage caused by chemotherapy. Each class of chemotherapeutic agents may have different mechanisms of action on cancer cells, with the end result being to affect the cell cycle. Histological studies on human ovaries have shown that chemotherapy can cause the loss of primordial follicles and ovarian atrophy; oocyte death due to induced apoptosis was identified as the main mechanism responsible for the loss of germ cells, but also injury to blood vessels and focal ovarian cortical fibrosis may contribute to generate the detrimental consequences of cytotoxic therapies [10]. Several fertility preservation options ranging from routine to experimental strategies are now available to counteract the treatment-related infertility risk. Both the American Society of Clinical Oncology (ASCO) and the European Society for Medical Oncology (ESMO) discuss the application of administration of GnRH analogues, ovarian transposition, surgical methods of fertility protection performed before anticancer treatment or oocyte in vitro maturation (IVM) and recommend the cryopreservation of oocyte and embryo as the mostly employed procedures able to guarantee motherhood in post-puberal female cancer survivors [11,12]. Although these cryopreservation procedures appear effective in terms of successful pregnancies in healthy women (nearly 45% for cryopreserved oocytes and 30% for embryos) [13], the preparative controlled ovarian stimulation is not suitable in cases of pre-puberal girls as well as in circumstances requiring urgent anticancer treatments as for hematologic malignancies, which cannot be retarded in relation to the time necessary to induce multifollicular growth.

Ovarian tissue cryostorage is a valuable option for fertility preservation in case of women who urgently need chemotherapy, as in neoadjuvant protocols [14]. The major advantage of using cryopreserved cortex includes its feasibility independent from the menstrual cycle, also in prepubertal girls, and without the need of a precise OR evaluation, as the ovarian cortex always contains a relevant number of follicles. Ovarian biopsy is usually performed by laparoscopy taking approximately one third of both ovaries: a sharp surgical blade is used to gently remove the medulla and cutting the remaining ovarian cortex into pieces of approximately 5 × 5 × 1–2 mm that allow cryoprotective agents (CPAs) to quickly penetrate into the tissue and reduce the damage exerted by low temperature on of the follicle pool. Although slow freezing has been the conventional cryopreservation technique for years, extensive loss of follicles and damage to stromal cells have been reported [15]. To optimize the freezing strategies, vitrification has been proposed as an alternative procedure; this method is based on the induction of ultra-rapid cooling in the presence of high concentration of cryoprotectants, and is apparently functional in preventing cell injury by producing a glass-like amorphic state, as well as in maintaining stromal integrity similar to fresh tissue [16,17,18]. Furthermore, Diaz-Garcia et al., evaluated the live birth rate following oocyte vitrification with respect to OTC and autologous transplantation in oncological patients undergoing gonadotoxic treatments, and reported that despite slightly higher results achieved after oocyte freezing, ovarian tissue vitrification should be considered an alternative, good option when oocyte cryostorage is not feasible [19]. Interestingly, a recently published systematic review and meta-analysis has reported a significantly greater proportion of intact stromal cells in vitrified tissue versus slow-frozen tissue, with no significant differences with respect to the proportion of intact primordial follicles, extent of DNA fragmentation, or mean primordial follicle density [20]. Conversely, some reports suggested that vitrification may induce changes in mRNA expression and delayed growth of follicles in vitro in human ovarian tissues [21,22].

More recently, utilization of slush nitrogen has been proposed to further improve vitrification efficacy; morphology, ultrastructure and viability of both follicles and stromal cells are apparently better preserved in defrosted components, as compared with the same procedure using liquid nitrogen [23,24]. However, the worldwide practice is still predominantly the slow freezing of ovarian tissue and only four children have been born after vitrification.

Ovarian tissue transplantation (OTT) after thawing can be performed either at heterotopic sites outside the pelvis (subcutaneous tissue of the forearm or of the lower abdomen, sovrapubic subperitoneal space, or subfascial space of the abdominal rectus or of the pectoralis muscle), or at orthotopic sites (the remnant atrophic ovary, the broad ligament, the serosa of Fallopian tubes, or the pelvic wall at the level of ovarian fossa) [25]. The grafting site may likely influence the efficacy of the procedure. Most pregnancies (more than 120) were reported after orthotropic grafting [26], which is by far more frequently adopted than heterotopic, and is preferred because it allows also spontaneous conception. After thawed ovarian tissue grafting, the transplanted tissue undergoes neovascularization due to the local production of proangiogenetic factors; as soon as the blood supply takes place, folliculogenesis begins inside the transplanted fragments; estrogenic levels begin slowly to rise, with a consequent decrease in circulating FSH. This process takes about 10–15 weeks to be clinically detectable, and after this time menstruation may restore. This happens in a fairly high proportion of cases, ranging from 67.3% to 93.7% [27,28,29]. Despite the high rate of follicular loss due to post-grafting ischemia, the number of primordial and primary follicles in the grafted tissue is so high that those who survive to ischemic death are enough to guarantee a remarkable endocrine activity for a few years [30,31]. The earliest case of ovarian restoration and live birth using cryopreserved human ovarian fragments was reported in 2000 [32] and, to date, more than 200 births have been reported after the autotransplantation of frozen ovarian tissue [33]. A recent meta-analysis of three centers has calculated a pregnancy rate of about 50% per patient [34] and a live birth rate of 25% [13]. Although a vast majority of women receiving autotransplantation (95%) experience the return of endocrine function, the average duration of ovarian endocrine function is approximately 2–5 years, being related to the OR at the time of OT cryostorage [35]. To increase the number of mature oocytes promptly originated after transplantation, the in vitro activation of dormant follicles (IVA) has been recently proposed [36]. The procedure includes two steps after tissue thawing: the first is based on the complete fragmentation of cortical biopsies in order to promote follicle growth and, at the same time, the progression from secondary to early antral stage by disrupting the Hippo signaling pathway [37]. The second step involves the in vitro culture of cortical pieces with a mix of both PTEN inhibitor and PI3K activator, in order to stimulate the activation of dormant primordial follicles [38]. This procedure has been reported by Suzuki et al., who successfully restored fertility in patients diagnosed with POI after auto-grafting of vitrified human ovarian tissue coupled with follicles obtained by the IVA procedure [39]. Interestingly, this fascinating strategy would fit mainly to women undergoing OTC and reimplantation in more advanced fertile age.

Although the main scientific societies (American Society for Reproductive Medicine and European Society of Human Reproduction and Embryology) have established that OTC has completed the experimental stage and should now be referred to as usual care [14,40], OTC still faces many challenges in clinical application, especially for cancer patients [41]. The frozen ovarian tissue, which is generally stored before the start of cancer treatment or after the remission induction phase, carries the risk of harboring metastasized malignant cells [42,43,44]. After thawing and subsequent transplantation of the OT, these micrometastases have shown the capacity to develop into recurrent tumors in the mouse model, indicating that the autotransplantation of frozen-thawed ovarian tissue could lead to the reintroduction of the malignancy [45]. With the rising number of OTT, the urgency to develop strategies directed at the elimination of malignant cells or to offer alternatives to autotransplantation has become even more evident [46]. A safe fertility restoration program based on OT cryopreservation and transplantation should necessarily take into account the contamination of malignant cells potentially leading to cancer recurrence after allografting.