Fertility Preservation before Therapy for Cancer New Perspectives

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FERTILITY PRESERVATION BEFORE THERAPY FOR CANCER NEW PERSPECTIVES Sohani Verma Senior Consultant Obstetrician and Gynaecologist, Infertility and IVF Specialist, Incharge IVF Lab, Indraprastha Apollo Hospitals, Sarita Vihar, New Delhi 110 076, India. e-mail: [email protected], [email protected] As the number of young and adult nulliparous cancer survivors is constantly increasing due to progress in the field of oncology, the adverse effect of the life saving cancer therapy on reproductive function is assuming greater importance. Appropriate strategies to spare fertility in all young cancer patients must be considered. Recent advances in assisted reproduction technology such as in-vitro fertilization (IVF), intra-cytoplasmic sperm injection (ICSI) and cryopreservation have revolutionized the options available to these patients for their fertility preservation. While sperm and embryo cryopreservation are now established procedures, oocyte (mature or immature) cryopreservation has limited application till date. Ovarian tissue cryopreservation although at present an experimental technique, is fast emerging out as the most promising future option for fertility preservation. There is need for the professionals dealing with young cancer patients to familiarize themselves with these developments and with the importance of describing and providing access to these options. All possible fertility preservation options should be offered and discussed before starting the therapy for cancer. A multidisciplinary approach involving a close liaison between oncology and assisted conception units is crucial for providing the proper guidance and support to the young vulnerable patients diagnosed with cancer and their families in deciding what is likely to be the most appropriate course of action for them. Key words: Cancer, Fertility preservation, New perspectives, Ovarian cryopreservation, Sperm banking.

INTRODUCTION

gametes and gonadal tissue” - although in early experimental stage at present, is fast emerging out as the most promising option for fertility preservation. The first human live birth following orthotopic transplantation of cryopreserved ovarian tissue in a 32 year old Belgian woman 7 years after banking her ovarian tissue before starting chemotherapy for Hodgkin’s lymphoma, has already been reported by Donnez, et al. in 2004[3].

OVER recent years improvements in the treatment of cancer have led to a significant increase in the long-term survival rates of patients, especially of younger ages[1]. As the number of both - childhood and adult cancer survivors is rising, attention has now focused on not just the quantity but also on their quality of life issues such as fertility and sexuality.

Several proven and potential fertility preservation options are now available for young cancer patients. However, since patients must utilize these strategies before cancer therapy is initiated, an awareness among the professionals dealing with the cancer patients peadiatricians, oncologist, chemotherapy and radiotherapy specialists and general physicians is essential to provide the opportunity before it is too late for them. A multidisciplinary approach and a close liaison between oncology unit and assisted conception unit (ACU) is extremely important to implement such options. Since many of these options are still experimental with no standard protocols and no established risk-benefit profile, detailed professional counselling of patients and their families, meticulous record keeping, long-term follow-up and further research in the subject are extremely essential.

The aggressive cancer therapy required for cure is known to have long-term psychological and health-related consequences, impaired hormonal responses and future infertility. At least 15% of young cancer survivors will have a high risk (95%) of early and irreversible gonadal failure, whereas others may have lesser extents of compromised reproductive capacity as a result of their cancer treatment[2]. In the past few years, the introduction of two new technologies in the field of reproductive science has revolutionized the opportunities to preserve reproductive options for both young men and women who are diagnosed with cancer. One of these “in-vitro fertilization (IVF)” along with “intracytoplasmic sperm injection (ICSI)” is already a well-established technology. Another “cryopreservation of 15

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POTENTIAL GONADOTOXICS

fertility in women and in approximately 50% of men [8]. Chemotherapy does not appear to have any significant lasting adverse effect on uterine function. Successful pregnancies with no increased risk of miscarriage and healthy offspring have been reported following treatment with multiagent chemotherapy regimens[9].

Overall chances of cure for childhood cancer is estimated to be approximately 70% and for specific cancers, for example leukaemias, lymphomas and germ cell tumors cure rate is nearly 100%[2]. These results are getting even better due to more aggressive cancer treatment - entailing however, among other side effects, increased gonadal toxicity and subsequent infertility.

RADIOTHERAPY INDUCED FERTILITY DAMAGE The nature of reproductive damage depends on the field of treatment, total dose and fractionation schedule of radiation therapy and the age and sex of the patient [10-12]. In males doses as low as 0.1-1.2 Gy radiation can damage dividing spermatogonia and disrupt cell morphology resulting in oligozoospermia[10,12]. Permanent azoospermia has been reported following single fraction irradiation with 4 Gy or 1.2 Gy fractionated. Leydig cells are more resistant to damage form radiotherapy than the germinal epithelium and normal potency is frequently present despite severe impairment of spermatogenesis[12]. Total body, abdominal or pelvic irradiation may cause ovarian and uterine damage [8,13]. The human oocyte is sensitive to radiation even at a dose of less than 2 Gy. In women, as the oocytes once destroyed do not regenerate, premature menopause may occur, although the risk is lower for younger women due to larger number of primordial follicles, than for the older women. 1 Gy radiation of the ovary can induce definitive menopause in a 40 years old patient, while 80% of women under 30 years of age will conserve menstruation up to 4 Gy [14].

Different cancers are associated with different risks of future fertility compromise. Those at maximum risks are often those, most intensively treated with consequent multiple toxicities. Different types of therapies and cancers most likely to cause a high risk of future fertility compromise, are shown in Table 1[4]. CHEMOTHERAPY INDUCED FERTILITY DAMAGE Males are more susceptible to subfertility following chemotherapy than females but females may be at risk of premature menopause. Treatment with some chemotherapeutic agents is particularly likely to result in gonadal toxicity. Those most likely to cause germ-cell damage are listed in Table 2. These gonadotoxic agents include alkylating agents specifically cyclophosphamide, procarbazine, cisplatinum, lumistine and carmostine. Oocyte loss induced by cytotoxic therapy has been shown to occur by apoptosis[5]. In a long-term follow-up study 89% of the males treated before puberty for Hodgkin’s disease with “ChIVPP” (Chlorambucil, vinblastine, pro carbazine, prednisolone), had evidence of severe damage to the germinal epithelium and recovery of spermatogenesis was thought to be unlikely [6]. Around 50% of the girls treated for Hodgkin’s disease pre-pubertally with six or more courses of ChIVPP had raised plasma gonadotrophin concentrations[6]. The use of ABVD (Adriamycin, bleomycine, vinblastine, dacarbazine), which does not contain alkylating agents or procarbazine, is significantly less gonadotoxic[7]. Current regimens with hybrid protocols are likely to preserve

However, even with the retention of ovarian function a severe depletion of the follicle store may occur with raised gonadotrophin levels and severely compromised fertility potential. Childbearing potential is further compromised in post-radiotherapy females due to possible radiation damage to the uterus. Uterine irradiation in childhood increases the incidence of nulliparity, spontaneous miscarriage and

Table 2: Gonadotoxicity agents.

of

Group

Proven gonadotoxicity

Alkylating agents

Cyclophosphamide Chlorambucil Melphalan Busulfan Carmustine Lomustine Mechlorethamine Procarbazine Cisplatin

- Ewing’s sarcoma.

Vinca alkaloids

Vinblastine

-

Antimetabolites

Cytosine arabinoside

Table 1: High risk of future potential fertility compromise associated with different cancers and their therapies. -

Total body irradiation.

-

Localized radiotherapy:pelvic or testicular.

-

High-dose chemotherapy, ‘conditioning’ for bone marrow / stem cell transplant.

-

Hodgkin’s disease:alkylating agent-based therapy. Soft tissue sarcoma:metastatic.

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¾ Ovarian transposition outside the pelvis first proposed more than 40 years ago[22], is an option for young women with gynaecological malignancies who require pelvic radiation therapy. Retention of the ovaries in their normal pelvic location will almost definitely produce sterility. Although benefit of such procedure is not universally accepted[23], in the largest series of ovarian transposition at the time of radical hysterectomy for cervical cancer, ovarin function was preserved in 79 (83%) of 95 patients [24]. Metastasis or recurrences, as well as benign cysts and further surgery in the transposed ovaries are a concern, however this is rare, with only 2 of 107 patients with cervical cancer reported in one series developing a recurrence in the ovaries[25], both these patients had lower uterine segment involvement and lymph-vascular space invasion (LVSI). A preoperative pelvic magnetic resonance image (MRI) is advisable on patients who are contemplating ovarian transposition. Ovarian transposition in properly selected and counselled patients gives them an opportunity to retain ovarian function as well as attempt future pregnancies. This should be done laparoscopically if a laparotomy is not essential for the treatment of the primary malignancy. The ovaries should be sutured to the peritoneum in the paracolic gutters above the pelvic brim. Both spontaneous pregnancies where patients had received pelvic radiation therapy with an intact uterus[26] and surrogate pregnancies involving patients who under-went a hysterectomy and pelvic radiation therapy have been reported[27].

intrauterine growth retardation[13,15]. The doses of pelvic irradiation >20 Gy are associated with high risk of midtrimester fetal loss[13]. The mechanism underlying these findings remains unclear but reduced elasticity of the uterine musculature and uterine vascular damage have been suggested[15,16]. Associating radiotherapy with chemotherapy leads to a synergy of gonadotoxic effects, for example, following bone-marrow grafting with purely alkylating conditioners, two thirds of young women recover their ovarian function, whereas associated total body irradiation almost always causes irreversible damage[14]. FERTILITY SPARING STRATEGIES ¾ Limitation of radiation exposure by shielding the testes and ovaries where possible must be practiced. Consideration should be given to use less gonadotoxic chemotherapy agents where applicable, without compromising the chances for cure. ¾ A number of studies have shown that gonadotrophin releasing hormone (GnRH) analogues inhibit chemotherapy induced ovarian follicle depletion in rodents by blocking gonadotrophin induction[16]. In one clinical study reported, the co-treatment of GnRH analogues and chemotherapy resulted in primary ovarian failure in only 1 of 44 (2.3%) compared with 26 of 45 (58%) in the group treated with chemotherapy alone[17]. The judicious use of GnRH analogues may play a role in the appropriate patient group, such as young women and children subjected to alkylating agent based chemotherapy for Hodgkin’s disease. However, reported results are inconsistent and lack of follicle stimulating hormone (FSH) and GnRH receptors on primordial follicles and oocytes does not make gonadal suppression an effective strategy of gonadal protection [18]. Clinical studies in men too have been inconclusive [19-21]. In men treated with sterilizing radiotherapy and chemotherapy for childhood cancer, effective gonadotrophin suppression with medroxyprogesterone acetate for at least three months did not result in restoration of spermatogenesis[19].

¾ The other options for young women with cervical cancer include excisional cone biopsy, radical abdominal trachelectomy and radical vaginal trache-lectomy (RVT). These options can only be used in a very select group of patients. However, nearly half of all patients younger than 40 years of age who might otherwise undergo a radical hysterectomy, may be candidates for these fertility-sparing procedures[28]. ¾ Excisional cervical cone biopsy alone without lymph node evaluation may be considered in patients with stage IA1 (<3 mm stromal invasion, micro invasive) cervical cancer and no LVSI. These patients have a 0.8% risk for pelvic node metastasis as compared to nearly 8% in patients with >3 mm stromal invasion or those with LVSI[29]. In the largest series reported[30], all 200 patients treated with a laser cone biopsy for cervical cancer stage IA1 and no LVSI, were alive without evidence of recurrence. The median follow-up in that groups of 200 patients was 117 months (range 72-420 months). It is essential that both negative endocervical margins and curettings be obtained. Patients

¾ Inhibition of the apoptosis induced by cytotoxic therapy has been explored as a mechanism for preventing ovarian failure. Treatment of mice oocytes with Sphingosine-1-Phosphate (S-1-P), a metabolite of ceramide is found to prevent chemotherapy induced apoptosis both in vitro and in-vivo conditions with pregnancy rate of 100%[5]. While S-1-P may herald promise of a new approach to preservation of ovarian function, further studies are necessary to explore the potential detrimental effects of such treatment on normal neurological function. 17

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who have both positive margins and curettings may have a 10% chance of having a lesion >3 mm (stage IA2) [31]. These patients require a more extensive procedure as well as evaluation of the pelvic lymphatics and are not candidates for cone biopsy alone.

acetate and megestrol acetate. Pre treatment evaluation of patients considering conservative hormonal therapy includes detailed history, physical examination, dilatation and curettage (D&C) and radiological imaging. A recent comprehensive review[41] identified a total of 81 patients treated conservatively from 1961 to 2003. There were 62 patients (76%) who responded to initial therapy and of these 47 patients (76%) did not show any evidence of recurrence during a median follow up period of 46 months. Remaining patients underwent hysterectomy at some point. The patients opting for conservative approach need to understand that there are very limited data and they must be willing to accept a small but undefined risk.

¾ The young women desirous of preserving their fertility, with more extensive disease confined to the cervix, can be the candidates for radical trachelectomy, which can be accomplished either abdominally or vaginally, as an alternative to radical hysterectomy with pelvic and paraaortic lymph node dissection or pelvic radiation with concurrent chemotherapy. The first RVT was performed by Daniel Dargent in 1987[32]. Proper selection and thorough gynaecological and colposcopic examination of patients is essential. A preoperative pelvic MRI is useful investigation. In general, RVT should be considered only for young patients who desire future fertility and have lesions smaller than 2 cm confined to the cervix. RVT involves a two-step procedure. A laparoscopic pelvic lymphadenectomy is performed. If the nodes are negative, then only the RVT is performed. Adenocarcinoma on histology and LVSI are relative contraindications[8]. The procedure of RVT is well described in the literature[33]. Pregnancies have been documented after RVT[34,35]. Earlier studies showed higher rate of late (>14 weeks) pregnancy losses and premature deliveries, however Dargent and colleagues have reported two procedures that have resulted in the reduction of late losses[34]. The first is the placement of a permanent cervical cerclage and the second is the closure of the cervical os in the third month of pregnancy - with these procedures late pregnancy losses in his series reduced from 50% to 10%. All patients need to be delivered by caesarean section due to presence of the permanent cerclage.

¾ Ovarian carcinoma although less common in premenopausal, these do develop in young women. The vast majority of these are malignant germ cell tumours and sex cord-stromal tumours. These have an overall excellent prognosis, frequently limited to one ovary and usually present at a very early stage without extra ovarian spread. Epithelial ovarian tumours in premenopausal women are often borderline tumours. Surgical staging is the mainstay of treatment but should not result in sterility. Most of these cases can be managed conservatively with preservation of the uterus and normal contralateral ovary. Chemotherapy should be administered as indicated in patients with ovarian carcinoma. The highest rates of amenorrhoea and premature ovarian failure are seen among older women and those who receive alkylating agents such as cyclophosphamide[42]. The current agents used in ovarian carcinoma-carboplatin and paclitaxel have lower rates of permanent ovarian failure. The recurrence and survival rates among the small number of patients with ovarian carcinoma treated conservatively reported in the literature appear to be comparable with those of patients undergone more aggressive surgical procedures[43]. As with all conservative strategies for treating cancer, patients need to understand the limited data and the undefined risk that they are assuming, as well as the intense follow-up that is required.

¾ The endometrial cancer although rarely found in women younger than 25 years age, its reported incidence is 1.2 24 per 100,000 in women aged 25-49 years[36]. Young patients who develop endometrial cancer often have some degree of hyperestrogenism, anovulation, obesity and lipid and carbohydrate imbalance. Nearly half are nulliparous and the vast majority present with abnormal menstrual bleeding[37-40]. Hysterectomy may not be an acceptable option for these women desirous of childbearing. Conservative approach that provides a chance at successful pregnancies and deliveries is possible in a small select group of such young patients with grade I endometriod adenocarcinoma of the uterus confined to the endometrium. The data on conservative management using progestational agents instead of surgery are very limited. Most commonly used progestational agents have been medroxyprogesterone Apollo Medicine, Vol. 3, No. 1, March 2006

NEW OPTIONS FOR FERTILITY PRESERVATION Men Cryopreservation of spermatozoa is now a well established, highly successful technique[44] and fertility preservation by sperm banking should be offered to all post-pubertal males diagnosed with cancer[4]. Spermatozoa are usually obtained from the ejaculate by masturbation. If a semen sample cannot be obtained, electro ejaculation under anaesthesia may be offered as an 18

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alternative to testicular biopsy[4]. Sperm can also be retrieved by epididymal aspiration or testicular biopsy in sexually mature males. Not infrequently semen sample produced by cancer patients at the time of diagnosis is of poor quality. However, with advances in assisted reproductive techniques, in particular intracytoplasmic sperm injection (ICSI), which involves the injection of a single spermatozoa directly into an oocyte, the problems of low numbers and poor motility sperm may be circumvented [45]. It is recognized that any child with testicular volume of greater than 4 mL (Tanner stage 2 or above) should be considered as potentially harbouring mature gametes[4]. Depending upon the number and quality of semen samples obtained, these can be cryopreserved and used later for intrauterine insemination (IUI), IVF or ICSI.

drugs are other agents, tried for super ovulation for IVF in patients with breast cancer[50]. In endometrial cancer patients, aromatase inhibitor agents may be the only safe choice for ovarian stimulation[51]. Mature oocyte cryopreservation ™ Superovulation with gonadotrophins followed by

harvesting of the mature metaphase II oocytes and cryopreserving these for later thawing and fertilization seems to be an attractive alternative for fertility preservation in post-pubertal females without partners. However, the technique is not proved as successful as for the sperm and embryos in humans and not many eggs can be harvested from one stimulated cycle. On average 5-10 oocytes may be harvested per patient with fewer than one baby born per 100 oocytes stored[52].

The cryopreservation of immature sperm, germ cells or immature testicular tissue for boys in whom mature sperm are not produced, has not yet been established as a successful clinical treatment, although live human births resulting from the transfer of embryos fertilized with immature spermatogenic cells have been reported[46].

Mature oocytes are intrinsically fragile cells undergoing division and blocked in the second meiosis metaphase. Exposure to low temperatures and to cryoprotectants can cause depolymerization of the microtubules, accompanied by chromosome dispersion, thereby increasing the risks of aneuploidy in the zygotes. Premature exocytosis of the cortical granules has the effect of hardening the zona pellucida, which may be responsible for the reduced fertilizability of cryopreserved oocytes. Oocyte freezing has enabled a few births recently[53] and the results seem to be getting further enhanced by improvements in oocyte freezing and thawing procedures[54]. Mature oocytes cryopreserved using controlled rate methods currently have around a 50% survival chance and a <10% chance of producing a pregnancy[55,56].

Women Embryo cryopreservation ™ In young sexually mature females with partners,

collection of mature oocyte for in-vitro fertilization and subsequent embryo cryopreservation is an established procedure with proven success. If a partner is not available, the insemination of mature oocytes using donor sperm to create embryos for cryopreservation could be considered but this option raises a number of issues, for example disposal in the event of the mother’s death, the creation of embryos for single women, etc. Once the patient has recovered, these frozen embryos can be reimplanted in the uterus during a spontaneous or stimulated menstrual cycle. Pregnancy rates are between 15-20% for three frozen transferred embryos [47]. The main drawback of embryo storage is the 3-5 weeks time required for super ovulation with gonadotrophins, which will delay the commencement of cancer therapy, and not many embryos can be obtained from one stimulation cycle.

Immature oocyte cryopreservation ™ Fully-grown, immature oocytes may be collected from

regularly menstruating women in whom the ovaries have received minimal or no stimulation with gonadotrophins[57] and cryopreserved using methods similar to those for mature oocytes[55]. Although these immature oocytes are more tolerant to freeze-thawing damage than mature metaphase-II oocytes, the disadvantage is that after thawing, the cells must be matured in-vitro before fertilization. In-vitro maturation (IVM) has proved highly successful in animals[58] but in humans, it is still problematic and clinical success rate is low[59,60]. The potential for detrimental effects upon chromosome movements during oocyte maturation after cryo preservation at an immature stage remains a concern[61]. However, in future with further improvements in culture conditions it may be a viable option.

Conventional superovulation with gonadotrophins raises concerns about their detrimental effect on hormone dependent cancers due to increased oestrogen levels such as for breast and endometrial cancer. Natural (unstimulated) cycle IVF had been suggested and performed in such patients but typically no more than a single embryo can be obtained[48]. Success with ovarian stimulation using tamoxifen with higher number of embryos and pregnancies in breast cancer patients has been reported[49]. Aromatase inhibitor 19

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Vitrification techniques

density, follicle survival after thawing and to search for evidence of any malignant cells by a qualified histopathologist. This information will enable the patient to make an informed decision in due course about the use of the tissue.

™ The term “vitrification” - literally “turned into glass” - is

defined as the conversion of a system from a fluid to a solid solely by an increase in viscosity without a phase change, without any crystallisation of water and therefore in the complete absence of ice[62].

The cryopreserved ovarian tissue has three potential options for its use in future (Fig. 1).

The success rate for conventionally cryopreserved human oocytes has not been impressive though resulting live births have been reported[63,64]. Vitrification methods use cryoprotectant in concentrations that are sufficient to prevent the crystallisation of ice altogether, and this technique using ethandiol has provided some rather encouraging results[65,66]. However, the concentrations of cryoprotectant required are very high and therefore potentially harmful to cells[62]. Further developments in vitrification mixtures with low concentrations that do not require rapid cooling and warming[67,68] are likely to lead to safe and practical vitrification systems. Vitrification also offers the possibility of preferred solution for the cryopreservation of whole organs (ovary and testes) and tissue slices[62], however it is still largely experimental at this stage and further optimization of the procedure is required.

•

The laparoscopic insertion of cortical biopsies inside the pelvis near to the fimbrial end of the fallopian tube raises the hope of fertility recovering spontaneously[76]. In September 2004, a team from Belgium reported the first birth of a human baby following orthotopic transplantation of cryopreserved ovarian tissue in a woman who became infertile after chemotherapy for breast cancer 7 years back[3].

Ovarian cryopreservation

Most of the ovarian cryopreservation and transplantation procedures reported so far have been the transplantation of small avascular ovarian cortical pieces[73,74]. These are grafted without vascular anastomosis and are completely dependent on the establishment of neovascularization after grafting. Consequently, the cells in the graft undergo significant

The limitations encountered by all other methods of fertility preservation led to the idea of ovarian tissue freezing. The cryopreservation of ovarian tissue is fast emerging out as the most promising new option for conserving the fecundity of young cancer patients from the sterilizing effects of chemotherapy and radiotherapy. The ovarian cortex contains very small primordial follicles comprising the ovarian reserve. The cryopreservation of ovarian cortex has been applied in animals with demonstrated fertility in a proportion of those into which the thawed tissue has been transplanted[69].

Cryopreserved ovarian tissue

Ovarian cortex can be removed surgically via laparoscopy using a purpose - designed biopter[70] which removes shallow circles of cortex of about 5 mm in diameter. Alternatively, a part or whole of an ovary can be resected, this can be processed in the laboratory to yield just the surface cortical layer of around 1.0-1.5 mm in thickness. For cryopreservation, the tissue is normally cut into strips upto 5 mm wide. These are equilibrated by rolling for 30 min at 4ºC in medium with 1.5-mol dimethylsulphoxide 1–1 as cryoprotectant using a controlled-rate freezer and a programme similar to that used for embryos and oocytes [71,72]. As this tissue is likely to be in storage for many years, it is important to be aware of its quality upon freezing. Hence, a small sample of the fresh tissue and a small testthaw sample should be processed to check for follicle Apollo Medicine, Vol. 3, No. 1, March 2006

Autrografting of the cryopreserved ovarian tissue at the “orthotopic” site is the only method by which “natural” fertility can be restored[73,74]. Dr. Roger Gosden and his associates in 1994 were first to demonstrate the successful births of healthy animals following autotransplantation of cryopreserved ovarian tissue[75]. Pregnancies have been obtained in this way in several animal species even upto more than two years after re-implantation of the frozen ovarian tissue.

Fig. 1. Cryopreserved ovarian tissue. 20

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ischaemic and reperfusion damage[69,77]. Ovarian vascular transplantation with intact fresh and frozenthawed ovaries has been successfully performed in animals[78,79]. In humans, the feasibility of cryopreserving intact ovary with its vascular pedicle for future vascular transplantation is currently being explored[80]. •

of isolation techniques and culture conditions, immature follicles have demonstrated extensive growth in-vitro [87] and live births following embryo transfers have been reported in animals[88-90]. In humans, the factors that initiate follicle growth are unknown and several months may be required for the primordial follicles to grow to antral sizes[91,92]. Current in-vitro growth protocols are inadequate to sustain this duration of culture.

Grafting the thawed ovarian tissue at a heterotopic site with a rich vascular bed may be an alternative option. Endocrine cycles should still be restored but ovarian stimulation, follicle aspiration and IVF will be needed to conceive. Heterotopic implants of ovarian tissue could be supported at many locations in the body provided they are easily visualized, accessible and do not have a hepatic circulation which may destroy steroid hormones from the graft, reducing pituitary negative feedback, leading to graft hyperstimulation[81]. Reported heterotopic sites have included the subcutaneous tissue of the left axilla and the abdominal wall[18]. First human embryo transfer using oocytes retrieval from cryopreserved ovarian tissue implanted at a heterotopic location beneath the abdominal skin 6 years later, was reported by Oktay, et al in 2004[18].

Overall the ovarian tissue cryopreservation is potentially the most promising fertility preservation strategy for children and young adult females diagnosed with cancer before starting their gonadotoxic chemo or radiotherapy. However, surgical procedure - laparoscopy or laparotomy is required to remove the ovarian tissue. Although considerable time will elapse before these cancer survivors may wish to use their stored gonadal tissue for fertility, allowing time for further improvements and developments of above mentioned techniques, this potential must be balanced against uncertainty of the benefits which might accrue, and the risks of surgical intervention required to obtain the gonadal samples, additional time needed and the psychological risks of raising the hopes and not being able to fulfill them later[4].

However, at present there are concerns and uncertaintities regarding how long the graft will survive and secondly whether the graft will contain malignant cells and thus reintroduce disease to patients in remission[82,83]. This second concern presents a significant risk in patients with bloodborn diseases such as leukaemia or where ovaries are involved in disease [84]. Nevertheless, in many cancers, such as Wilm’s tumour and Hodgkin’s disease, metastasis do not invade the ovary and frozen-banked tissue from these patients should carry low risk of inducing relapse. •

•

PROTOCOL FOR OVARIAN CRYOPRESERVATION [4] Although still a research procedure, ovarian cryopreservation creates the opportunity to elaborate longterm strategies and many centres all over the world since mid 1990’s are banking the frozen ovarian tissue[93]. The multidisciplinary working group convened by the British Fertility Society in 2003 published a series of recommendations and guidelines to define a strategy for fertility preservation including ovarian cryopreservation[4].

Transplantation into a different in-vivo environment “xenografting” is an alternative option to autografting frozen-banked tissue. Ovine, feline and primate ovarian tissue grafted beneath the renal capsules of immune deficient mice were found to revascularize and follicle growth were recorded[85,86]. In theory these antral follicles could be aspirated and the oocytes harvested, matured and fertilized in-vitro. The procedure could prove useful for endangered species, transgenic animals and rare breeds but for obvious ethical and moral implications, xenografting is unfeasible for human clinical application.

COUNSELLING Consideration of fertility preservation is a quality of life issue at a time of intense stress for young patients and their families. Nevertheless, an open discussion of fertility issues is usually embraced and may even provide reassurance to the patient and family facing treatment for cancer that there is hope for a quality future. Independent counselling by a dedicated clinical nurse or practitioner(s) with reproductive medicine experience, preferably given over several days with written information is recommended. Although ongoing support may be indicated throughout the period of diagnosis and treatment, the contribution of a psychosocial specialist is particularly important in relation to fertility issue at the beginning when crucial decisions have to be made.

In-vitro maturation (IVM) represents the third and last option for the use of cryopreserved ovarian tissue. Follicle isolation from ovarian tissue for in-vitromaturation allows malignant cells to be ‘cleaned’ from the banked material of cancer patients. Using a variety 21

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All other possible alternative options including chances of spontaneous pregnancy, embryo or oocyte cryopreservation, donor oocyte programme, surrogacy if uterine function is affected must be informed and discussed. A structured detailed discussion of the rationale behind the ovarian cryopreservation technique and its current lack of a successful track record, technical limitations and future prospects must be undertaken and well documented. The counselling should include a personalized risk and benefit assessment according to the nature of the condition presented, its treatment, chances of survival and chances of infertility resulting. INDICATIONS FOR OFFERING OVARIAN TISSUE CRYOPRESERVATION

• • •

A statement of understanding of the experimental nature of the procedures and their potential hazards.

•

An acceptance of the need to be screened for HIV, Hepatitis B&C.

•

A statement that consent to treatment can not be given at that time and that separate written consent may be obtained from the individual at a later date after proper counselling in an assisted conception units.

•

A statement that future fertility treatment is not assured .

•

Parents, supervising medical staff or family physicians should be asked to inform the ACU in the event of the death of the patient.

Pre-cryopreservation screening

The chance of survival, the risk of sub-fertility and the age of the patient need to be taken into account. However, the following category should be included: •

•

Those having high-dose chemotherapy involving alkayting agents or total body irradiation (TBI). Those having direct pelvic irradiation Those having cycles of MOPP or CHOPP for Hodgkins disease. Those who are HIV and Hepatitis B&C negative.

SEEKING CONSENT There should be structured interviews and the consent should involve if possible a trained psychosocial specialist, oncologist and reproductive medicine specialist. Consent has to be dynamic and interactive process based on giving the information in a way that the person can understand and think about. This is a particular challenge at a time when this task competes with plans to start treatment with fertility damaging, but life-saving agents. Ideally, multiple opportunities should be made available within a short period of time.

•

The recording of height, weight and body mass index.

•

Pelvic or vaginal ultrasound for ovarian volume, its activity and uterine dimensions.

•

Serum luteinizing hormone (LH), follicle stimulating hormone (FSH), Progesterone, oestradiol, testosterone, Inhibin and thyroid function tests (cycle timed when appropriate).

•

HIV, Hepatitis B & C screening.

•

Recording of the diagnosis and stage of disease.

•

Recording of potentially gonadotoxic treatment to date (cumulative dose).

Post cryopreservation record • • • • •

In UK, gamete cryopreservation is governed by Human Fertilization and Embryo act 1990[93]. The age for giving consent is 18 years in England and Wales and 16 years in Scotland. Below that parents can give the consent in the “best interests of the child”.

Further follow-up • •

The consent should include: • • • • •

•

Written consent to obtain germ-cell tissue. Written consent to cryopreserve the germ-cell tissue. Written consent to dispose off the tissue in the event of death or incapacitation. A written agreement to offer part of the tissue obtained for research. A written agreement to donate the tissue for research in the event of death.

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The decision and clinical outcome in all patients counselled. Any data on the quality of germ-cell tissue sample and gametes obtained. Any peri-operative complications of tissue retrieval. Counselling given. The time taken from counselling to tissue retrieval.

•

Regular contact with the storage facility should be maintained. A register of all patients, with appropriate consent, should be maintained Endocrine assessments should be made in collaboration with the late-effects clinic and an assisted conception unit Information about log-term access to psychological support should be provided.

CONCLUSION Improved survival rates in cancer patients bring new awareness and interest in the “quality of life issues” such as 22

Theme Symposium

reproduction. Since all strategies for fertility preservation must be utilized before the therapy for cancer is initiated, a high level of awareness of these technologies is essential among oncologists, radiation and chemotherapists and other professionals treating cancer patients. The strategy should be tailored according to the patient’s age, presence of a partner, type of malignant disease, therapeutic agent and time interval available.

Squifflet J, Martinez Mandrid B, Van Langendonckt A, et al. Live birth after orthotopic transplantation of cryopreserved ovarian tissue. Lancet, published online September 24, 2004. 4. Multidisciplinary Working Group convened by the British Fertility Society. A strategy for fertility services for survivors of childhood cancer. Human Fertility 2003; 6,2: A1-A40. 5. Morita Y, Perez GI, Paris F, et al. Oocyte apoptosis is suppressed by disruption of acid sphingomyelinase gene or by sphingosine-1-phosphate therapy. Nat Med 2000; 6: 1109-1114.

Limitation of radiation exposure by shielding of the testes and ovaries should be practiced in all cases possible. Fertility sparing strategies such as ovarian transposition, conservative surgery for ovarian cancer, progestins for early endometrial cancer and radical vaginal trachelectomy (RVT) for cancer cervix early stage - should be considered in all young cancer patients wherever feasible.

6. Mackie EJ, Radford M, Shalet SM, et al. Gonadal function following chemotherapy for childhood Hodgkin’s disease. Med Pediatre Oncol 1996; 27: 74-78. 7. Viviani S, Sanoro A, Ragni G, et al. Gonadal toxicity after combination chemotherapy for Hodgkin’s disease: comparative results of MOPP vs ABVD. Eur J Cancer Clin Oncol 1985; 21: 601-605.

Sperm banking should be offered to all sexually mature boys at risk of infertility due to cancer therapy. Recent breakthrough in the field of assisted reproduction technologies (IVF with ICSI), cryobiology (cryopreservation of gametes and gonadal tissue) and autotransplantation of cryopreserved ovarian tissue have unfolded a totally new horizon. While semen and embryo cryopreservation are now established procedures, oocyte (mature or immature) cryopreservation has limited application. Ovarian tissue cryopreservation at present, is an experimental technique, although successful pregnancy following its autotransplantation in human has recently been reported[3].

8. Wallace WHB, Thomson AB, Kelsey TW, et al. the radiosensitivity of the human oocyte. Hum Reprod 2003; 18: 117-121. 9. Nicholson HS, Byme J, et al. Fertility and pregnancy after treatment for cancer during childhood or adolescence. Cancer 1993; 71: 3392-3399. 10. Leiper AD, Grant DB, Chessells JM, et al. Gonadal function after testicular radiation for acute lymphoblastic leukaemia. Arch Dis Child 1986; 61: 53-56. 11. Speiser B, Rubin P, Casarett G, et al. Azoospermia following lower truncal irradiation in Hodgkin’s disease. Cancer 1973; 32: 692-696.

Ovarian cryopreservation is intended to create future opportunities for these patients. The rapidly advancing experimental techniques for harvesting and cryopreserving the gonadal tissue should be discussed and embarked on, without unrealistic expectations. Close liaison between oncology unit and assisted conception unit is extremely important. Patient commitment and the intense multidisciplinary nature of treatment must be recognised. Psychological support and counselling is critical in helping the young patients and their families in making difficult decisions and coping with the treatment and its late effects. Medical record keeping, long term follow-up and prospective physical and mental health research in this highly sensitive area are vital to improve the quality of life for the increasing number of cancer survivors.

12. Shalet SM, Tsatsoulis A, Whitehead E, et al. Vulnerability of the human leyding cell to radiation damage is dependent upon age. J Endocrinol 1989; 120: 161-165. 13. Critchley HOD, Wallace WHB, Shalet SM, et al. Abdominal irradiation in childhood: the potential for pregnancy. Br J Obstet Gynecol 1992; 99: 392-394. 14. Salle B and Lornage J. Fertility preservation techniques in 2004. In: Updates in Infertility Treatment 2004 Filicori Marco (Ed). Medimond International Proceedings Division, Bologna, Italy. 2004, 127-137. 15. Sanders JE, Hawley J, Levy W, et al. Pregnancies following high-dose cyclophosphamide with or without high-dose busulfan or total-body irradiation and bone marrow transplantation. Blood 1996; 87: 3045-3052. 16. Critchley HOD, Bath LE and Wallace WHB, et al. Radiation damage to the uterus - review of the effects of treatment of childhood cancer. Hum fertile 2002; 5: 61-66.

REFERENCES 1. Boring CC, Squires TS, Tong T, Montgomery S, et al. Cancer Statistics. CA Cancer J Clin 1994; 44: 7-26.

17. Blumenfeld Z, Avivi I, Linn S, et al. Prevention of irreversible chemotherapy-induced ovarian damage in young women with lymphoma by a Gonadotrophinreleasing hormone agonist in parallel to chemotherapy. Hum Reprod 1996; 11: 1620-1626.

2. Wallace WH, Blacklay A, Eiser C, Davies H, Hawkins M, Levitt GA, Jenney ME, et al. Developing strategies for long-term follow-up of survivors of childhood cancer. British Med J 2001; 323: 271-274.

18. Oktay K, Buyuk E, Veeck L, Zaninovic N, Xu K, Takeuchi T, opsahl M, Rosenwaks Z, et al. Embryo development after

3. Donnez J, Dollmans M, Demylle D, Jadoul P, Pirard C, 23

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Theme Symposium heterotopic transplantation of cryopreserved ovarian tissue. The Lancet 2004; 363(9412): 837-840.

Operative Techniques in Gynacologic Surgery Gerhenson D, Fowler M. (eds.). Philadelphia: WB Saunders. 1997, 187-199.

19. Thomson AB, Anderson RA, Irvine DS, et al. Investigation of suppression of the hypothalamic-pituitary-gonadal axis to restore spermatogenesis in Azoospermia men treated for childhood cancer. Hum Reprod 2002; 17: 1715-1723.

34. Mathevet P, Laszlo de Kaszon E, Dargent D, et al. Fertility preservation in early cervical cancer. Gynaecol Obstet Fertil 2003; 31: 706-712.

20. Masala A, Faedda R, Alagna S, et al. Use of testosterone to prevent cyclophosphamide-induced azoospermia. Ann Intern Med 1997; 126: 292-295.

35. Bernardini M, Barrett J, Seawood G, et al. Pregnancy outcomes in patients after radical trachelectomy. Am J Obstet Gynaecol 2003; 189: 1378-1382.

21. Shetty G, Meistrich ML. Hormonal approaches to preservation and restoration of male infertility after cancer treatment. J National cancer Inst 2005; 34: 36-39.

36. Jemal A, Murray T, Ward E, et al. Cancer statistics, 2005. CA Cancer J Clin 2005; 55: 10-30. 37. Crissman JD, Azoury RS, Barnes AE, et al. Endometrial carcinoma in women 40 years of age or younger. Obstet Gynaecol 1981; 6: 699-704.

22. McCall ML, Keaty EC, Thompson JD, et al. Conservation of ovarian tissue in the treatment of carcinoma of the cervix with radical surgery. Am J Obstet Gynaecol 1958; 75: 590600.

38. Gallup DG, Stock RJ, et al. Adenocarcinoma of the endometrium in women 40 years of age or younger. Obstet Gynaecol 1984; 64: 417-420.

23. Anderson B, LaPolla J, Turner D, et al. Ovarian transposition in cervical cancer. Gynaecol Oncol 1993; 49: 206-214.

39. Evans-Metcalf ER, Brooks SE, reale FR, et al. Profile of women 45 years of age and younger with endometrial cancer. Obstet Gynaecol 1998; 91: 349-354.

24. Morice P, Juncker L, Rey A, et al. Ovarian transposition for patients with cervical carcinoma treated by radiosurgical combination. Fertil Steril 2000; 74: 743-748.

40. Geisler HE, Huber CP, Rogers S, et al. Carcinoma of the endometrium in premenopausal women. Am J Obstet Gynaecol 1969; 104: 657-663.

25. Morice P, Haie-Meder C, Pautier P, et al. Ovarian metastasis on transposed ovary in patients treated for squamous cell carcinoma of the uterine cervix: report of two cases and surgical implications. Gynaecol Oncol 2001; 83: 605-607.

41. Ramirez PT, Frumovitz M, Bodurka DC, et al. Hormonal therapy for the management of grade-I endometrial adenocarcinoma: A literature review. Gynaecol Oncol 2004; 95: 133-138.

26. Morice P, Thiam-Ba R, Castaigne D, et al. Fertility results after ovarian transposition for pelvic malignancies treated by external irradiation or brachytherapy. Hum Reprod 1998; 13: 660-663.

42. Meirow D, Nugent D, et al. The effects of radiotherapy and chemotherapy on female reproduction. Hum Reprod Update 2001; 7: 535-543. 43. Schilder JM, Thompson AM, DePriest PD, et al. Outcome of reproductive age women with stage IA or IC invasive epithelial ovarian cancer treated with fertility-sparing therapy. Gynaecol Oncol 2002; 87: 1-7.

27. Giacalone PL, Laffargue F, Benos P, et al. Successful in vitro fertilization-surrogate pregnancy in a patient with ovarian transposition who had undergone chemotherapy and pelvic irradiation. Fertil Steril 2001; 76: 388-389.

44. Sanger WG, Olson JH, Sherman JK, et al. Semen cryobanking for men with cancer : criteria change. Fertile Steril 1992; 58: 1024-1027.

28. Sonoda Y, Abu-Rustum NR, Gerignani ML, et al. A fertilitysparing alternative to radical hysterectomy: How many patients may be eligible? Gynaecol Oncol 2004; 95: 534538.

45. Aboulghar MA, Mansour RT, Serour GI, et al. Fertilization and pregnancy rates after intracytoplasmic sperm injection using ejaculate semen and surgically retrieved sperm. Fertil Steril 1997; 68: 108-1011.

29. Benedet JL, Anderson GH, et al. Stage IA carcinoma of the cervix revisited. Obstet Gynaecol 1996; 87: 1052-1059. 30. Ueda M, Ueki K, Kanemura M, et al. Conservative excisional laser conization for early invasive cervical cancer. Gynaecol Oncol 2004; 95: 231-234.

46. Tesarik J, Mendoza C, Testart J. Viable embryos from injection of round spermatids into oocytes. N Engl J Med 1995; 333: 525.

31. Roman LD, Felix JC, Muderspach LI, et al. Risk of residual invasive disease in women with microinvasive squamous cancer in a conization specimen. Obstet Gynaecol 1997; 90: 759-764.

47. Edgar DH, Bourne H, Speirs AL, McBain JC. A quantitative analysis of the impact of cryopreservation on the implantation potential of human early cleavage stage embryos. Hum Reprod 2000; 15: 175-179.

32. Dargent D, Brun JL, Roy M, et al. The radical trachelectomy: An alternative to radical hysterectomy in the treatment of invasive cancers that develop on the external os of the uterine cervix. J Obstet Gynaecol 1994; 2: 285-292.

48. Omland AK, Fedorcsak P, Storeng R, Dale PO, Abyholm T, Tanbo T, et al. Natural cycle IVF in unexplained, endometriosis-associated and tubal factor infertility. Hum Reprod 2001; 16: 2587-2592. 49. Oktay K, Buyuk E, Davis O, Yermakova I, Veeck L,

33. Plante M, Roy M, et al. Radical trachelectomy. In: Apollo Medicine, Vol. 3, No. 1, March 2006

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Theme Symposium Rsenwaks Z, et al. Fertility preservation in breast cancer patients: IVF and embryo cryopreservation after ovarian stimulation with tamoxifen. Human Reproduction 2003; 18,1: 90-95.

63. Kyono K, Fuchinoue K, Yagi A, Nakajo Y, Yamashita A, Kumagai S, et al. Successful pregnancy and delivery after transfer of a single blastocyst derived from a vitrification mature human oocyte. Fertile Steril 2005; 84(4): 1017.

50. Fisher SA, Reid RL, Van Vugt DA, Casper RF, et al. A randomized double-blind comparison of the effects of Clomiphene citrate and the aromatase inhibitor letrozole on ovulatory function in normal women. Fertil Steril 2002; 78: 280-285.

64. Paynter SJ, et al. Current status of cryopreservation of human unfertilized oocytes. Hum Reprod Update 2000; 6: 449-456. 65. Kuleshova L, Gianaroli L, Magli C, Ferraretti A, Trounson A, et al. Birth following vitrification of a small number of human oocytes. Hum Reprod 1999; 14: 3077-3079.

51. Oktay K, Sonmerzer M, et al. Ovarian tissue banking for cancer patients: fertility preservation, not just ovarian cryopreservation. Hum Reprod 2004; 19(3): 477-480.

66. Kuleshova LL, Lopata A, et al. Vitrification can be more favorable than slow cooling. Fertil Steril 2002; 78: 449-454.

52. Porcu E, Fabbri R, Seracchiolo R, et al. Birth of a healthy female after intracytoplasmic sperm injection of cryopreserved human oocytes. Fertil Steril 1997; 68: 724-726.

67. Armitage WJ, Hall SC, Routledge C, et al. Recovery of endothelial function after vitrification of cornea at –110ºC. Investigative Ophthalmology & Visual Science 2002; 43: 2160-2164.

53. Porcu E, Fabbri R, Ciotti PM, Petracchi S, Seracchioli R, Flamigni C, et al. Ongoing pregnancy after intracytoplasmic sperm injection of testicular spermatozoa into cryopreserved human oocytes. Am J Obstet Gynaecol 1999; 180:1044-1045.

68. Fahy GM, Wowk B, Wu J, Paynter S, et al. Improved vitrification solutions based on the predictability of vitrification solution toxicity. Cryobiology 2004; 48: 22-35. 69. Baird DT, Webb R, Campbell BK, Harkness LM, Gosden RG, et al. Long-term ovarian function in sheep after ovariectomy and transplantation of autografts stored at – 196ºC. Endocrinology 1999; 140: 462-471.

54. Porcu E, Fabbri R, Ciotti PM, Petracchi S, Seracchioli R, Flamigni C, et al. Ongoing pregnancy after intracytoplasmic sperm injection of epididymal spermatozoa into cryopreserved human oocytes. J Assist Reprod Genet 1999; 16: 283-285.

70. Meirow D, Fasouliotis SJ, Nugent D, Schenker JG, Gosden RG, Rutherford AJ, et al. A laparoscopic technique for obtaining ovarian cortical biopsy specimens for fertility conservation in patients with cancer. Fertil Steril 1999; 71: 948-951.

55. Tucker MJ, Morton PC, Wright G, Sweitzer CL, Massey JB, et al. Clinical application of human egg cryopreservation. Hum Reprod 1998; 13: 3156-3159. 56. Porcu E, Fabbri R, Damiano G, Giunchi S, Fratto R, Ciotti PM, Venturoli S, Flamigni C, et al. Clinical experience and applications of oocyte cryopreservation. Molecular and Cellular Endocrin 2000; 169: 33-37.

71. Newton H, Aaubard Y, Rutherford A, Sharma V, Gosden R, et al. Low temperature storage and grafting of human ovarian tissue. Hum Reprod 1996; 11: 1487-1491. 72. Newton H, Fisher J, Arnold JR, Pegg DE, Faddy MJ, Gosden RG, et al. Permeation of human ovarian tissue with cryoprotective agents in preparation of cryopreservation. Hum Reprod 1998; 13: 376-380.

57. Mikkelsen AL, Smith SD, Lindenberg S, et al. In vitro maturation of human oocytes from regularly menstruating women may be successful without follicle stimulating hormone priming. Hum Reprod 1999; 14: 1847-1851.

73. Gosden RG, Aubard Y, et al. Transplantation of ovarian and testicular tissue. Medical Intelligence Unit 1996; RG Landes Co, Austin, Texas.

58. Trounsen A, Wood C and Kausche A. In vitro maturation and the fertilization and development competence of oocytes recovered from untreated polycystic ovarian patients. Fertil Steril 1994; 62: 353-362.

74. Nugent D, Meirow D, Brook P, et al. Transplantation in reproductive medicine: previous experience, present knowledge and future prospects. Hum Reprod Update 1997; 3: 267-280.

59. Cha KY, Koo JJ, Ko JJ, et al. Pregnancy after in vitro fertilization of human follicular oocytes collected from nonstimulated cycles, their culture in vitro and their transfer in a donor oocyte program. Fertil Steril 1991; 55: 109-113.

75. Gosden RG, Baird DT, Wade JC, Eb R, et al. Restoration of fertility to oophorectomized sheep by ovarian autografts stored at –196ºC. Hum Reprod 1994; 9(4): 597-603.

60. Trounsen A, Anderies C Janes G. Maturation of human oocytes in vitro and their developmental competence. Reprod 2001; 121: 51-75.

76. Aubard Y, Piver P, Cogni Y, Fereaux V, Poulin N, Driancourt MA, et al. Orthotopic and heterotopic autografts of frozen-thawed ovarian cortex in sheep. Hum Reprod 1999; 14: 2149-2154.

61. Park SE, Son WY, Lee SH, Lee KA, Ko JJ, Cha KY, et al. Chromosome and spindle configurations of human oocytes matured in vitro after cryopreservation at the germinal vesicle stage. Fertil Steril 1997; 68: 920-926.

77. Baird DT, Webb R, Campbell BK, Harkness LM, Gosden RG, et al. Long-term ovarian function in sheep after ovariectomy and transplantation of autografts stored at 196ºC. Endocrin 1999; 140: 462-471.

62. Pegg David E, et al. The role of vitrification techniques of cryopreservation in reproductive medicine. Hum Fertil 2005; 8,4: 231-239.

78. Wang X, Chen H, Yin H, Kim SS, Tan SL, Gosden RG, 25

Apollo Medicine, Vol. 3, No. 1, March 2006

Theme Symposium et al. Fertility after intact ovary transplantation. Nature 2002; 415: 385.

development in cryopreserved marmoset ovarian tissue after transplantation. Hum Reprod 1995; 10: 2334-2338.

79. Yin H, Wang X, Kim SS, Chen H, Tan SL, Gosden RG, et al. Transplantation of intact rat gonads using vascular anastomosis: effects of cryopreservation, ischaemia and genotype. Hum Reprod 2003; 18: 1165-1172.

87. Hartshorne GM, et al. In vitro culture of ovarian follicles. Rev Reprod 1997; 2: 94-104. 88. Spears N, Boland NI, Murray AA, Gosden RG, et al. Mouse oocytes derived from in vitro grown primary ovarian follicles are fertile. Hum Reprod 1994; 9: 527-532.

80. Martinez-Madrid B, Dolmans M, Langendonckt AV, Defrere S, Donnez J, et al. Freeze-thawing intact human ovary with its vascular pedicle with a passive cooling device. Fertil Steril 2004; 82,5: 1390-1394.

89. Cortvindt R, Smitz J, van Steirteghem AC, et al. In vitro maturation, fertilization and embryo development of immature oocytes from early prenatal follicles from prepubertal mice in a simplified culture system. Hum Reprod 1996; 11: 2656-2666.

81. Biskind GR, Biskind MS, et al. Experimental ovarian tumours in rats. Am J Clin Path 1949; 19: 501-520. 82. Gosden RG, Rutherford AJ, Norfolk DR, et al. Transmission of malignant cells in ovarian grafts. Hum Reprod 1997; 12: 403.

90. Cortvrindt R, Smitz J, Van Steirteghem AC, et al. A morphological and functional study of the effect of slow freezing followed by complete in vitro maturation of primary mouse ovarian follicles. Hum Reprod 1996; 11: 2648-2655.

83. Shaw J, Trounson A, et al. Oncological implications in the replacement of ovarian tissue. Hum Reprod 1997; 12: 403405.

91. Gougeon A, et al. Dynamics of follicular growth in the human: a model from preliminary results. Hum Reprod 1986; 1: 81-87.

84. Shaw JM, Bowles J, Koopman P, et al. Fresh and cryopreserved ovarian tissue samples from donors with lymphoma transmit the cancer to graft recipients. Hum Reprod 1996; 11: 1668-1673.

92. Gougeon A, et al. Regulation of ovarian follicular development in primates: Facts and hypotheses. Endocrine Rev 1996; 17: 121-155.

85. Gosden RG, Boulton MI, grant K, Webb R, et al. Follicular development from ovarian xenografts in SCID mice. J Reprod Fertil 1994; 101: 619-623.

93. Poirot C, Vacher-Lavenue MC, Helardol P, et al. Human ovarian tissue cryopreservation: indications and feasibility. Hum Reprod 2002; 17: 1447-1452.

86. Candy CJ, Wood MJ, Whittingham DG, et al. Follicular

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