FERTILITY PRESERVATION
STRATEGIES
Due to significant improvements in cancer treatments, patients affected by oncologic disease are living longer, fuller lives. Cancer treatments have also drawbacks and patients (or parents in case of children) must be informed of the long-term side effects of oncological treatments. However, ionizing radiation and most of alkylating agents, which are used for gonadotoxic chemotherapy regimens, can often induce premature ovarian failure, rendering the patient infertile. As results, the fertility potential of reproductive-age women affected by cancer has become an increasing focus for those who counsel and treat such patients (32).
American Society of Clinical Oncology (ASCO) releases guidelines suggesting dealing with cancer patients, fertility preservation issue as soon as possible after diagnosis and oncological disease staging in order to have time to determine the best strategies for fertility preservation (33). The strategy must be individualized in each case depending on patient’s age, type and stage of the cancer, therapeutic plan to be followed, foreseeable long-term effects, possibility or impossibility of delaying the start of treatment, whether or
not the patient has a partner/spouse, biology of the tumour and potential for metastasis in the ovary.
Nowadays, different fertility preservation approaches are available.
FIG.3.1CRYOPRESERVATION OPTIONS FOR ADULT AND PREPUBERTAL FEMALES AIMING AT PRESERVING FERTILITY POTENTIAL (.
3.1 FERTILITY PRESERVATION STRATEGIES. 3.1.1 Embryo cryopreservation.
Embryo cryopreservation is currently the only highly successful method of preserving fertility for female cancer patients. The protocol for embryo banking is similar to an in vitro fertilization (IVF) cycle done for patients with infertility with the exception of the embryo transfer. The female patient undergoes controlled ovarian stimulation (COS)
using gonadotropin injections to promote multifollicular growth. Dosing of the protocol is determined by assessing ovarian reserve using the same methods used for female infertility patients: basal serum FSH level, antral follicle count, and/or serum AMH level (33). Following COS an oocyte retrieval procedure is performed, typically under conscious sedation using transvaginal ultrasound-guided needle aspiration. The oocytes are then fertilized in the laboratory and the embryos created are frozen for future use.
Reported survival rates per thawed embryo range from 35% to 90%, implantation rates from 8% to 30%, and cumulative pregnancy rates of 30–40% (34).
One of the limitations of embryo cryopreservation is that a partner or sperm donor is required (35). Moreover, this procedure may not be an option for women with highly aggressive malignancies including leukaemia, some lymphomas, and sarcomas, which warrant immediate cancer treatment.
In Italy, freezing embryos is forbidden by law 40 of 2004
3.1.2. Oocyte cryopreservation
The first pregnancy obtained from a cryopreserved oocyte was reported in 1986 (36). Oocyte cryopreservation represents an important strategy for cancer patients, in countries such as Italy, where embryo cryopreservation is forbidden, but also for those patients who for ethical-religious reasons do not accept freezing of embryos. This technique also presents the advantage of not requiring the presence of a partner at the time of treatment. Moreover oocytes freezing is no more considered, by scientific community, as an experimental practice but as a well established one (37).
• Postmenarchal women up to the age of 38 years with a sufficient ovarian reserve, who receive chemotherapy or another treatment which could lead to a significant chance of premature ovarian insufficiency or loss of ovarian function
• The time until the start of chemotherapy is at least 2 weeks
The procedure consists, firstly, in ovarian stimulation to induce multiple follicles growth. Stimulation can be started during menstruation performing a classic GnRH antagonists protocol (as this is associated with reduced risk of ovarian hyperstimulation syndrome) or in other menstrual cycles phases using simultaneously GnRH antagonists and recombinant FSH. In the case of estrogen-dependent tumours, stimulation can be combined with letrozole daily which is administered at the same time as the gonadotrophin. Following COS, an oocyte retrieval procedure is performed using transvaginal ultrasound-guided needle aspiration (38).
Unfertilised oocytes are preserved by slow freezing or vitrification. Both techniques use cryoprotectants or substances which reduce stress due to the variation of osmotic solutes in the intra-and extracellular consequent of cooler temperature. The difference between the two methods consists in type and concentration of cryoprotectants and in the speed of cooling rate (39). Slow freezing method is a standard operating procedure in most IVF centres. Oocyte slow freezing traditionally cools to −7°C at −1 to −2°C/min, seeded at −7°C, and further cooled to −30°C to −35°C at −0.3°C/min, then free falling to −50°C before plunging into liquid nitrogen, it took about 3 h for the whole freezing procedure. To date, there is not enough evidence to show that such slow cooling is necessary. Several different solutes have been used to protect human oocytes against freezing damage, including dimethylsulfoxide (DMSO), glycerol, and both ethylene and propylene glycol.
Vitrification uses high concentrations of cryoprotectants (1.0–1.6 mol Ethylene Glycol and DMSO) and rapid cooling (−1,500°C/min) that solidify without the formation of ice crystals.
According to the current data, vitrification appears to be more effective. The introduction and refinement of vitrification has minimized structural damage to oocytes and has improved survival rates, currently in the range 87%–97% (about a 15% improvement compared with slow freezing) (40). However, the overall live birth-rate per cryopreserved oocyte is approximately 2%, which is much lower than the rate using fresh oocytes. In a randomized study, 600 patients are enrolled. They receive either fresh donor oocytes or frozen oocytes post vitrification and found no difference in the pregnancy rate per transfer (55.4% vs. 55.6%) or ongoing pregnancy rate per transfer (49.1% vs. 48.3%) (40).
Significant risks of oocyte cryopreservation are ovarian hyperstimulation syndrome (OHSS) or the collection of immature oocytes with a low fertilisation potential. Moreover, this procedure may not be an option for women with highly aggressive malignancies including leukaemia, some lymphomas, and sarcomas, which warrant immediate cancer treatment.
3.1.3 Cryopreservation of immature oocytes
In vitro maturation offers another feasible alternative for women avoiding ovarian
stimulation. The process involves aspiration of immature oocytes after minimal to no stimulatory medication, followed by meiotic maturation in vitro from the germinal vesicle to the metaphase II stage. Matured oocytes can then be cryopreserved. Although oocytes are usually collected in the window of the pre-ovulatory follicular phase, IVM has also
contributed time flexibility to cancer patients by successfully maturing oocytes retrieved during the luteal phase. Finally, IVM of oocytes aspirated from antral follicles of harvested ovarian tissue may be an option for prepubertal females. This technique has been performed experimentally and with good success in girls as young as 5 years. Caution must therefore be exercised in extrapolating the data for both efficiency and safety to the young cancer patient population (41).
Finally, given the low numbers of live births reported after IVM with cryopreservation, and the limited data and long-term follow-up of offspring, caution should once again be exercised in routinely recommending this technique.
3.1.4 Ovarian tissue cryopreservation
Transplantation of frozen-thawn ovarian cortex has shown to be a new promising method for recovery of ovarian function and in some cases sufficient to restore fertility.
Indications and requirements are (42):
• Girls and women up to the age of ca. 35–37 years and with an age-appropriate ovarian reserve who receive chemotherapy or another treatment which could lead to a significant chance of premature ovarian insufficiency
• With oncological disease: exclusion of ovarian metastases using appropriate diagnostic imaging
• Exclusion of an oncological disease which is associated with a high risk of ovarian metastases (haematological neoplasias, metastatic breast cancer, ovarian cancer, etc.) • The time until the start of chemotherapy is at least 3 days
• Low risk intubation of the patient and surgery is possible (caution: mediastinal tumours in patients with Hodgkin’s lymphoma)
Removal of the ovarian tissue is performed laparoscopically where possible. The amount of tissue removed depends on the expected probability of losing all egg cells. Histological examination of a reference biopsy (to exclude tumour cells, proof of follicle presence) is necessary. Transport from operating theatre to laboratory: rapid transport of the removed tissue is performed in transport medium on ice. However, transport from the place of removal to the tissue bank is also possible over a longer period of time (20 h) (43). The most efficient method of tissue cryopreservation is currently the slow freezing technique. Reimplantation ca be orthotopic or heterothopic. The first one is performed putting tissue on ovarian surface or in a peritoneal window near the ovary, the second one in subcutaneous tissue of the abdomen or forearm. Orthotopical transplantation has the greatest chance of success. It is still unclear which exact site should be used for the transplant, whether a spontaneous pregnancy or IVF should be given priority and how the patient should be treated after the transplant. The transplantation should in most cases be performed 2 years after the end of treatment at the earliest, in agreement with the responsible oncologists, when the risk of relapse has significantly decreased.
Spontaneous pregnancies occurred. Successful teams (44) have achieved a pregnancy rate of ca. 30% per transplantation up to now, although the birth rate is lower. However, other teams report lower success rates, so it can be assumed that the success depends on the correct indication for cryopreservation, the age of the patient, the freezing technique and the transplantation technique.
One limitation common to many tissue transplants is the potential for ischemia within the tissue before it can become revascularized. Such ischemia may result in follicle atresia, thereby compromising the long-term function of the transplanted ovarian tissue. Reports have indicated that most follicles survive the freeze–thaw cycle. However, up to two-thirds are lost following transplantation. Approaches to prevent such ischemia may include varying the size of tissue transplants or selecting a site of implantation known to be responsive to angiogenesis. An alternative approach may be to deliver angiogenesis promoting factors, such as VEGF, to the implantation site at the time of surgery. Research in the area of regenerative medicine has demonstrated the ability to improve neovascularisation into tissues using such techniques, and applying these techniques to cortical strip transplantation may reduce follicle loss due to ischemia (45).
The main concern regarding ovarian transplantation is the risk of reintroducing malignant cells to the patient. This risk varies with cancer type, activity, and stage, as well as the mass of malignant cells transferred. With the exception of some haematological malignancies and some advanced stage solid tumours, most malignant tumours that occur in reproductive age women do not metastasize to the ovaries (43).
3.1.5 Ovarian suppression
It has been hypothesized that suppressing the ovarian function, with the use of gonadotropins realising hormone agonists (Gn-RH), transiently during chemotherapy could prevent ovarian follicle destruction by maintaining the follicles dormant. However, the pool of primordial follicles is normally non-proliferating. Those follicles lack FSH receptors and
their initial recruitment is not controlled by gonadotropins, therefore hormonal manipulation by suppressing gonadotropins release is not likely to affect them (46).
After an initial gonadotropin release (flare-up effect), GnRHa bring about a downregulation of the GnRH receptor, followed by hypogonadism. The flare-up effect of the GnRHa takes about 1 week; they should therefore be administered at least 1 week before the start of chemotherapy. If the flare-up needs to be reduced, a GnRH-antagonist can be administered once a day for 6 days at the same time as the GnRHa depot injection (47). Whether the fertility preserving effect of GnRHa can thereby be improved has yet to be proven. The effect of the GnRHa should continue for at least 1–2 weeks after the last chemotherapy cycle.
The vast majority of available studies having investigating gonadal protection by gonadotropin-releasing hormone analogues (GnRHa) agonists or antagonists during chemotherapy have been small, retrospective and uncontrolled. Twelve studies carried out between 1966 and 2008 showed that out of 234 patients who received chemotherapy, 59% of cases had premature ovarian failure (POF) versus 9% after a combination of chemotherapy with a GnRHa (n = 345). In 2009 and 2010, three meta-analyses were published addressing the co-treatment GnRHa during chemotherapy to reduce ovarian damage. Clowse et al. and Ben- Aharon et al. included 8, 16 studies, respectively, including those with retrospective controls. Clowse revealed that GnRHa are effective in preserving ovarian function and Ben-Aharon revealed that GnRHa are effective in reducing amenorrhoea. Bedaiwy et al. only included prospective randomized studies (n = 7) with 173 patients receiving GnRHa and 167 control patients. On the whole, the evidence that GnRHa have a protective effect on the ovaries is becoming more established (44).
One possible side effect of GnRH analogues is menopausal symptoms. These symptoms are delayed, but are also possible with chemotherapy alone. Treatment with GnRHa for over 6 months leads to a loss of bone mass (47). In case of severe symptoms and in case of long term use (6 month), add-back therapies, using low estrogens dosages, can be considered. However, data concerning the influence of add-back therapies on the fertility preserving action of GnRHa are not available.
3.1.6 Ovarian transposition
Ovarian transposition is recommended in case of radiotherapy to the pelvis, which would lead to a significant chance of premature ovarian insufficiency. It consists in surgical transposition, (laparoscopic or laparotomic performs) of ovaries outside the irradiation field which may require section of the utero-ligament and Falloppian tube. The ovary is anchored, as high as possible, to the anterior abdominal wall, laterally in the paracolic gutter. Titanium clips are placed on the two opposite borders of the ovary to allow radiological identification prior to radiotherapy.
Radiotherapy with two Gray leads to a loss of ca. 50% of the primordial follicles. The chance of premature ovarian insufficiency occurring in women aged 20 years is almost 100% if they receive radiotherapy with 15 Gray. The mobilised ovary is usually transposed craniolaterally, fixed and marked with clips in order to achieve the greatest distance possible from the main irradiated area. As loss of ovarian function can occur despite transposition, additional cryopreservation of the ovarian tissue is recommended. According to the published literature, there is a success rate of up to 85% with this technique in patients with regular ovulatory cycles, and also in patients under the age of 40 after
radiotherapy. Unspecific post-operative abdominal discomfort has been described, which was a result of ovarian cysts or peritoneal adhesions in most cases (48).
3.1.7 Fertility “sparing” surgery
Conservative surgery aiming at preserving reproductive organs offers the opportunity to preserve fertility potential and to achieve pregnancy naturally in some cases. Indications for fertility-sparing surgery include a well-differentiated low-grade tumour in its early stages or with low malignant potential ( cervical cancer stage 1A1, 1A2, 1B1, borderline ovarian tumours, ovarian epithelial cancer stage 1, malignant ovarian germ cell tumours/sex cord stromal tumours, endometrial carcinoma grade1, stage1A) (44).
The most established fertility preservation sparing procedure in women is the radical trachelectomy described first by Dargent in 1994 (49). It is currently offered to women with early stage invasive cervical cancer having the desire to preserve fertility. Approximately 500 cases have been reported most of them from Europe, Japan, U.S.A, Canada and China. The classical treatment of cervix cancer otherwise includes radical hysterectomy with bilateral pelvic lymphadenectomy in early stage disease or chemo and radiotherapy for advanced stages. The global utilization of fertility-sparing surgery is currently unknown. Due to improvement in screening programs worldwide, cervical cancer is becoming less common in many countries. In a recent survey, the European Society of Gynaecological Oncology (ESGO) Task Force for Fertility Preservation in Gynaecologic Cancer investigated the numbers and eligibility of gynaecologic cancer patients for fertility-sparing treatment in selected gynaecologic oncology centres across Europe. The study included data from the year 2007 at 14 ESGO-accredited centres in Austria, Belgium,
Czech Republic, Germany, Greece, The Netherlands and Turkey. The numbers of patients eligible for conservative management in this survey were small, on average 14–15 per year in ESGO-accredited centres, and only 7–8 of those actually underwent fertility-sparing surgical treatment. This study raises concerns on the need to centralize referrals for fertility sparing treatments in young women with gynaecological cancer at accredited units aiming at ensuring thus a sufficient number of patients to maintain the quality of care for the patients at a referral centre (50).
3.2 FUTURE DEVELOPMENT
Methods for the in vitro culture of cryopreserved tissue or follicles are in relatively early stages of development. In order to successfully translate the system to the human, the optimal techniques for culture must be identified for isolation techniques, matrix conditions, media composition, assessment of the developmental state of the follicle and oocyte, and for determination of the timing of oocyte IVM.
Two approaches to in vitro follicle maturation have been investigated to date (51): - Organ culture: whole slices of ovarian cortical tissue are cultured intact retains the organizational structure of the ovarian tissue and maintains the interactions between the follicle and surrounding stromal cells. This method has supported growth of human primordial follicles to the secondary follicle stage, and has supported follicle survival for up to 4 weeks. The main challenge with in vitro organ culture, as in transplantation, is preventing atresia due to ischemia in the interior of the tissue, since there is no possibility of revascularization in the in vitro environment. Varying the size and geometry of the cultured tissue pieces or slices has been shown to improve follicle survival. A second
disadvantage to organ culture is the inability to observe and monitor follicles during culture by light microscopy due to the thickness of the tissue.
- In follicle maturation: Culture of isolated follicles allows for the individual monitoring and tracking of each follicle to assess its developmental state. The IFM approach involves in vitro follicle growth (IVFG), or the in vitro culture of immature follicles, followed by IVM of the oocyte within the follicle. In IFM systems, follicles are isolated from the ovary using a mechanical or enzymatic approach. Follicles are then cultured for a period of time during which the growth, steroid production, and morphology of each follicle can be monitored. Several IFM systems have produced live mouse pups following culture of immature follicles. Systems have also been demonstrated in humans and non-human primates to support follicle growth and development. The development of culture systems for human follicles is in the early stages, however, and to date, no meiotically competent oocytes have been produced.
