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Cryopreservation of ovarian tissue

Potential "reproductive insurance" for women at risk of early ovarian failure

Carl E Wood, Jillian M Shaw and Alan O Trounson

Cryopreservation of ovarian tissue is a potential alternative or addition to cryopreservation of embryos or oocytes for sustaining fertility in women at risk of premature ovarian failure. Grafts of cryopreserved ovarian tissue have resulted in live-born mice and sheep. While human applications are in their infancy, frozen human ovarian tissue grafted to the kidney capsules of immunodeficient mice has produced normally developing ovarian follicles.
(MJA 1997; 166: 366-369)


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Introduction - Oocyte cryopreservation - Embryo cryopreservation - Ovarian tissue cryopreservation - Conclusion - References - Authors' details

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Introduction

 Premature ovarian failure may occur in women with genetic diseases (such as Turner's syndrome), after chemotherapy or radiotherapy for malignant disease, in severe or recurrent ovarian disease (such as cysts, benign tumours or endometriomas), or after removal of the ovaries to treat endometriosis, ovarian pain or genital cancer. For women at risk of premature ovarian failure, there are now three possibilities for preserving their fertility: cryopreservation of their oocytes, embryos, and, most recently, ovarian tissue. This article provides an overview of these procedures, highlighting the recent developments in ovarian tissue cryopreservation.  

Oocyte cryopreservation

 Oocytes can be collected when either mature or immature. Cryopreservation of mature oocytes, although successful in the mouse,1-3 has met with little success in most other species, including man.4,5 All cryopreservation procedures in humans have led to impaired oocyte survival and subsequent reduced rates of fertilisation and embryo cleavage, with the result that fewer than 2% of oocytes are capable of producing a term pregnancy.

For example, when Friedler and colleagues published a review of oocyte cryopreservation in 1988,6 fewer than 20% of oocytes were able to be fertilised and even fewer developed as embryos after preservation by both slow cooling and vitrification (cooling in a concentrated cryoprotectant solution that forms a glassy [vitreous] solid). More recent reports on freezing (both slow7-11 and rapid12-14 ) of human oocytes show little, if any, progress in the proportion that can be fertilised normally after thawing (see Box). Despite claims of advances and the establishment of egg banks for women with cancer, no recent births have been reported.

The difficulties in cryopreserving human oocytes are related to their large volume and variable membrane permeability. This makes it difficult to achieve sufficient dehydration during cooling to prevent intracellular ice formation, without disrupting other cytoplasmic and nuclear components.

It has been documented that cooling an oocyte on its own,13,17 exposure to cryoprotectants13,18 and cryopreservation can increase the incidence of spindle anomalies,8,17 initiate parthogenetic activation (development without a paternal genome)10 and stimulate release of cortical granules (which are usually released at fertilisation, causing the zona to harden, reducing the likelihood of polyspermy).19 Cooling and cryopreservation can also increase the incidence of chromosomal loss from the meiotic spindle, as the temperature-sensitive spindle, which holds the chromosomes in place, disaggregates and reforms abnormally on rewarming.17 Successful fertilisation cannot occur in any of these circumstances.

Fertilisation failure can be partially corrected with intracytoplasmic sperm injection, but this does not resolve the problem of reduced cleavage or reduced developmental competence.10,11 Given that the probability of pregnancy and birth is, optimistically, around 10%-15% for a normally fertilised non-frozen oocyte, it is likely that up to 100 unfertilised oocytes will have to be cryopreserved in order to ensure a pregnancy. As mature oocytes are available for only a limited time in each menstrual cycle, and rarely in large numbers, this is simply not feasible. While it is also possible to cryopreserve immature human oocytes,20,21 this offers no advantage over cryopreserving mature oocytes at present, because of the difficulty of obtaining viable oocytes after in-vitro maturation. However, considerable research is being directed to cryopreserving oocytes of domestic animal species, and this may ultimately benefit human oocyte freezing.  

Embryo cryopreservation

 Cryopreservation of embryos is reasonably successful.22-25 The pregnancy rate after transfer of two to three cryopreserved embryos is around 15%-25%, and the probability of pregnancy accumulates with repeated transfers.

However, there are associated problems: obtaining oocytes for fertilisation is technically difficult, limiting the number that can be used; there is a need for a male partner or sperm donor; unmarried couples may be excluded by government regulations or legal restrictions; and there may be significant ethical problems in disposing any unwanted embryos. Unforeseen circumstances, such as death, divorce or other family problems, may prevent embryos being implanted into the natural mother.

Further, recovering oocytes to generate embryos usually requires ultrasound-guided needle aspiration of the leading follicle(s) in several successive menstrual cycles and ovarian stimulation to induce superovulation. Ovarian stimulation involves raising blood levels of oestrogen up to 20-fold above normal and may not be appropriate in women with oestrogen-sensitive cancers of the breast, ovarian epithelial tumours or severe endometriosis. In addition, in women with cancer there is a risk of transferring cancer cells together with the frozen thawed embryos; the embryos should be denuded of all other cells before transfer to the uterus.  

Ovarian tissue cryopreservation

 Although still in its infancy, ovarian tissue cryopreservation is a viable alternative to cryopreservation of oocytes or embryos. The ovarian cortex of young women contains several hundred thousand primordial follicles;26 even small pieces (1 mm3) may contain several hundred follicles. Although it has not yet been demonstrated that cryopreserved ovarian tissues can restore fertility in humans, live young have been obtained from fresh and frozen ovarian tissue grafts in mice2,27 and sheep.28 Cryopreserved ovarian tissue from marmosets grafted into immunodeficient mice develops apparently normal antral follicles.29

Human studies: Human ovarian cortical tissue, both fresh and frozen, grafted to the kidney capsule of immunodeficient mice also results in apparently normal developing follicles.30 A month after implanting a small piece of frozen thawed human ovarian tissue into these mice, the tissue contained numerous follicles which had been recruited into their growth phase.28 These increased in diameter from 37 µm, with few granulosa cells, to 85 µm, with one or two complete layers of granulosa cells (Figure). A large proportion of the follicles in the graft had been recruited. A similar study has reported successful storage and grafting of human ovarian tissue in immunodeficient mice.31

Advantages of ovarian cryopreservation: Ovarian cryo preservation has the advantage that wedges of ovarian tissue (about 2 cm2 in area) can be collected by laparoscopy from each ovary at any stage of the menstrual cycle without compromising a woman's health or fertility. Unlike embryo and oocyte cryopreservation, collection of ovarian cortical material does not delay hysterectomy and oophorectomy for severe endometriosis and does not involve use of "fertility drugs". The amount of ovarian tissue needed to restore regular menses and fertility after ovarian failure or removal is not currently known, but ovulation can occur with as few as 100 oocytes (in women and mice) to 400 oocytes (in cattle) remaining in the ovary.24,32,33

Applications of ovarian cryopreservation: Ovarian tissue cryopreservation might benefit patients with recurrent ovarian cysts or severe endometriosis, who are at risk of premature menopause. It could be combined with embryo cryo preservation, with cortical ovarian strips (1 mm x 10 mm) taken at the time of egg retrieval in an in-vitro fertilisation treatment cycle. Used alone, ovarian cryopreservation might be of particular benefit to those women, such as single women, who are unable to avail themselves of embryo cryo preservation. Oocyte preservation might be an option if women have access to a reproductive unit with an established record of pregnancies after this procedure. At present, these do not exist.

The value of ovarian tissue grafting for cancer patients is debatable. Viruses, including HIV34-36 and hepatitis virus,37,38 and cancers39 can be transmitted by grafts, and cancers have been known to recur in patients in remission after the replacement of autologous cryopreserved bone marrow.40-42 It is therefore possible that bloodborne cancers such as leukaemia, systemic cancers such as lymphoma and metastasising cancers could be transmitted by ovarian tissue grafts. Shaw et al.43 have shown that when small pieces (around 1 mm3 ) of ovarian tissue obtained from donor mice with lymphoma were grafted into normal healthy recipient mice, 13 of 14 recipients developed the lymphoma. This occurred with both fresh and cryopreserved ovarian grafts, highlighting a significant risk for clinical applications of the procedure.

Separating primordial follicles from stromal tissue or cancer cells is currently not possible. Polymerase chain reaction (PCR) techniques have increased the sensitivity of tests to detect the presence of remnant cancer cells,40,44,45 and it may be possible to culture tissue in vitro under conditions aimed at eliminating cancer cells.42 However, it is unlikely that cancer cells can be flushed out of an ovary or removed from the graft by cytotoxic drugs at the time of replacement, because of the deleterious effect on the graft.

The alternative of removing primordial follicles from the graft and using matured eggs for in-vitro fertilisation, or replacing follicles in the patients, is currently not feasible as the enzymatic procedure used to obtain human primordial follicles46 results in separation of the oocyte and its surrounding support cells in culture.47 Future advances in collection and culture procedures for primordial follicles may allow in-vitro maturation of follicles in humans, as a live birth has recently been reported for mouse primordial follicles matured and inseminated in vitro and returned to the recipient as an embryo.48

Although treatment for cancer may affect fertility and produce premature menopause, it is known that many young women treated for cancer recover their fertility and bear children. The incidence of congenital and other malformations in these children is not statistically different from that in the general population,49 indicating that the follicles that survive cancer therapy are normal. However, cancer therapy may deplete the germ cell population. A study of 2283 long term female survivors of childhood or adolescent cancer found they were less likely to become pregnant than their sibling controls (relative fertility, 0.85; 95% confidence interval, 0.78-0.92).50 In females, radiation therapy below the diaphragm had the most marked effect, depressing fertility by about 25%, while alkylating agents administered alone had no apparent effect. Infertility was most marked after treatment for Hodgkin's disease (relative fertility, 0.77) and soft tissue sarcoma (relative fertility, 0.82) and apparently unaffected after treatment for melanoma, Wilms' tumour and female genital cancer. As there is a risk of transferring cancer cells with ovarian grafts and as follicles which survive cancer therapy appear normal, patients with cancer that may spread systemically should wait until full remission before storing ovarian tissue.

Ethical issues: Transplantation of cryopreserved ovarian tissue may raise new ethical issues. It could be transplanted into women beyond the age of the menopause as a form of hormone replacement therapy. It could also facilitate older women having access to their own "younger eggs", which have been stored for 10 or more years. At present, IVF units in Australia limit the use of donor eggs to women within the reproductive age span, a generous upper limit being healthy women up to 52 years of age. Storage of the woman's own ovary from a younger age may avoid the use of donor eggs at an older age.

Ovarian tissue could not be transplanted into other women on medical grounds, as it would require use of drugs such as cyclosporin to prevent rejection, increasing the risk of malignant disease. The use of such drugs for transplantation of organs on grounds other than a threat to a person's life was not accepted by an ethics committee at the Epworth Hospital, Melbourne, when tubal transplantation was proposed to assist in the treatment of infertility.  

Conclusion

 Patients with endometriosis or other conditions which may perturb their fertility, but which carry little risk of transmitting cancer cells or infectious agents, may choose to have oocytes or ovarian tissue collected and frozen. Those with a partner could also have embryos frozen. Current evidence suggests that ovarian tissue should not be collected from patients with a malignant cancer until they have reached full remission. Patients can return to have their frozen eggs, embryos or ovarian tissue thawed and replaced if they enter menopause or suffer infertility before they are ready to have children. Return of cryopreserved ovarian tissue has the potential to restore the patient's menstrual cycles, allowing natural conception and pregnancy without the need for assisted conception techniques. Unlike oocyte and embryo cryopreservation, ovarian tissue can be collected before or after puberty and at any stage of the menstrual cycle. At the moment, the efficacy of cryopreservation of oocytes is very low, while that of embryos is reasonable and that of ovarian tissue is unknown.  

References
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(Received 8 May, accepted 14 Nov, 1996)  

Authors' details

Monash IVF, Epworth Hospital, Melbourne, VIC.
Carl E Wood, FRCS, FRACOG, Director; and Emeritus Professor, Monash University, Melbourne, VIC.
Institute for Reproduction and Development, Monash University, Melbourne, VIC.
Jillian M Shaw, PhD, Research Fellow. Alan O Trounson, PhD, Deputy Director.
Reprints: Professor E C Wood, 284 High Street, Ashburton, VIC 3147.

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