Best Practice & Research Clinical Obstetrics & Gynaecology
Volume 24, Issue 1 , Pages 113-126, February 2010

Orthotopic and heterotopic ovarian tissue transplantation

  • Murat Sonmezer, MD (Associate professor)

      Affiliations

    • Ankara University School of Medicine Department of Obstetrics and Gynecology, Ankara, Turkey
    • Ankara University Center for Research on Human Reproduction, Ankara, Turkey
    • Tel.: 90 312 5957027; fax: +90 312 32003553.
    • ,
  • Kutluk Oktay, MD FACOG (Professor, Director)

      Affiliations

    • Laboratory of Molecular Reproduction and Fertility Preservation Department of Obstetrics, Gynecology, Westchester Medical Center-New York Medical College, Valhalla, NY, USA
    • Corresponding Author InformationCorresponding author. Tel.: +1 212 7461600; fax: +1 212 9944499.

published online 07 October 2009.

Article Outline

Although still experimental, cryopreservation and transplantation techniques for ovarian tissue have been well described, and a number of successful human pregnancies have occurred. Ovarian cryopreservation is the only fertility preservation procedure that can be offered to prepubertal children, and when cytotoxic treatment is urgent. There are two main approaches for autotransplantation of human ovarian tissue. In the heterotopic transplantation, cortical fragments can be grafted subcutaneously at various sites whereas in orthotopic transplantation cortical pieces are transplanted into its original location. Both approaches have their own advantages and disadvantages. While natural pregnancy can occur in orthotopic transplantation, heterotopic transplantation may be indicated if the pelvis is not suitable for transplantation due to previous radiation or severe scar formation. Furthermore, tissue monitoring may be easier in the heterotopic site. In this article, we reviewed the indications, limitations, risks and transplantation techniques for ovarian tissue.

Key words: cryopreservation, fertility preservation, ovarian tissue, transplantation

 

Cancer is a major health problem in both developed and developing countries. Recent data estimate that 692,000 women and 745,000 men have been afflicted with cancer in 2007 in the United States.1 In women, cancer incidence rates for all sites increased by 0.3% per year from 1987 to 2003. However, there have been remarkable improvements in survival rates for various cancers, as a result of the developments in current treatment modalities, along with the ability to detect tumours in the early stages by well-established screening programmes. Furthermore, a cure is now possible for many childhood and adult cancers, especially for leukaemias and lymphomas. By the year 2010, one in every 250 person is estimated to have survived childhood malignancies.2 In addition to malignant diseases, increasing number of non-malignant systemic conditions has also been successfully treated with chemotherapy or haematopoietic stem cell transplantation.3

As increasing number of women survive cancer each year, a growing segment of patients inevitably face the long-term sequel of cancer treatment that dramatically compromise their quality of life. Increased awareness of the impact of various cytotoxic treatments on gonadal function has now resulted in a surge in the number of patients seeking help to preserve their fertility. Cryopreservation of embryos is a standard technique for fertility preservation when there is adequate time for ovarian stimulation. If the patient has no partner or unwilling to use donor sperm, oocytes can be frozen instead. Although cryopreservation and transplantation techniques have been well described for ovarian tissue, currently the experience with this procedure is limited. Nevertheless, at present, it is the only fertility preservation procedure that can be offered to prepubertal children and can be implemented without any delay in treatment. In this article, the reader will find a comprehensive review of ovarian cryopreservation and transplantation techniques.

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Ovarian tissue cryopreservation: indications 

Studies with fresh ovarian transplants date back to the beginning of the 20th century.4 Cryopreservation of ovarian tissue and transplantation began in animal models in 1950s after the discovery of cryoprotective agents.5 Early studies showed limited success with only 10% of the primordial follicles surviving the freeze–thaw procedure, because of the availability of a poor cryoprotectant agent ‘glycerol’, and the absence of automated cryopreservation systems.6 Additional cryoprotectants became available during 1990s, including propandiol, dimethylsulphoxide and ethylene glycol, leading to successful ovarian tissue cryopreservation (OTC), transplantation and resumption of fertility in animals.7, 8 In the second half of 1980s, heterotopic transpositon of human ovary with microsurgical anastomosis has been reported.9 In the first report of orthotopic ovarian transplantation with frozen banked ovarian tissue, menopause was temporarily reversed in a 29-year-old woman suffering from surgical menopause.10 After declaration of the first embryo from frozen–thawed ovarian fragments transplanted in a heterotopic site in 200411, subsequent live births were reported using this technique.12, 13

There is a growing list of indications for OTC for fertility preservation (Table 1). The indications now include not only neoplastic diseases but also non-neoplastic conditions requiring chemotherapy, radiotherapy or haematopoietic stem cell transplantation. Oophorectomy for benign ovarian tumours and for BRCA germline mutations can also be indications for OTC.

Table 1. Indications for ovarian tissue cryopreservation.
Cancer in children
Hodgkin and non-Hodgkin lymphoma
Leukemias
Ewing sarcoma
Wilms tumor
Neuroblastoma
Genital rhabdomyosarcoma
Pelvic osteosarcoma
Breast cancer
Infiltrative ductal histological subtype
Stage I–III
Cancer of the cervix
Autoimmune and hematological diseases treated with chemotherapy or HSCTa
Surgery for benign ovarian disease
Endometriosis
Benign ovarian lesions
Patients receiving pelvic radiation
Solid organ tumors presenting in the pelvis
Ewing sarcoma
Osteosarcoma
Tumors of the spinal cord
Retroperitoneal sarcoma
Rectal cancer
Benign bone tumors
Prophylactic oophorectomy
BRCA-I/II positive patients
Patients undergoing surgery for gynecological cancers

aIncludes genetic, hematological, and autoimmune disorders.

Although the clinical indications are similar for ovarian tissue and oocyte cryopreservation, there are fewer logistical restrictions with the latter. OTC has broader applications and, in theory, a greater source of oocytes compared with oocyte cryopreservation as ovarian cortex may contain tens of thousands of oocytes. Several livebirths have been reported with oocyte freezing; however, the number of successful pregnancy reports has been growing from frozen–thawed ovarian tissue transplantation (Table 2).

Table 2. Embryo development and pregnancies reported after transplantation of frozen thawed ovarian tissue.
AuthorYearTransplantation siteCryo indicationIVF/spontaneousAge at ovarian cryo.Age at tx.Outcome
Oktay [10]2004HeterotopicBreast cancerIVF3036Embryo development
Donnez [12]2004OrthotopicHodgkin's diseaseSpontaneous2531Healthy live birth
Meirow [13]2004OrthotopicHodgkin's diseaseIVF2628Healthy live birth
Demeestere [40]2006Orthotopic/heterotopicHodgkin's diseaseSpontaneous2429One miscarriage at 7 weeks, one healthy live birth
Oktay [48]2006HeterotopicHodgkin's diseaseSpontaneous2832Healthy live birth
Rosendahl [49]2006Orthotopic/heterotopicHodgkin's diseaseIVF from heterotopic site2830Biochemical pregnancy
Anderson [41]2008OrthotophicNon Hodgkin's lympohomaIVF3234Ebryo development
Anderson [41]2008Orthotopic/heterotopicHodgkin's diseaseIVF2527Clinical pregnancy
Anderson [41]2008OrthotopicHodgkin's diseasIVF2628Healthy live birth
Anderson [41]2008OrthotopicEwings sarkomuIVF2730Healthy live birth
Silber [42]a2008OrthotopicIdiopathic premature ovarian failureSpontaneous1428Ongoing pregnancy

Tx: transplantation cryo: cryopreservation.

aFresh and frozen thawed ovarian cortical strips are used.

Childhood cancers 

Cancer ranks as the second leading cause of death in children between the ages of 1 and 14 years. Cure rates and life expectancy have dramatically improved for many childhood cancers. Despite increased survival rates, they are certainly not immune to the gonadotoxic effects of various cancer treatments.

Children have the highest number of primordial follicle reserve and the greatest benefit from OTC is expected in this group.14 As an advantage, no ovarian stimulation is required in OTC and therefore time restrictions are fewer, and no concerns exist as to the risk of stimulating hormone-sensitive cancers following ovarian stimulation.3 Since it avoids ethical concerns of ovarian stimulation and oocyte retrieval in children, cryopreservation of the ovarian tissue is the only acceptable option for prepubertal children undergoing any type of gonadatoxic therapy. In addition, this is the only fertility preservation technique that can reverse hormonal functions.

Cancers in adults 

Breast cancer is the most common female cancer seen during reproductive ages. In 2007, as many as 182,460 new cases were estimated to be diagnosed with breast cancer, accounting for more than a quarter of all female cancers.1 Approximately, 25% of breast cancer cases occur before menopause, and 15% occur under the age of 45. More than 90% of all breast cancers are diagnosed at a local or regional disease stage, with corresponding 5-year survival rates of 97% and 79%, respectively.15 With improved cure rates from breast cancer, a greater attention has been focussed on the long-term adverse effects of breast cancer treatment, which can compromise the quality of life of breast cancer survivor. Along with the increase in the number of women who delay first childbirth beyond the age of 35, more liberal use of adjuvant chemotherapy has resulted in a large proportion of young women with breast cancer facing infertility and premature ovarian failure. The incidence of chemotherapy-induced amenorrhoea has been reported as 68% with classic oral CMF, while it ranges between 0% and 96% with antracyline-based regimens.16 When considering OTC, it should be underlined that the risk of ovarian involvement seems low in early-stage and locally advanced breast cancer.

Each year, nearly 500,000 women are afflicted by cervical cancer worldwide.17 Almost half of them are under the age of 35. Patients with advanced-stage disease and those with early-stage disease with high-risk factors receiving pelvic or pelvic/para-aortic radiation therapy are at high risk for developing ovarian failure.18 In the case of ovarian stimulation in patients with cervical cancer, there is risk of bleeding from the cancerous cervix during oocyte retrieval. Ovarian tissue can be harvested in selected patients for cryopreservation during primary cancer surgery; however, the risk of ovarian involvement has to be considered. Squamous cell cancer of the cervix, which is the most encountered subtype, rarely metastasises to the ovaries, whereas adenocarcarcinoma of the cervix involves ovaries at a rate as high as 12%.19 Patients receiving only pelvic radiotherapy can benefit from ovarian transposition; however, success rates vary greatly because of the possibility of vascular damage during the procedure. As such, patients receiving pelvic radiation therapy for any pelvic tumour such as rectal cancer, solid organ tumours presenting in the pelvis, osteosarcoma and tumours of the spinal cord can also resort to fertility preservation technologies including OTC.

Autoimmune diseases 

A number of autoimmune diseases can affect women of reproductive age. Cytotoxic treatment has been used effectively to treat various autoimmune diseases, including systemic lupus erythematosus, steroid-resistant glomerulonephritis, Behcet's disease, inflammatory bowel diseases and pemphigus vulgaris.20 Cortical fragments of ovary can be harvested to cryopreserve for possible future use in order to preserve fertility.

Patients undergoing haematopietic stem cell transplantation 

Autologous or allogeneic haematopietic stem cell transplantation (HSCT) has become an important therapeutic tool in the management of some malignant and non-malignant systemic diseases. Diseases associated with genetically abnormal stem cells, some autoimmune diseases unresponsive to immunosuppressive therapy and diseases associated with the deficiency of bone marrow stem cell products are among the non-malignant diseases treated with HSCT.21, 22 HSCT has also been used to treat breast cancer, multiple myeloma and lymphoma. If there are time restrictions for ovulation induction, or if the patient is single or a child, ovarian tissue can be frozen in these patients to preserve fertility.

Others 

The cumulative lifetime risk of developing ovarian cancer is ∼ 60% in the presence of BRCA-1 mutation, and 10–20% in women with BRCA-2 mutation. Furthermore, lifetime risk of breast cancer in female carriers of BRCA-1 mutation is 80–90%. Despite the fact that the risk of peritoneal cancer cannot be totally eliminated in BRCA-positive patients, prophylactic oophorectomy is suggested as soon as childbearing is completed or by the age 35–40 years to decrease the risk of both breast and ovarian cancer.23 Cortical pieces of ovarian tissue can be harvested to freeze for future use to preserve fertility.

There is a risk that ovarian reserve can be compromised by any type of ovarian surgery for benign ovarian diseases, mostly including endometriosis and benign neoplastic ovarian lesions.24 Despite that follicular growth appears lower with frozen/thawed tissue, functional ovarian cortex surrounding benign ovarian cysts transplanted into the subcutaneous space of severe combined immunodeficient disease (SCID) mice can sustain ovarian tissue function.25 Healthy pieces of cortical ovarian tissue can be isolated and frozen in these patients for possible future use. A combination of OTC and in vitro maturation can be implemented to maximise the success rate of the procedure.

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Xenografting studies of human ovarian tissue 

Cortical fragments of human ovarian tissue can be xenotransplanted into T- and B-cell-deficient SCID mice.26, 27 This animal model was first used by Gosden to observe follicle development in xenografted sheep and cat ovarian tissue.27 Subsequently, follicle development, ovulation, corpus luteum formation and metaphase II oocytes were demonstrated by several groups after gonadotropin stimulation of xenografted human ovarian tissue in SCID mice.28, 29, 30

In a recent study, it was demonstrated that cryopreservation and xenotransplantation did not appear to greatly affect the human primordial/primary follicle ultrastructure.31 Interestingly, in frozen–thawed xenografts, secondary human ovarian follicles presented a well-preserved ultrastructure, but asynchrony between oocytes and granulosa cell development was detected. On the contrary, a previous study demonstrated that immature oocytes in human ovarian tissue xenotransplanted into SCID mice grew to maturity, whereas many oocytes, grown in host animals and further matured in vitro, showed aberrant microtubule organisation and chromatin patterns.32

In a prospective study, the effect of a gonadotropin-releasing hormone (GnRH) agonist on the number of follicles in different developmental stages in cryopreserved human ovarian grafts transplanted into gonadotropin-stimulated or unstimulated SCID mice were investigated.33 GnRH agonist treatment did not prevent primordial follicle depletion after the xenografting of ovarian tissue in SCID mice with or without gonadotropin stimulation. Furthermore, GnRHa caused an additional loss of follicles if administered during the critical neo-vascularisation period after the transplantation. The same group also found that prolonged gonadotropin stimulation significantly reduces primordial follicles in xenografts of cryopreserved human ovarian tissue.34 Even though ovariectomy may improve the development of follicles after xenotransplantation of cryopreserved human ovarian grafts, exogenous use of gonadotropins seems essential for improved follicle survival in recipient SCID mice.35

In summary, even though human ovarian xenografts provided a model to study human ovarian tissue autotransplantation, their use as a means to use banked ovarian tissue is in question. Concerns regarding cross-species retroviral infections should be addressed. Moreover, this technique will require large numbers of animals to be killed since only very small fragments of ovarian tissue can be xenografted. This may not only make the technique impractical, but may also raise further ethical concerns.

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Human ovary transplantation 

There are two main approaches for autotransplantation of human ovarian tissue. In the heterotopic transplantation, cortical fragments can be grafted subcutaneously at various sites; such as forearm and abdominal wall. In orthotopic transplantation, ovarian cortical fragments are transplanted into its original location, on the remaining ovary or near the infundibulopelvic ligaments or ovarian fossa. Both the approaches have their own advantages and disadvantages. A laparoscopic approach makes ovarian tissue harvest a less invasive procedure.*20, 36 Using modern effective cryoprotectant agents such as ethylene glycol, dimethyl sulphoxide (DMSO) and propanediol, successful live births were reported from cryopreserved ovarian tissues in a number of species. In 2000, resumption of ovarian endocrine function was demonstrated after orthotopic transplantation of frozen–thawed ovarian tissue.10, 37 First primate pregnancy was reported in a monkey in 2004 from fresh ovarian transplants grafted into a heterotopic location without any surgical connection to major blood vessels.38 These are followed by reports of live births from orthotopic transplants of frozen–thawed ovarian tissues.12, 13

Despite many patients having had cryopreserved ovarian tissue over the world, only a small fraction of patients have undergone transplantation of cryopreserved ovarian cortical tissue. In our experience, only three of 59 women (5.1%) underwent transplantation, two with the heterotopic (abdominal wall) and one with the orthotopic technique.39 The majority of patients had either haematologic malignancies (45.7%) or breast cancer (22%). No complications occurred and no histological evidence of cancer was found in the harvested tissue. Fifty-six of 59 patients have not yet used their ovarian tissue. The reasons for non-utilisation were social/personal, being still under treatment and death. Of interest, one woman with a heterotopic transplant conceived spontaneously and delivered. Of the three transplants, one ceased function after 9 months and two are still functioning at up to 7 years' follow-up.

Orthotopic transplantation 

In 2000, we reported the first case of a laparoscopic orthotopic transplantation of frozen–thawed ovarian cortical strips in a 27-year-old woman.10 Frozen–thawed ovarian fragments were sutured to two triangular frames made from an absorbable cellulose membrane. Ovarian tissue was transplanted beneath the left pelvic peritoneum of the ovarian fossa using laparoscopy (Fig. 1). To improve vascularisation, aspirin (80mg per day p.o.) and follicle-stimulating hormone (FSH; 150IU per day i.m.) were given for a week following the operation. The patient was stimulated with daily menopausal gonadotropins 15 weeks after grafting and was gradually increased from 150 to 675IU per day. Ovulation was confirmed by elevated progesterone levels, ultrasonographic demonstration of a corpus luteum, free fluid in the cul-de-sac and change in endometrial pattern on ultrasound. Ovarian function continued for 9 months in this patient.

  • View full-size image.
  • Fig. 1. 

    a. Retrograde loading of the graft that was reconstructed by stringing the ovarian tissue between two strips of Surgicel. b. Placement of the base suture through the upper peritoneal edge. By pulling on this suture, the graft is flattened against the vascular pelvic wall.

Permission for Figure 1a and b: Oktay K, Aydin BA, Karlikaya G. A technique for laparoscopic transplantation of frozen-banked ovarian tissue Fertility and Sterility Fertil Steril. 2001 Jun; 75(6):1212–6.

Thereafter, another case of orthotopic transplantation of frozen–thawed ovarian tissue was reported by Radford et al. in a 36 year-old woman with stage IIIB nodular sclerosing Hodgkin's lymphoma.37 Before administering high-dose chemotherapy for third recurrence of the disease, the right ovary had been cryopreserved. Nineteen months later, after she had experienced premature ovarian failure, two ovarian cortical strips were thawed and transplanted onto the left ovary and another to the site of the removed ovary. Seven months after transplantation, the patient reported resolution of the menopausal symptoms. Five weeks later, serum oestradiol rose up to 352pmoll−1 and pelvic ultrasonography showed a 10-mm thick endometrium and a 20-mm follicle on the right side. However, progesterone levels were never found over 2nmoll−1, and no ovulation was detected. As in our case, gonadotropin levels were in the post-menopausal range 9 months after the transplantation. Cortex of the frozen–thawed ovary, as well as biopsy samples of the retained left ovary, did not show evidence of Hodgkin's lymphoma. However, none of these cases can be considered as ‘ideal’ to judge the performance of this procedure, because the cryopreserved tissue was previously compromised due to ovarian surgery or chemo- and radiotherapy.

First human pregnancies and healthy live births from frozen–thawed human ovarian tissues were claimed in lymphoma survivors who were transplanted to the orthotopic site.12, 13 In the first case, the patient received a combination chemotherapy, including chlorethamine, vincristine, procarbazine, prednisone, doxorubicin, bleomycin and vinblastine. This combination chemotherapy commonly causes ovarian failure in 20% of the women aged 25 years or younger. Since ovarian tissue fragments were grafted onto pre-existing right ovary, concerns remained about whether the oocyte fertilised had ovulated from the pre-existing ovary or from the transplanted graft. The second live birth was reported by Meirow in 2005. In this case, ovarian tissue was harvested after administration of a second-line conventional chemotherapy regimen, before treatment with high-dose chemotherapy for Hodgkin's lymphoma. The patient's menses ceased after the high-dose chemotherapy, and premature ovarian failure was confirmed by high levels of FSH and leutinising hormone (LH) (40–104IUl−1), and undetectable levels of anti-Müllerian hormone (AMH) and inhibin B levels. When the patient was free of disease, strips of frozen–thawed ovarian tissue were transplanted to the left ovary and small fragments were injected into the right ovary. Eight months after transplantation, the patient resumed menstruation. AMH and inhibin B levels elevated. One month later, after transplantation, the patient conceived after transfer of a four-cell embryo using in vitro fertilisation.

Demeestere and associates reported a spontaneous pregnancy after orthotopic and heterotopic transplantation of cryopreserved ovarian tissue in a 29-year-old woman previously treated by conditioning therapy for bone marrow transplantation (BMT) for Hodgkin's disease.40 Andersen et al. reported a series of six women who underwent orthotopic and heterotopic autotransplantation tissue fragments into two pockets created on either side of the ovary or into subperitoneal pockets.41 Ovarian function was restored in all women and, two women became pregnant and delivered a healthy baby each using natural-cycle in vitro fertilisation (IVF). Silber et al. reported eight cases of ovarian transplantation with fresh or frozen-thawed ovarian tissue in monozygotic twins. Seven had fresh cortical tissue transplant, one of whom received a second frozen–thawed transplant.42 One patient underwent fresh microvascular transplant. Six conceived naturally, one of whom conceived again after a frozen–thawed secondary transplant.

In a meta-analysis, Bedaiwy et al.43 idendified 46 women who underwent ovarian transplantation, which was performed to treat premature ovarian failure (POF) in 27 women, to prevent POF in 15 women, to treat infertility in two and was accidental in one. Resumption of ovarian function was established with a median time of 120 days (range: 60–244 days) in 23 women with FSH >30 at the time of transplantation. Among them, four women had recurrent ovarian failure within 6 months. The authors noted that there are insufficient data to evaluate the long-term ovarian function (>12 months). Authors found that fresh grafts had an increased likelihood of return of ovarian function and a decreased likelihood for recurrent ovarian failure compared with cryopreserved grafts. In 25 women who sought pregnancy, eight women had nine pregnancies at 12 months, giving a cumulative pregnancy rate of 37%.

The advantage of orthotopic transplantation is that a natural pregnancy can occur; however, the procedure requires abdominal surgery and general anaesthesia. However, orthotopic location is not preferred when the risk of ovarian metastasis is high because tissue monitoring may be more difficult.

Heterotopic transplantation 

Using a heterotopic site for autotransplantation of human tissues is a well-known concept and has been used for grafting fresh or frozen–thawed parathyroid tissue following total parathyroidectomy for more than 30 years.44 In this technique, general anaesthesia or abdominal surgery is not required, follicle monitoring and, when necessary, removal of transplanted tissue is easier in a subcutaneous site. Various body sites can be used to graft ovarian pieces, subcutaneous space above the brachioradialis facia of the forearm or under rectus sheet in lower abdomen.20

Transposition of fresh human ovary by microsurgical anastomosis was first described by Leporrier et al. before pelvic irradiation to treat Hodgkin's disease.9 One year after the procedure, puncture of the ovarian compartment produced a mature oocyte specimen. In 2004, Hilders et al. reported a successful ovarian transplantation to the left upper arm using microsurgical anastomosis in a patient with cervical carcinoma.45 After transplantation, adequate arterial and venous blood flow was demonstrated and ovarian cycles remained regular for more than 1 year.

Heterotopic ovarian transplantation with fresh ovarian cortical pieces was first reported in a 35-year-old woman with stage III squamous cervical carcinoma prior to pelvic radiation in 2001.46 Following laparoscopic oophorectomy, premature ovarian failure was confirmed by elevated FSH and LH levels. Six weeks after transplantation of ovarian tissue, the patient noticed a painless swelling at the site of the transplantation. The oestradiol levels increased, and ultrasound revealed four antral and one dominant follicle. Restoration of stromal function was also demonstrated by normal testosterone levels. After controlled ovarian stimulation, two immature oocytes and one in metaphase I were retrieved. The metaphase I oocyte underwent in vitro maturation, but fertilization did not occur after intracytoplasmic sperm injection (ICSI). In another patient who underwent oophorectomy due to recurrent benign ovarian cysts, fresh ovarian tissue was transplanted subcutaneously to the forearm46 (Fig. 2). Resumption of menstruation and spontaneous ovulation occurred as early as 3 months after transplantation; however, the cycle length varied from 14 to 45 days, and the graft stopped functioning after 3 years.

Permission for Figure 2: A technique for transplantation of ovarian cortical strips to the forearm. Oktay K, Buyuk E, Rosenwaks Z, Rucinski J. Fertil Steril. 2003 Jul; 80(1):193–8.

Other investigators also confirmed that fresh or frozen–thawed heterotopic ovarian transplantation restores ovarian function; however, the grafts have a limited life span.47 Demeestere and associates performed orthotopic transplantation of cryopreserved ovarian tissue at ovarian and peritoneal sites, together with a heterotopic transplantation at the abdominal subcutaneous site in a 29-year-old woman previously treated with BMT for Hodgkin's disease.40 They observed that ovarian reserve markers progressively improved within 5 months after the transplantation. Follicular development was observed at all transplantation sites but was predominant at the ovarian site. The patient became spontaneously pregnant following the sixth cycle, but she later miscarried.

For the first time, an embryo was generated in 2004, using oocytes retrieved from subcutaneously transplanted frozen–thawed ovarian cortical pieces.11 This was a breast cancer survivor, whose ovarian cortical pieces were transplanted 6 years later, resulting in reversal of menopause. In 2006, we reported a spontaneous pregnancy after heterotopic ovarian transplantation to the abdominal region.48 As premature ovarian failure was diagnosed by high FSH levels, the occurrence of this pregnancy cannot readily be explained. In the same year, Rosendhal et al. reported a biochemical pregnancy after heterotopical autotransplantation of cryopreserved ovarian cortical tissue.49 Following experiments in mice, we speculated that the transplanted tissue can induce growth of remaining ovarian follicles.50

Heterotopic transplantation may be indicated if the pelvis is not suitable for transplantation due to previous radiation or severe scar formation. Furthermore, easy tissue monitoring is an advantage of heterotopic transplantation. However, there are still numerous challenges to perfecting the heterotopic ovarian transplants as the oocyte maturation process appears to occur differently than in the orthotopic environment. It remains to be clarified whether this delay in oocyte maturation is due to diminished blood flow or differences in local milieu.

Even though the ovarian grafts have limited life span and these patients behave as poor responders to ovulation induction, endocrine function can be restored using either orthotopic or heterotopic transplantation of frozen–thawed ovarian tissue. Despite only a few pregnancies being reported using this technology, it holds future promise for preservation of fertility of the patients exposed to chemoradiotherapy, radiation or radical ovarian surgery.

Whole ovary cryopreservation 

Cryopreservation of whole human ovary is a challenging issue. First, human ovary is larger and more complex than the ovaries of the animals; and second, it may be challenging to devise a cryopreservation protocol that will optimally preserve both the ovarian follicles and vasculature structures.

Bedaiwy et al. suggested that inferior epigastric vessels can be the most suitable heterotopic site for vascular anastomosis, and end-to-end anastomosis yields the highest patency rate of vascularised grafts.51 In another study, the same researchers assessed the immediate post-thawing injury to the human ovary that was cryopreserved either as a whole with its vascular pedicle or as ovarian cortical strips in two patients (46 and 44 years old), who agreed to donate their ovaries for experimental research after undergoing hysterectomy.52 In both the patients, one of the harvested ovaries was sectioned in small pieces and cryopreserved by slow freezing. The other ovary was cryopreserved intact with its vascular pedicle. Tissues from non-cryopreserved ovaries in the two cases served as control specimens. The overall viability of the primordial follicles was 75% and 78% in intact frozen–thawed ovaries and 81% and 83% in ovarian cortical strips in the 46- and 44-year-old patient, respectively. Comparable primordial follicle counts, absence of features of necrosis, mean values of apoptosis and weak Bcl-2 and p53 protein expressions were observed in both the intact frozen–thawed ovary and the frozen–thawed ovarian cortical strips. It is however hard to draw any conclusion from that study as primordial follicle density is extremely low in women in their mid-40s and as the assessment was based on morphology alone.

Several authors demonstrated that cryopreservation of intact human ovary with its vascular pedicle is not associated with any signs of apoptosis or ultrastructural alterations in any cell types.53 However, there has been no case of successful ovarian transplantation with whole-frozen ovarian tissue and no study reported on the functionality of ovaries frozen intact. Research on cryopreserving whole human ovary is continuing and, although challenging, whole ovary vascular transplantation may become a clinically viable option.

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Potential risks associated with ovarian tissue cryopreservation: reseeding primary tumour 

One of the greatest concerns with OTC is the risk of re-implanting an occult tumour with frozen–thawed ovarian pieces. The risk of ovarian involvement according to different types has been reported in detail previously.3 When there is a high risk of ovarian metastasis, OTC for the purposes of future autotransplantation should not be performed. Most of the tumours seen during reproductive years have a relatively low risk of ovarian involvement. Exceptions to this include blood-borne malignancies, such as leukaemias, as well as Burkitt's lymphoma and neuroblastoma.

Based on clinical experience, ovarian involvement is extremely rare in Hodgkin's patients. A recent human study demonstrated that none of 26 patients with lymphoma had evidence of ovarian involvement by histology and immunohistochemistry.54 In squamous cell cervical cancer, ovarian involvement is <1.0%, whereas it is reported to be between 1.7% and 12.5% in adenocarcinoma of the cervix. With the exception of advanced-stage breast cancer, the risk of ovarian metastasis is negligible. When there is a high risk of ovarian involvement, or if ovarian micrometastases are found, to eliminate the risk of re-implanting tumour cells, techniques such as in vitro maturation of primordial follicles or ovarian tissue xenografting may become options for these patients, as more research into these methods is leading to greater knowledge of the risks and benefits. Regardless of the primary cancer, a detailed histological assessment for micrometastases must be performed on portions of the harvested tissue prior to cryopreservation so as to avoid transplanting tissue that contains cancer. Molecular markers can also be used to detect an extremely small number of metastatic cells in the tissue when such markers are available. A recent study showed that highly sensitive real-time reverse transcription polymerase chain reaction (RT-PCR) was positive in one patient with chronic myeloid leukaemia, despite negative results in conventional histological examination.55

Patients with high-risk cancers either should not be given the option of ovarian autotransplantation or ovarian tissue harvest should be performed after the first round of chemotherapy in order to clear any neoplastic cells residing in the ovary. However, it should be stressed that the ovarian reserve and effectiveness of assisted reproductive technologies diminish with each round of chemotherapy administered.56 Further experimental studies in other cancers will aid researches in clarifying the most debated concerns with respect to ovarian cryopreservation.

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Malignant transformation 

Ovarian tissue transplantation should not be performed for women with ovarian cancer, but, in theory, immature oocytes can be collected from cryopreserved tissue. Retrieval of immature oocytes followed by in vitro maturation and vitrification was reported in women with borderline ovarian malignancy to preserve fertility.57 The risks are less evident in women at high risk of developing ovarian cancer, such as those with BRCA mutations who undergo prophylactic oophorectomy. Although the risk cannot be totally eliminated, prophylactic oophorectomy is usually offered to decrease the risk of developing ovarian cancer or as a part of a treatment plan for breast cancer when childbearing is completed or by ages 35–40 years.58 The incidence of occult ovarian metastasis ranges between 2% and 18.5% in BRCA carriers, and a detailed histological analysis of the ovarian cortical pieces is mandatory.

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Future options 

In vitro maturation of primordial follicles and ovarian tissue xenotransplantation may 1 day become common applications in conjunction with the cryopreservation of ovarian tissue. Primordial follicles can be isolated from fresh and cryopreserved human ovarian tissue to be grown in vitro; however, there has been no report of clinical success with that approach.59, 60 A recent study showed that isolated fresh human follicles are able to survive and grow after xenografting. Indeed, these techniques also carry additional risks. As mentioned with oocyte in vitro maturation, epigenetic abnormalities secondary to incomplete methylation imprints in immature oocytes may be of paramount concern.61, 62

Xenotransplantation would avoid the potential risk of cancer re-seeding with ovarian transplantation in high-risk cases, but transmission of prions and animal viruses accompanying the retrieved oocytes must be considered. In addition, with this technology, large numbers of animals have to be killed to obtain adequate tissue, since only small ovarian pieces can be xenografted, raising further ethical concerns. Regardless of the application, the practitioner should have a candid and thorough discussion with the patient regarding all the available options and make it clear that most fertility preservation options are currently experimental.

Practice points

 


•The indications of ovarian tissue cryopreservation have expanded to include both malignant and various non-malignant benign diseases treated with cytotoxic treatment

•It is the only fertility preservation option for prepubertal girls and for women who face the high likelihood of diminished ovarian reserve requiring immediate treatment

•The risk of ovarian involvement by tumour cells and the risk of ovarian failure derived by cytotoxic treatment should be assessed before embarking on harvesting ovarian tissue; however, that risk is minimal for most cancer types

•Laparoscopy is a minimally invasive surgery to harvest ovarian tissue, which enables patients to receive chemotherapy or radiotherapy without any significant delay of cancer treatment

•Heterotopic transplantation may be indicated if the pelvis is not suitable for transplantation due to previous radiation or severe scar formation. Orthotopic location may restore natural fertility

•Due to the limited life span of ovarian grafts, the transplantation procedure should only be performed when the patient is ready to conceive

•Due to loss of great majority of primordial follicles during neo-vascularisation injury, the cryopreservation of ovarian tissue is more likely to succeed in patients aged <35 years but this may change with the improvement of cryopreservation and transplantation techniques

Research agenda

 


•Further studies are required to improve follicle survival after transplantation of frozen–thawed ovarian pieces

•Larger clinical studies are needed

•Ovarian cryopreservation methods need to be improved

•The mechanism behind spontaneous recovery of ovarian function after highly gonadotoxic chemotherapy and a possible role of ovarian transplantation in facilitating this recovery deserve further exploration

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Conflict of interest statement 

Both of the authors declare that there are no financial or personal relationships with other people or organisations.

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Acknowledgement 

Kutluk Oktay is supported by NICHD grant no HD053112.

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References 

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PII: S1521-6934(09)00116-3

doi:10.1016/j.bpobgyn.2009.09.002

Best Practice & Research Clinical Obstetrics & Gynaecology
Volume 24, Issue 1 , Pages 113-126, February 2010

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