Whole abdomen and pelvic radiation greater or equal to 15 Gy in pre pubertal or 10 Gy in post pubertal girls especially in combination with any alkylating agents
Craniospinal radiation
Total Body irradiation as part of conditioning for bone marrow transplant
High dose chemotherapy:
Cyclophosphamide 120–200 mg/kg as conditioning for BMT
Cyclophosphamide >5 g/m2
Cisplatin
Ifosfamide >60 mg/m2
Melphalan 140–210 mg/m2
Busulphan 8–16 mg/kg
Thiotepa
Procarbazine
BCNU/CCNU
As programs progress and more clinical data becomes available, we believe that ovarian tissue cryopreservation will be increasingly offered to non-malignant diseases such as females at risk of premature ovarian failure: i.e. Turner syndrome, family history of premature ovarian insufficiency, benign autoimmune diseases requiring gonadotoxic chemotherapy or patients needing bone marrow transplantation for benign haematological diseases such as sickle cell anaemia and thalassaemia major.
Following referral it is mandatory to have a thorough pre-treatment consultation by a multidisciplinary team that specializes in fertility preservation. This team should include an oncologist, a reproductive endocrinologist, a surgeon, specialized nurse and a paediatrician and social worker as required. The cancer diagnosis, the chemotherapeutic agents and their doses, radiotherapy field and scattered radiation doses, as well as the patient’s age should to be taken into consideration before deciding on the need and the extent for the ovarian tissue cryopreservation.
Preoperative Workup
Preoperative workup of the patient is performed to exclude ovarian pathology and possible pelvic metastases (e.g. gynecologic ultrasonography, CT scans or MRI) [3]. During laparoscopy, a meticulous inspection of both ovaries is performed to look for malignancy [4–6]. Ovarian activity is measured by either measuring the antral follicular count by ultrasound scan or testing patient’s blood for ovarian reserve markers, i.e. Anti Mullerian Hormone (AMH) or Follicle Stimulating hormone (FSH).
Ovarian cortical tissue harvesting is commonly performed as a laparoscopic procedure under general anaesthesia. Three or four entry puncture sites are used: 12 mm intraumbilical trocar , two 5 mm trocars in the left and right iliac fossae. A fourth 12 mm suprapubic trocar can be used if oophorectomy is performed to allow atraumatic ovarian removal with an endobag. If open surgery is planned for the patient, ovarian biopsies should be taken at the time of the surgery. Other procedures requiring a general anaesthetic can be performed at the same time such insertion of Hickman catheter prior to starting chemotherapy.
Ovarian biopsies are taken either with laparoscopic forceps or scissors. It is important to minimize as much as possible the use of diathermy so as not to damage the ovarian tissue. If bleeding does occur, this is best controlled with small, precise mirco-bipolar diathermy. Care should also be taken to minimize trauma to the ovarian tissue and the prompt removal of the tissue to the laboratory team.
The ovarian cortical tissue size harvested or number of biopsies taken is dependent on the estimated gonadotoxic effects of the planned chemotherapy and/or radiotherapy regimen, varying from 20 biopsies (10 biopsies from each ovary) or 2 cortical strips measuring 15 mm long and 5 mm wide taken from each ovary. When a high-risk chemotherapy or radiotherapy is planned and risk of premature ovarian failure is high (60–80 %), oophorectomy should be considered (Fig. 7.1). Ovarian tissue cryopreservation can also be combined with intraoperative retrieval of immature oocytes from the contralateral ovary in cases of oophorectomy or from both ovaries if only biopsies are taken. Retrieved oocytes are then subjected to in vitro maturation and cryopreservation of either mature oocytes or embryos.
Fig. 7.1
Shows the different ovarian tissue harvesting options: biopsy, partial ovarian cortex resection, unilateral oophorectomy. The amount of the ovarian cortical tissue harvested is tailored to the gonadotoxicity degree of the anti-cancer treatment and the ovarian size
Processing the Ovarian Tissue
Preparation of the ovarian tissue, cryopreservation and thawing must ensure follicle viability and integrity of tissue compartments and cell-to cell contacts [7]. Two well-established methods can be used: rapid vitrification and slow freezing of finely dissected thin slices of ovarian cortex tissue [8–11]. Both freezing methods are acceptable with good survival rates and comparable tissue viability and morphology integrity rates. Several recent reports have shown improved outcomes after vitrification of ovarian tissue as compared to slow freeze technique in preserving granulosa cells and ovarian stroma [12, 13]. However, until clinical outcome data show improved outcome of the vitrification protocol, we think the slow freeze protocol should be adopted. In the upcoming sections the slow freeze protocol used in our Unit will be described.
Ovarian Dissection
The cooled ovary should be transported from the operating theatre to the laboratory within 30 min of procurement and then tissue processing is immediately commenced within a clean facility in the laboratory. Antral follicles are aspirated at the beginning and fluid is carefully assessed for the presence of oocytes. In addition, the remaining media left after ovarian tissue dissection is ideally given to embryologists at the end of the procedure to check for potential immature oocytes. If immature oocytes are found then they undergo in vitro maturation (IVM) and the resultant mature eggs are vitrified. The ovary is immersed in a petri dish in cooled Leibovitz 15 medium and is bi-valved using sterile scalpel and forceps or tweezers (Fig. 7.2). The inner medulla tissue is dissected away leaving the thin outer layer of the cortex that contains the majority of follicles [9, 14]. Our practice is to have two staff working together to minimise damage to tissue by reducing the overall processing time and reducing the length of ischaemia before starting the freezing procedure. The cortical tissue slices are all trimmed to within a standard thickness of 1–2 mm to ensure that cryoprotectants adequately diffuse into the ovarian tissue during the cryoprotection procedure prior to freezing. In addition, after autografting, thin slice grafts are more likely to survive the post transplantation ischemic stage, by more efficient simple diffusion before neovascularisation and perfusion are adequately established [10, 15, 16]. The slices are then cut in small pieces of 1 mm width and 5 mm length. Other groups reported variable sizes of the slices. There seems to be no difference in results in the different sizes: the most critical factor is the slice thinness. Slices are then placed in fresh Leibovitz medium until cryoprotection.
Fig. 7.2
Ovarian tissue processing in the laboratory as demonstrated for whole ovary removal. (a) The ovary is bi-valved. (b) The medulla is cut and carefully removed and the 1 mm thickness outermost cortex is achieved. (c) The ovarian cortex slices before freezing
Quality Assessment (After Dissection)
A standard practice in centres performing ovarian tissue cryopreservation is to perform routine tests to ensure primordial follicles are present in the ovarian tissue slices and that they are morphologically normal. At this stage, these tests primarily include light microscopy examination to assess the percentage of primordial follicles found in fresh (or pre-processed) ovarian tissue slices [10, 17–20]. Hence, a specimen should be taken during dissection from the ovarian cortex and sent off for histopathological evaluation by light microscopy (×400 magnification) after fixation in Bouin’s solution and using haematoxylin/eosin staining to check for the presence of follicles and morphology of the tissue. Samples are also tested using standard trypan blue viability staining methodology. Another specimen is placed in 4 % formaldehyde and sent for pathological evaluation for the presence of malignant cells, prior to cryopreservation. Other samples are also taken and stored separately for post-thaw testing before reimplantation in the future. All histological tests are ideally performed by the same specialised consultant pathologist using the same protocols.
Cryoprotection
To minimise damage due to normally lethal intracellular ice crystal formation and build-up of extracellular salt concentration, the tissue is quickly transferred after dissection into test tubes containing 8 ml of pre-cooled cryoprotectant medium and labelled. We use a mixture containing 1.5 M penetrating cryoprotectant medium in order to dehydrate cells and increase solute concentration around the cells. We use the ethylene glycol as an effective cryoprotectant for ovarian tissue as was shown in several works [10, 11, 15]. The non-penetrating sugar, sucrose (0.1 M) is also added to the medium since it is known to act as an osmotic buffer and is an added precaution against excessive osmotic swelling during cryoprotectant removal and thus minimises freezing injury [21]. The tissue is incubated on a shaking plate at 2–8 °C for exactly one hour. Three to five tissue slices are then quickly transferred to cryovials with 1 ml of cooled cryoprotectant. The permeation of isolated cells with cryoprotectants is relatively fast, but diffusion through multicellular tissues such as ovarian cortical tissue slices is much slower i.e. exposure to cryoprotectant is prolonged to ensure adequate concentrations in the centre of the tissue. However, this has to be balanced with the fact that cells on the tissue surface may be at risk of excessive toxicity. Cryoprotection time periods should therefore be carefully standardised and strictly adhered to.
Freezing
The freezing protocol of slow freezing has long been well established and optimised designs for ovarian tissue cryopreservation are very similar [3, 10, 14, 15]. As the temperature is cooled, ice crystals growth is initiated by ice crystal nucleation induction (i.e. ‘seeding’). As the crystals grow the water in the solution is turned to solid state, increasing the solute concentration and this draws water out of the cells [22]. More water can be incorporated into ice at lower temperatures but the rate at which water can leave a cell also falls as temperature is lowered. The effectiveness therefore depends upon equilibrium between the rate at which water can leave a cell and the rate at which it is converted into ice. The rates used by our programme classically involve an initial cooling rate of approximately −2 °C/min down to −9 °C with then a hold for 15 min to perform ‘seeding’. We achieve this by using long handled metal forceps dipped in liquid nitrogen and gripping the cryovials at the meniscus. This is followed by a 5 min soaking hold period. Then a slower rate of cooling −0.3 °C/min is programmed to reduce temperature down to −40 °C. The final stage of the freezing program is a faster cooling stage of −10 °C/min down to −140 °C. All tissue is frozen in the same programmable Sylab freezer (Ice Cube) using the same program.
Storage
The cryovials containing the tissue are then sealed inside an additional sterile cryogenic bag, which is labeled and placed in a protective cardboard box and then transferred to liquid nitrogen storage into the predetermined quarantine rack position. Many establishments then plunge their ovarian tissue directly into liquid nitrogen, however, we prefer to store tissue in vapour phase nitrogen (at approximately −170 °C) rather than in liquid nitrogen (at −196 °C ) in order to minimise risk of cross-contamination of tissue packaging from bacteria or viruses surviving in the nitrogen tank [23]. This does not affect the quality of tissue storage, since below −130 °C, the glass transition temperature of water, no biological or physical changes take place and therefore below this temperature tissue may be safely stored [22].
Thawing Out
Thawing techniques are a standard amongst the different ovarian processing units [10]. The rapid thawing process is performed within the clean room facility in the laboratory whilst the patient is in operating theatre in order to minimise ischaemic time. The relevant cryovials are removed from the nitrogen storage freezer and held in air for 30 s. The cryovial is then placed in a water bath at 30 °C for 3 min. The cryovial is then transferred to the cleanroom microbiological safety cabinet for three-stage serial dilution of the ethylene glycol, each stage taking 5 min and performed on a shaker at room temperature. The ovarian slices then remain in a test tube with a final mixture of 1 ml sucrose, 1 ml serum substitute supplement and 8 ml Leibovitz medium. The same protocol is also used for all specimens used for testing.
Post Thawing
Quality Assessment
Specimens of the ovarian cortex are thawed out approximately 1 month prior to the planned auto-transplantation using the same protocol as for tissue slices for clinical use. They are then sent off for the same histopathologic evaluation of the presence, viability and morphology of follicles as was performed following dissection, involving the same histological techniques and trypan blue staining [17–20]. Published studies consistently show there is only minimal difference between results for fresh and for frozen-thawed tissue [10, 17]. The number of follicles counted, however, will depend upon age of donor [17, 22] and whether any previous treatment of chemo- or radiotherapy has been given prior to procurement of ovarian tissue.
Screening for Micrometastases
The major concern with ovarian tissue banking is the possibility of re-seeding a tumour, harboured within the ovarian slices, after auto-grafting the frozen thawed tissue slices. Tissue samples are therefore carefully screened for the presence of malignant cells (micrometastases) by histology and specific immunohistochemical or molecular tests such as polymerase chain reaction (PCR) testing and real-time PCR to detect molecular markers that would indicate presence of cancer cells [3, 6, 24]. The various tumours are categorised into three groups according to their risk of ovarian micro-metastasis [6, 25] and this should be taken into account upon the initial consultation as part of informed consent. Testing should be performed approximately 1 month prior to planned transplantation using the up-to-date licensed methods available at the time of re-implantation, thus taking advantage of the fact that diagnostic methods for detecting minimal residual disease are likely to be improving with time. When tissue shows evidence of malignant cells (i.e. any positive test result) , the patient is advised against future auto-transplantation of ovarian cortical tissue slices. Instead, in the future these patients may benefit from later advances and possible breakthroughs in the field of In Vitro Maturation (IVM) of ovarian tissue, in vitro maturation of follicles or isolated follicle transplantation, which probably do not pose any risk to transmission of tumour cells [26]. A combined immature oocyte recovery for IVM, in addition to the ovarian tissue cryopreservation may be of critical importance in these patients.
Auto-transplantation
Auto-grafting of the tissue is done when the patient is in remission and old enough and ready to start a family. The tissue is commonly transplanted in the orthotopic position (i.e. into its natural site such as the ovarian hilum or medulla or nearby structures such as the broad ligament or the pelvic sidewall). This has been shown to offer the potential for spontaneous pregnancy without the need for primary and immediate resort to IVF [10, 15, 27–29]. Alternatively, heterotopic transplantation (e.g. non-native ectopic location such as the arm or abdomen) has certain advantages [9, 30, 31] such as easier follicular monitoring and egg retrieval for IVF as well as a closer monitoring for cancer recurrence following auto-grafting. Nevertheless, orthotopic transplantation has been shown to result in less follicle loss and more effective revascularisation and is believed to be more effective [31]. Orthotopic autotransplantation can be performed either by laparotomy or laparoscopy. Some advocate [32] performing it in two stages: in the first stage laparoscopy is performed 7 days before the re-implantation procedure, aiming at creating a peritoneal window at the ovarian hilum, broad ligament or the pelvic sidewall. The goal of this procedure is to induce neovascularization and the formation of granulation tissue in the area where the ovarian slices are to be re-implanted. It is believed that by this neoangiogenesis, the ischemic phase after the transplantation is shorter. A second stage laparoscopy follows after about a week during which the ovarian slices are transplanted into the peritoneal furrow. The slices can either be sutured to the ovarian medulla or left unsutured into the peritoneal window. Pregnancies were reported after auto-grafting of varying sizes of ovarian slices [33] as well as after micro-organ transplantation of thinned ovarian slices [15].
Stay updated, free articles. Join our Telegram channel
Full access? Get Clinical Tree
