Introduction

Recent reports state that cancer incidence in children, adolescents and young adults has seen a slight increase since the 1970s [1], but death rates in patients aged 0–19 years have continued to fall. Current 5-year overall survival estimates for childhood cancer exceed 83% (around 90% for most childhood hematological malignancies), translating into a growing population of adult survivors of childhood cancer [1, 2]. It is widely known that most cancer treatments like chemotherapy, radiotherapy and bone marrow transplantation are highly toxic to the gonads, putting girls and women of reproductive age at risk of premature ovarian insufficiency (POI) and subsequent infertility [3–8]. Moreover, non-oncological hematological diseases (thalassemia, aplastic anemia), autoimmune disorders (rheumatoid arthritis, systemic lupus erythematosus) [9–11] and other ovarian pathologies [8] often require treatment that may impair future fertility, exponentially increasing the number of women likely to suffer from iatrogenic menopause or POI.

Fertility preservation has emerged as a field of growing interest thanks to increasingly effective cancer screening and treatments. For young women with good prospects of surviving cancer, fertility preservation counseling before gonadotoxic treatment is imperative to offer them a potential chance of future childbearing. Several fertility preservation strategies are available for patients at risk of experiencing POI. Those that have proven most effective include: (i) embryo and oocyte cryopreservation, and (ii) ovarian tissue cryopreservation [2, 8]. Although the latter is still considered experimental, it remains the only legitimate option for prepubertal patients and women requiring immediate cancer treatment [2, 8]

Ovarian tissue cryopreservation and transplantation has gained popularity over the past decade due to its success in restoring not only fertility [8] but also ovarian endocrine function [8, 12]. Moreover, in a large series of more than 300 cases of transplantation, no evidence of reseeding malignant cells was found [12]. Indeed, after the first reported live birth in 2004 [13] and the second in 2005 [14], the number of babies born has shown a logarithmic increase, reaching more than 130 live births worldwide [8], with several ongoing pregnancies [7, 13–23].

Indications for Ovarian Tissue Cryopreservation

In contrast to oocyte and embryo cryopreservation, ovarian tissue cryopreservation for the purposes of autotransplantation does not require ovarian stimulation and can therefore be performed immediately, with no delay in cancer treatment. Furthermore, it does not require sexual maturity, which makes it the only fertility preservation method available to prepubertal girls at risk of treatment-induced POI [7, 8, 24–26]. It also restores general ovarian endocrine function [8, 12].

The most frequent indications for ovarian tissue cryopreservation in most centers are hematological malignancies, especially Hodgkin’s lymphoma and leukemia, followed by breast cancer (Figure 23.1) [17, 21, 23, 27, 28]. However, benign indications for ovarian tissue cryopreservation are also on the increase, with nonmalignant conditions also requiring treatment that may result in POI, such as systemic disorders, autoimmune diseases, bilateral benign gynecological conditions [20], borderline ovarian tumors [29], endometriosis [30] and genetic disorders like Turner syndrome [31, 32].

Figure 23.1 Indications for ovarian tissue cryopreservation Indications for ovarian tissue cryopreservation in adult women (A) and prepubertal and adolescent girls (B) in our center.

Certain selection criteria clearly need to be applied before contemplating ovarian tissue cryopreservation, as first stressed by Wallace et al. [33] and very recently emphasized by Donnez and Dolmans [8, 34], the most important being age less than 35 years (when the ovarian reserve is still relatively high), a realistic chance of surviving for 5 years, and at least a 50% risk of POI [7, 33, 35].

Harvesting Human Ovarian Tissue

Ovarian tissue cryopreservation is undertaken before chemotherapy. Written informed consent is obtained from the patient or her parents if she is under 18 years of age. It is always considered an emergency and ovarian biopsy is performed as soon as possible to avoid any delay to the start of chemotherapy.

The amount of ovarian tissue to be harvested for cryopreservation purposes varies according to the risk of POI and existing ovarian volume. Oophorectomy is usually indicated in case of pelvic radiotherapy or total body irradiation and in very young girls due to the small size of the ovaries. Otherwise, four to five fragments of tissue (10 × 5 × 1 mm) are typically recovered by laparoscopy and then processed for freezing [36]. With regard to biopsy thickness, based on the results of earlier studies [37–39], it is recommended that a 1–1.5 mm thick piece of ovarian cortex be taken [19]. This is considered to be of paramount importance, since superficial or very thin biopsies may not contain primordial follicles in the removed cortex, as primordial follicles are generally found at a distance of 0.8 mm from the mesothelium (Figure 23.2). These harvesting techniques and amounts of tissue recovered proved to be least harmful for the ovarian reserve. Indeed, taking multiple biopsies from one ovary has not been shown to compromise future hormone production [37], while unilateral oophorectomy precipitates menopause by no more than 1–2 years [40, 41].

Figure 23.2 Histology of an ovarian tissue biopsy Hematoxylin-eosin staining of an ovarian tissue biopsy of human origin showing follicles found at around 1000 µm distance from the surface/mesothelium

Processing and Freezing Human Ovarian Tissue

Harvested ovarian tissue biopsies need processing prior to cryopreservation. After collection, the tissue is transported to a clean room in order to prepare samples for freezing under laminar flow. Excess medullary tissue should be removed, and clean ovarian cortex cut into fragments of around 5−10 × 3−4 × 1 mm in strictly sterile conditions. More importantly, because the amount of tissue recovered from prepubertal patients may well be limited, medullary tissue should be cryopreserved too, since it is also a source of ovarian follicles [42]. Once cut into fragments, the samples are placed in cryovials containing freezing medium with cryoprotectant solution for storage.

Methods for ovarian tissue cryopreservation include vitrification and slow-freezing, with the latter currently the most widely accepted and implemented [43]. Slow-freezing involves exposing ovarian tissue to a solution containing low concentrations of cryoprotective agent, followed by slow-rate cooling generating extracellular ice formation (seeding) in the initial phase, which then draws out intracellular water. The slow-cooling procedure ends when the temperature is between −30°C and −80°C, after which samples are plunged into liquid nitrogen (−196°C) for storage. On the other hand, vitrification is an ultrarapid freezing technique that uses high concentrations of cryoprotectant in order to remove intracellular water before cooling starts. To achieve a glass-like state without ice crystal formation, samples can be directly plunged into liquid nitrogen for long-term storage.

Although vitrification is considered to be a more convenient method, and a number of studies have reported some advantages compared to slow-freezing, there is no evidence of its superiority in terms of clinical outcomes. Conversely, all but two live births to date have been achieved using slow-freezing for ovarian tissue cryopreservation [44]. However, since there are fewer reports on ovarian tissue cryopreservation with vitrification, only an approximate comparison can be made. Furthermore, in a review in 2011, Amorim et al. compared slow-freezing with vitrification and found that interspecies differences in ovarian morphology, as well as the diversity of freeze/thawing or warming solutions and equipment used, constitute serious limitations to the comparability of published data [45], making it almost impossible to make any conclusive statements at present. Studies must now establish whether ovarian tissue cryopreservation would benefit from switching to vitrification, like oocyte cryopreservation, or if slow-freezing should remain the gold standard thanks to all the successful clinical outcomes obtained with its use [8].

With the exception of countries like Israel [46], Denmark and Norway [23], among others, ovarian tissue cryopreservation remains experimental, but encouraging new data may prompt reconsideration of this designation in the future. Its experimental status is currently undergoing evaluation in the United States [24], as success rates of ovarian tissue transplantation are now similar to those achieved by other assisted reproductive technologies.

Ovarian Tissue Transplantation

Ovarian tissue transplantation is a procedure that involves thawing of previously recovered ovarian cortical pieces and surgically grafting the fragments back to patients. Described techniques include both orthotopic (pelvic cavity) and heterotopic (outside the pelvic cavity) sites [7].

Orthotopic Ovarian Tissue Transplantation

Orthotopic transplantation involves grafting ovarian cortical fragments to the exposed medulla of the denuded ovary or a specially created peritoneal site [20]. Orthotopic transplantation takes advantage of the anatomical position of the ovaries for possible natural conception if other conditions are met: permeable fallopian tubes, no male factor issues and restoration of ovarian function after transplantation.

The majority of reported orthotopic transplantations are carried out by minimally invasive surgery. The choice of grafting site and decision to graft in one or more locations depend on whether or not the patient previously underwent complete unilateral or bilateral oophorectomy [20]. Indeed, if at least one ovary is present, ovarian tissue may be transplanted both to the ovary after decortication and to a newly created peritoneal window. On the other hand, if no ovaries remain or what is left is severely atrophic or nonfunctional (due to the effects of high doses of radiotherapy), the only alternative for orthotopic transplantation is use of a peritoneal window [20, 47].

There are a number of variations in surgical techniques between leading teams in the field of ovarian tissue cryopreservation and transplantation worldwide.

Donnez’s Technique

When the first live birth after orthotopic transplantation of cryopreserved ovarian tissue was reported in a patient with iatrogenic POI by our team in 2004, it was a landmark in the history of ovarian tissue transplantation [13]. Our technique applied a two-step laparoscopic approach. The first laparoscopy was performed 7 days before reimplantation to create a peritoneal window by means of a large incision (1–2 cm²) using scissors just beneath the right ovarian hilus, followed by bipolar coagulation of the edges of the window. The goal was to induce angiogenesis and neovascularization in this area through formation of granulation tissue. The second laparoscopy was performed 7 days after creation of the peritoneal window and fragments of frozen-thawed ovarian tissue were pushed into the furrow created by this window, very close to the ovarian vessels and fimbria. No suture was used.

Several variations have been introduced since the first successful orthotopic transplantation, which are detailed in earlier reports [47]. There are different approaches that may be applied depending on the presence or not of the ovaries, and the amount of tissue available for thawing and autotransplantation:

1. 
   

   a. If at least one ovary is present, decortication of the ovary should be performed first. A relatively large piece of ovarian cortex (1–2 cm) is removed by means of scissors to have access to the medulla and its vascular network (Figure 23.3A). Strictly adhering to microsurgical techniques and principles, ovarian cortical pieces are then secured with 7/0 propylene stitches, or simply placed on the medulla and fixed with Interceed® and/or fibrin glue (Figure 23.3B–C).

Figure 23.3 Ovarian tissue transplantation techniques according to Donnez In patients with at least one remaining ovary: (A) Previously decorticated ovary with frozen thawed ovarian tissue fragments placed inside. (B) Fixed Interceed® covering the ovarian graft. (C) Addition of fibrin glue in order to further secure the graft. In oophorectomized patients: (D) Creation of a peritoneal window and placement of the ovarian tissue fragments inside. (E) Interceed® covering the graft. (F) Addition of fibrin glue to secure the graft. (G–H) If enough tissue is available and one ovary remains in place, transplantation can be performed in both sites (H)

1. 
   

   b. If both ovaries are absent, a peritoneal window may be created in two steps to induce angiogenesis before the grafting procedure, as in the case described earlier [13], or in one step [20]. The incision for this peritoneal window is made on the anterior leaf of the broad ligament in an area where a vascular network is visible (retroperitoneal vessels). Vessels may be easily localized by transillumination using the optical light of the instrument. Fragments are placed in the window and subsequently covered with Interceed®, the edges of which are fixed with fibrin glue (Figure 23.3D–E).

   
   
2. 
   

   c. A third option for patients with one or two ovaries still in place is grafting the tissue to both orthotopic sites simultaneously (if there is enough ovarian tissue), namely to the denuded ovary and the peritoneal window [48] (Figure 23.3G–H). With this type of transplantation, it is of utmost importance to be circumspect with amounts of tissue used, in case further reimplantations are needed in the same patient. It is recommended that only one-third of a patient’s cryopreserved tissue be thawed and grafted for each transplant.

Fixing ovarian tissue fragments to any of the grafting sites using direct stitches is not advised. Indeed, potential induction of inflammatory reactions leading to fibrosis could negatively impact the quality of follicles present at the time of grafting. In addition, considerably more handling and manipulation of thawed ovarian fragments are required when stitches are placed in such small pieces of tissue, causing further mechanical damage to follicles.

Silber’s Technique

The technique first described by Silber in 2005 [49] (Figure 23.4A) is transplantation by minilaparotomy through a 3.5-cm incision above the pubis [49–51]. Any remaining medulla from ovarian sections to be grafted should be removed before reimplantation. The cortex of each streak ovary is resected under magnification, exposing the entire raw surface of the medulla. Hemostasis inside the medulla should be meticulously controlled using microbipolar forceps and continuous irrigation with heparin-treated saline in order to prevent formation of a hematoma beneath the graft. At the same time, care should be taken to avoid impairing revascularization by minimizing cauterization. A section of ovarian cortex is laid over the raw medulla of each ovary and sutured to the medulla with 9/0 nylon interrupted stitches.

Figure 23.4 Ovarian tissue transplantation according to other teams (A) Transplantation by minilaparotomy. Cortex of each streak ovary is resected exposing the entire raw surface of the medulla. A section of ovarian cortex is laid over the raw medulla of each ovary and sutured to the medulla with 9/0 nylon interrupted stitches. Adapted from: Silber et al. Hum. Reprod. 2008. (B) Three pairs of transverse incisions made in the ovary through the tunica albuginea. With blunt dissection, cavities formed beneath the cortex for each strip. Each piece of thawed ovarian tissue gently placed in the cavities, and the incisions were closed with 4/0 Vicryl sutures. Adapted from: Meirow et al. N Engl J Med 2005. (C) Schematic representation of how the cortical strips were transplanted to the remaining post-menopausal ovary. Two incisions were made on each side of the ovary, where the strips were positioned next to one another with the cortical side facing out of the ovary.

Adapted from: Andersen et al. Hum. Reprod. 2008

Meirow’s Technique

Meirow’s transplantation procedure, first reported in 2005 [14] (Figure 23.4B), involves making three pairs of 5-mm transverse incisions in the ovary through the tunica albuginea. Blunt dissection is used to create cavities beneath the cortex for each of the strips of thawed ovarian tissue (1.5 × 0.5 cm in area; 0.1–0.2 cm in thickness), which are gently placed inside the cavities. The incisions are closed with 4/0 Vicryl sutures.

Andersen’s Technique

Andersen’s grafting procedure entails a combined laparoscopy/mini-laparotomy and was first described in 2008 [52] (Figure 23.4C). Ovarian cortical tissue fragments are transplanted into subcortical pockets in the remaining follicle-depleted ovary in all patients. The ovary is mobilized laparoscopically and made accessible through a 5-cm abdominal incision. Longitudinal incisions are made in the ovarian cortex to create two pockets, one on each side of the ovary, and the fragments are aligned next to one another inside the pockets.

Ovarian Tissue Transplantation Outcomes

After orthotopic reimplantation of frozen-thawed ovarian cortical fragments, ovarian endocrine activity is reinstated in almost all cases (95%) [8, 12, 28]. Even though it is difficult to determine the life span of grafted tissue, the mean duration of ovarian function after transplantation is 4–5 years, but it can persist for up to 7 years. Duration of function in grafts depends on a number of factors, including age at cryopreservation, follicle density and quantity of grafted tissue used. Indeed, women giving birth after ovarian tissue transplantation were shown to be significantly younger at the time of cryopreservation than those who failed to conceive despite a desire to do so [12].

The first pregnancy issuing from this procedure was reported in 2004 [27]. Pregnancy and live birth rates have continued to rise steadily, showing an exponential increase over the past few years (Figure 23.5). Indeed, the number of live births as of mid-2017 exceeded 130 [8]. Since the number of reimplantations performed worldwide is not known, in order to calculate the outcomes of ovarian tissue transplantation, data collection was based on patients from five major centers (n = 111 patients), yielding a pregnancy rate of 29% and a live birth rate of 23% [43]. These rates were further validated in a larger series of patients, with pregnancy and live birth rates of 33% and 25%, respectively [21). In our series of 22 women undergoing ovarian tissue reimplantation, the live birth rate was 41% (9 of 22) [8]. One woman in our institution had her ovarian tissue cryopreserved at the age of 17 due to a neuroectodermal tumor, and gave birth three times after one orthotopic ovarian tissue transplantation procedure [19], making her one of the three women in the world to have had three babies from a single graft.

Figure 23.5 Live births after ovarian tissue reimplantation Number of live births after orthotopic transplantation since the first reported case in 2004.

Adapted from Donnez J. and Dolmans M. N Engl J Med 2017

Challenges of the Early Post-grafting Period

Despite increasing success and better clinical outcomes of ovarian tissue transplantation, there is still room for improvement. Since it is an avascular grafting procedure, one of the biggest challenges in ovarian tissue transplantation is the early post-grafting period [58].

The size of fragments typically cryobanked ranges from 1–10 mm in length to 1–1.5 mm in width, which excludes the presence of any distinguishable blood vessels for further reanastomosis during grafting. Despite the absence of anastomosis, ovarian tissue transplantation without any surgical connection to major blood vessels allows restoration of ovarian function and fertility in patients, as previously stated. However, graft behavior depends on reestablishment of vascularization, with implants exposed to ischemic damage during the initial post-transplantation phase until they become fully vascularized.