Ovarian tissue cryopreservation and transplantation in cancer patients




Advances in the diagnosis and treatment of childhood, adolescent and adult cancer have greatly increased the life expectancy of premenopausal women with cancer.


The ovaries are very sensitive to cytotoxic treatment, especially to alkylating agents.


The only established method of fertility preservation is embryo cryopreservation according to the Ethics Committee of the American Society for Reproductive Medicine (2005), but this option requires the patient to be of pubertal age, have a partner or use donor sperm and be able to undergo a cycle of ovarian stimulation, which is not possible when the chemotherapy has to be initiated immediately or when stimulation is contraindicated, according to the type of cancer.


For patients who need immediate chemotherapy, cryopreservation of ovarian tissue is the only possible alternative.


This article reports the techniques and results of orthotopic transplantation of cryopreserved ovarian tissue. Among almost 30 cases reported in the literature, six live births have been achieved to date.


According to previous reports, more or less 700 000 new cancers are expected every year in the female population in the United States ; of these women, 8% will be under the age of 40. By 2010, it is estimated that one in every 250 people in the adult population will be childhood cancer survivors.


Advances in the diagnosis and treatment of childhood, adolescent and adult cancer have greatly increased the life expectancy of premenopausal women with cancer. Indeed, aggressive chemotherapy and radiotherapy, as well as bone marrow transplantation, can cure more than 90% of girls affected by childhood malignancies , but have resulted in a growing population of adolescent and adult long-term survivors of childhood malignancies , who may experience infertility problems due to induced premature ovarian failure (POF).


Gonadotoxicity of chemotherapy ( Table 1 )




  • 1.

    The ovaries are very sensitive to cytotoxic treatment, especially to alkylating agents (e.g., cyclophosphamide, busulfan, melphalan, chlorambucil, dacarbazine, procarbazine, ifosfamide, thiotepa and nitrogen mustard), which are classified as high risk for gonadal dysfunction (for review, see ref ).



    Table 1

    Cytotoxic agents according to degree of gonadotoxicity.












    High risk Intermediate risk Low/no risk
    Cyclophosphamide
    Busulfan
    Melphalan
    Cholarambucil
    Dacarbazine
    Procarbazine
    Ifosfamide
    Thiotepa
    Nitrogen mustard
    Adriamycin (Doxorubicin)
    Cisplatin
    Carboplatin
    Methotrexate
    Bleomycin
    5-Fluorouracil
    Actinomycin D
    Mercaptopurine
    Vincristine


  • 2.

    Follicular destruction induced by alkylating agents generally results in loss of both endocrine and reproductive function, depending on the dose and age of the patient. Indeed, Larsen et al. reported a fourfold increased risk of POF in teenagers treated for cancer, and a risk increased by a factor of 27 in women between 21 and 25 years of age. Complete amenorrhoea was reported after a dose of 5 g of cyclophosphamide in women over 40 years of age, and after doses of 9 g and 20 g in women of 30–40 and 20–30 years of age respectively. A combination of various chemotherapeutic agents further increases gonadal toxicity. After MOPP/ABV hybrid chemotherapy, Schilsky et al. found that amenorrhoea developed in 89% and 20% of patients over and under 25 years of age at the time of treatment, respectively. The median age of patients who became amenorrhoeic after therapy was significantly higher than that of patients who maintained normal menses (26 years vs. 20 years; p = 0.008).


  • 3.

    Cyclophosphamide is the agent most commonly implicated in causing damage to oocytes and granulosa cells in a dose-dependent manner.


  • 4.

    Procarbazine has recently been described as high risk for inducing premature menopause. Ten years after treatment, the actuarial risk of premature menopause was 84% after high cumulative doses (>8–4 g m −2 ), and 15% after low doses (≤4.2 g m −2 ) of procarbazine. The authors concluded that as long as alkylating agents are used to treat Hodgkin’s lymphoma, premature menopause will remain a frequent adverse effect of treatment, with various clinical implications.





Radiotherapy


Abdominal ionising radiation associated with alkylating agents often induces POF, rendering patients infertile in almost 100% of cases. Indeed, for radiotherapy, it has been stated that a dose of 5–20 Gy administered to the ovary is sufficient to completely impair gonadal function , whatever the age of the patient. The dose of radiation required to destroy 50% of the oocyte reserve has been found to be <2 Gy. Moreover, uterine irradiation at a young age reduces adult uterine volume. Radiation doses between 14 and 30 Gy have been reported to result in uterine dysfunction. The practitioner should be aware of this effect of radiotherapy on the uterus, which could interfere with the implantation capacity of embryos.




Radiotherapy


Abdominal ionising radiation associated with alkylating agents often induces POF, rendering patients infertile in almost 100% of cases. Indeed, for radiotherapy, it has been stated that a dose of 5–20 Gy administered to the ovary is sufficient to completely impair gonadal function , whatever the age of the patient. The dose of radiation required to destroy 50% of the oocyte reserve has been found to be <2 Gy. Moreover, uterine irradiation at a young age reduces adult uterine volume. Radiation doses between 14 and 30 Gy have been reported to result in uterine dysfunction. The practitioner should be aware of this effect of radiotherapy on the uterus, which could interfere with the implantation capacity of embryos.




Chemotherapy and total body irradiation before bone marrow transplantation


Intensive chemotherapy and/or total body irradiation (TBI) required before bone marrow transplantation (BMT) constitute the treatment combination presenting the greatest risk of premature ovarian failure. Indeed, these high doses of chemotherapy (commonly using the highly cytotoxic cyclophosphamide/busulfan regimen) and/or radiotherapy lead to subsequent ovarian failure in almost all cases, children and adults alike. The risk of premature ovarian failure was estimated to be 92% in the study by Meirow and Nugent , and 100% in an earlier study by Teinturier et al. Teinturier et al. actually reported 0% of ovarian recovery after busulfan treatment before BMT. A large retrospective survey of pregnancy outcomes after haematopoietic stem cell transplantation (HSCT) (peripheral blood or BMT) involving 37 362 patients revealed that only 0.6% of patients conceived after autologous or allogeneic SCT. It is thus obvious that high doses of alkylating agents, irradiation and advancing age increase the risk of gonadal damage.


Cryopreservation should not be reserved solely for women with malignant disease. Indeed, HSCT has been increasingly used in recent decades for non-cancerous diseases, such as benign haematological disease (sickle cell anaemia, thalassaemia major and aplastic anaemia) and autoimmune diseases previously unresponsive to immunosuppressive therapy (systemic lupus erythematosus (SLE) and autoimmune thrombocytopaenia). Other benign diseases, such as recurrent ovarian endometriosis or recurrent ovarian mucinous cysts, are also indications for ovarian cryopreservation. Patients undergoing oophorectomy for prophylaxis may potentially benefit from ovarian cryopreservation too. The indications for cryopreservation of ovarian tissue in case of malignant and non-malignant disease are summarised in Table 2 .



Table 2

Indications for cryopreservation of ovarian tissue in case of malignant and non-malignant disease.













































































A. Malignant
a) Extrapelvic diseases
Bone cancer (osteosarcoma – Ewing’s sarcoma)
Breast cancer
Melanoma
Neuroblastoma
Bowel malignancy
b) Pelvic diseases
– Non-gynecological malignancy
Pelvic sarcoma
Sacroblastoma
Rhabdomyosarcoma
Sacral tumors
Rectosigmoid tumors
– Gynecological malignancy
Early cervical carcinoma
Early vaginal carcinoma
Early vulvar carcinoma
Selected cases of ovarian carcinoma (stage IA)
Borderline ovarian tumors
c) Systemic diseases
Hodgkin’s disease
Non-Hodgkin’s lymphoma
Leukemia
Medulloblastoma
B. Non-malignant
a) Uni/bilateral oophorectomy
Benign ovarian tumors
Severe and recurrent endometriosis
BRCA-1 or BRCA-2 mutation carriers
b) Risk of premature menopause
Turner’s syndrome
Family history
Benign diseases requiring chemotherapy: autoimmune diseases (systemic lupus erythematosus, rheumatoid arthritis, Behçet’s disease, Wegener’s granulomatosis)
c) Bone marrow transplantation
Benign hematological diseases: sickle cell anemia, thalassemia major, aplastic anemia
Autoimmune diseases unresponsive to immunosuppressive therapy




Fertility preservation in cancer patients: different cryopreservation options


The only established method of fertility preservation is embryo cryopreservation, according to the Ethics Committee of the American Society for Reproductive Medicine , but this option requires the patient to be of pubertal age, have a partner or use donor sperm and be able to undergo a cycle of ovarian stimulation, which is not possible when chemotherapy has to be initiated immediately or when stimulation is contraindicated according to the type of cancer.


Ovarian tissue cryopreservation


For patients who need immediate chemotherapy, cryopreservation of ovarian tissue is the only possible alternative. The main aim of this strategy is to reimplant cortical ovarian tissue into the pelvic cavity (orthotopic site) or a heterotopic site such as the forearm or abdominal wall once treatment is completed and the patient is free of disease.


Human ovarian tissue can be successfully cryopreserved, showing good survival and function after thawing. After reviewing all relevant studies since 1996, Hovatta concluded that adequate penetration of cryoprotectant through the stroma and granulosa cells to the oocytes is necessary for satisfactory results. Ice crystal formation must also be minimised by choosing optimal freezing and thawing rates. The choice of cryoprotectant with maximum permeation capacity but minimum toxicity and ice crystal formation potential is specific to each cell and tissue type. In the ovary, it is a compromise between the stroma, the follicular cells and the oocytes. On the basis of current knowledge, the standard method for human ovarian cryopreservation is slow-programmed freezing using human serum albumin-containing medium and propanediol, dimethylsulphoxide (DMSO) or ethylene glycol as a cryoprotectant, combined or not with sucrose.


Autotransplantation of cryopreserved human ovarian tissue


Reported cases of autotransplantation of cryopreserved ovarian tissue, either to an orthotopic or heterotopic site, are summarised on the ISFP (International Society for Fertility Preservation) website ( www.isfp-fertility.org ), detailing, in each case, the age of the patient before freezing, whether she received chemotherapy before freezing, the indications for cryopreservation, the graft site and size, the interval before recovery of ovarian function after grafting and the outcome of transplantation.


Orthotopic autotransplantation of cryopreserved human ovarian tissue


In theory, natural pregnancy may be achieved via orthotopic tissue transplantation if the fallopian tubes remain intact.


In 2000, Oktay and Karlikaya reported laparoscopic transplantation of frozen–thawed ovarian tissue in a 29-year-old patient, who had undergone bilateral oophorectomy for intractable menorrhagia. The reason for this bilateral oophorectomy was not made clear and data on restoration of ovarian function were never described for some unknown reason.


Radford et al. reported a patient with a history of Hodgkin’s disease treated by chemotherapy. Ovarian tissue was biopsied and cryopreserved 4 years after chemotherapy. Histological section of the ovarian cortical tissue revealed only a few primordial follicles due to the previous chemotherapy. Eight months after reimplantation, oestradiol was detected and follicle-stimulating hormone (FSH) and luteinising hormone (LH) levels decreased but, 1 month later, FSH and LH concentrations returned definitively to menopausal levels.


We reported the first successful transplantation of cryopreserved ovarian tissue resulting in a pregnancy and live birth. In 1997, a 25-year-old woman presented with clinical stage IV Hodgkin’s lymphoma. Ovarian tissue cryopreservation was undertaken before chemotherapy. After laparoscopy, the patient received MOPP/ABV hybrid chemotherapy (chlorethamine, vincristine, procarbazine, prednisone, doxorubicin, bleomycin and vinblastine) from August 1997 to February 1998, followed by supradiaphragmatic radiotherapy (38 Gy). According to Schilsky et al , the risk of POF after such a regimen in a woman aged 26 years is more than 90%, while according to Wallace et al. and Lobo , the risk of subfertility after Hodgkin’s treatment with alkylating agents is more than 80%. Indeed, not only the type of drug and dose, but also age, are important factors when evaluating the risk of POF after chemotherapy. In 2003, once the patient had been declared completely free of disease, transplantation was carried out (see Donnez et al. for techniques). A large strip and 35 small cubes of frozen–thawed ovarian tissue were implanted into a furrow created by the peritoneal window very close to the ovarian vessels and fimbria on the right side ( Fig. 1 ). Four months after transplantation, a laparoscopy was carried out to check the viability of the orthotopic graft and re-implant the remaining 32 ovarian cortical cubes. From 5 months to 9 months after re-implantation, concentrations of FSH, 17 β-oestradiol and progesterone showed the occurrence of ovulatory cycles. At 11 months, the patient became pregnant and subsequently delivered a healthy baby. This birth was announced in the Lancet in September 2004.




Fig. 1


A large strip and 35 small cubes of frozen-thawed ovarian tissue were implanted in a furrow created by the peritoneal window very close to the ovarian vessels and fimbria.


In 2006, restoration of ovarian function after orthotopic (intra-ovarian and para-ovarian) transplantation of cryopreserved ovarian tissue was reported in a woman treated by BMT for a non-cancerous disease (sickle cell anaemia). Vaginal echography and sequential measurement of FSH, LH, 17 β-oestradiol and progesterone concentrations revealed the onset of an ovulatory cycle 4½ months after re-implantation of ovarian tissue.


We applied a technique similar to that used by Silber et al. for the transplantation of fresh ovarian cortex between monozygotic twins in a woman who had also undergone BMT and two regimens of alkylating agents in 2000 for non-Hodgkin’s lymphoma. Cryopreservation of ovarian tissue was carried out 1 year after first-line chemotherapy. One ovary was removed and biopsies of cortical ovarian tissue revealed the presence of histologically normal primordial follicles. Six ovarian cortical pieces measuring 10 × 4–5 mm were then grafted onto the remaining ovary after the cortex of this ovary had been removed ( Fig. 2 ). The goal was to ‘rebuild’ an ovary. It was 5 months before a mature follicle (21 mm) developed and an increase in oestradiol levels (194 pg ml −1 ) was noted. The patient experienced an ovulatory cycle every 5 weeks, the preovulatory oestradiol level reaching values between 210 and 356 pg ml −1 .




Fig. 2


Ovarian cortical pieces measuring 4–5 mm to 1 cm in size were grafted onto the remaining ovary after the cortex of this ovary had been removed. a: Cortex of the remaining ovary was removed. b: Cortical pieces were sutured with 7-0 stitches.


In total, nine transplantations have been carried out in our department, according to the previously described technique.


Analysis of these cases raises some important points for discussion. First of all, in all cases, it took between 4½ months and 5 months after reimplantation before a follicle could be seen. The process of folliculogenesis takes ∼4–6 months, during which time the oocyte and surrounding somatic cells undergo a series of changes that eventually result in the development of a large antral follicle, capable of producing a mature oocyte. Thus, the appearance of the first follicle originating from the grafted tissue 5 months after re-implantation, proved by laparoscopy in one case, is totally consistent with the expected time course. This time interval between implantation of cortical tissue and the first oestradiol peak is also consistent with data obtained from sheep and humans, although some variations may be observed. Indeed, as discussed in a recent review , the delay between transplantation and follicular development was found to vary from 6 weeks to 8 months. Such a variation could be explained by a difference in follicular reserve at the time of cryopreservation.


Another very interesting finding is the persistence of relatively high FSH levels during the follicular phase. FSH levels remained as high as 25 mIU ml −1 during the follicular phase until ovulation, and then decreased to less than 15 mIU ml −1 during the luteal phase ( Fig. 3 ). This may constitute an argument against the use of gonadotropin injections. The relatively high FSH levels may be explained by the relatively low number of surviving primordial follicles in the graft. The patient should be considered a poor responder, with reduced inhibin B secretion. These results are in agreement with those obtained in sheep by Campbell et al.




Fig. 3


Mean FSH and 17β-oestradiol levels (±standard deviation) in the 7 cases of frozen-thawed ovarian tissue transplantation. It took between 4 and 6 ½ months after transplantation before a rise in oestradiol and a drop in FSH were observed. (Note that, in the last 4 cases, FSH and oestradiol values at 1 month post-transplantation were not taken into account, as the patients were under GnRH agonist down-regulation).


A further significant observation is the return to an FSH level of >35 mIU ml −1 immediately after each menstrual bleed, which supports the theory suggested by Baird et al. that some inhibitory mechanisms, such as inhibin B or anti-Müllerian hormone (AMH) normally produced by developing follicles in intact human ovaries, are probably almost non-existent in transplanted tissue. After transplantation, the patient would have been regarded a poor responder because, of the 500–1000 primordial follicles that would have been transplanted, more than 50% would have been lost owing to hypoxia. This raises the question of the evaluation of the ovarian reserve. There is a lack of data on the ovarian reserve in cancer. Qu et al , Gook et al. and Schmidt et al. have all demonstrated an unequal distribution of primordial follicles in ovarian cortex.


In 2005, Meirow et al. also published a live birth after orthotopic autotransplantation of cryopreserved ovarian tissue in a patient with POF after chemotherapy. Eight months after orthotopic transplantation, the patient spontaneously menstruated. The rise in AMH and increased inhibin B levels were consistent with the presence of early growing follicles and ovulation, respectively. Nine months after transplantation, the patient experienced a second spontaneous menstrual period. After a modified natural cycle, a single mature oocyte was retrieved and fertilised. Two days later, a four-cell embryo was transferred. The patient became pregnant from this embryo transfer and delivered a healthy infant weighing 3000 g. The possibility that the oocyte was derived from the native ovary is highly unlikely, given the consistent evidence of POF after high-dose chemotherapy in this patient, from whom ovarian tissue was harvested after administration of a first-line conventional chemotherapy regimen, prior to second-line high-dose chemotherapy.


Demeestere et al. recently reported a pregnancy after natural conception in a woman who had undergone orthotopic and heterotopic transplantation of cryopreserved ovarian tissue. They observed follicular development in all three transplantation sites: large follicles in the ovarian site, only one dominant follicle in the peritoneal site and follicles <13 mm in size in the heterotopic site. Detectable hCG levels and ultrasonography confirmed the presence of a viable intrauterine pregnancy. Unfortunately, this pregnancy, obtained by natural conception, ended in miscarriage at 7 weeks due to aneuploidy. Thereafter, this patient underwent a second transplantation, became pregnant and delivered a healthy baby.


Andersen et al. reported a series of six women (some already described in the paper by Schmidt et al. ). Orthotopic autotransplantation was performed in all patients, two of whom received additional heterotopic transplants. These authors also observed restoration of ovarian function in all women within 20 weeks. Four of the six women conceived following assisted reproduction. One woman miscarried in gestational week 7 and another had a positive hCG test but no clinical pregnancy. Two became pregnant after follicle puncture and ovum pick-up during natural cycles. Each delivered a healthy baby. Both these women had undergone orthotopic transplantation of cryopreserved ovarian tissue to the remaining ovary.

Only gold members can continue reading. Log In or Register to continue

Nov 9, 2017 | Posted by in OBSTETRICS | Comments Off on Ovarian tissue cryopreservation and transplantation in cancer patients
Premium Wordpress Themes by UFO Themes