SALVAGING FERTILITY

Gamete cryopreservation and ovarian tissue transplantation

By Giuseppe Del Priore, MD, MPH, John J. Zhang, MD, and James A. Grifo, MD, PhD

A cancer diagnosis no longer necessarily ends a woman's life— or her future fertility. Experts offer an update on two ways of salvaging potential fertility.

Although there is still no "cure" for many cancers, steady improvements in early detection and treatment have greatly increased survival after a cancer diagnosis. Even victims of advanced ovarian cancer can now expect to survive an average of 5 years.¹ Improved cancer treatment and the social phenomenon of later childbearing present us with an enviable problem: More and more women with cancer are now considering fertility preservation as part of their treatment planning.

In this review, we will discuss reproductive options for patients who are about to undergo potentially fertility impairing cancer treatments. In the January 2002 issue, we discussed some of a cancer patient's reproductive-preserving surgical options (for example, the option of radical trachelectomy for women with cervical cancer). Now we'll focus on what's on the horizon for gamete and ovarian tissue preservation and the potential advantages and disadvantage of each.^2,3

Ovary transplantation

For 100 years or more, patients and physicians have pondered the possibility of ovarian tissue transplantation.⁴ But the first reported—albeit unconfirmed—success circa 1900 seemed to owe more to luck than anything else. Supposedly the surgeon removed both ovaries but retained the right fallopian tube, then transplanted a small piece of the patient's diseased ovary back into the retained tube. The woman's menstrual function returned and she subsequently became pregnant.

All further attempts at the time to replicate the operation in other women failed. Although these early efforts didn't fulfill their promise in humans, all aspects of ovarian transplantation have succeeded in animals. These include fresh and frozen transplants with successful folliculogenesis, resumption of normal endocrine function, normal conception, and birth. The more recent history of reproductive organ transplantation includes well-documented research proceeding in incremental steps from in vitro and animal models to human tissue and patients.^5-7 However, these are cutting edge and evolving technologies that should be reserved for a limited number of patients who qualify under IRB-approved experimental protocols at the present time.^8,9 (See figure.)

Ultrasound image shows ovarian follicle development beneath the forearm skin after transplantation of ovarian cortical strips to a woman's forearm. The lump seen in photo of forearm itself represents developing dominant ovarian follicle. (Both photos courtesy of Kutluk Oktay, MD © Kutluk Oktay.)

In 1966, one of the first successful animal reports involved dogs. Researchers isolated and divided the vessels around the canine uterus, then re-anastomosed them en bloc, restoring uterine function; pregnancy subsequently resulted.¹⁰ In the era before the advent of in vitro fertilization or other assisted reproductive technologies (ART), a key reason for en bloc transplants was the need to preserve the tubes and uterus. Since then, variations on the utero-ovarian transplant in rats and other animals have continued with good success rates.¹¹ Successes in animal experiments include transplants of cortical strips, heterotopic (abnormally anatomically located), orthotopic (normally anatomically located), whole ovary with native vascular pedicles, and fresh or frozen tissue.^{12, 13}

Is success measured only by live births? Unfortunately, all transplanted ovarian tissue to date has functioned for only a limited time, although the degree of success varies with different animals. A finite functioning period is therefore likely to be the fate of the initial human transplant as well. Another potential concern is that the measures of success we've mentioned— folliculogenesis, hormone production, etc.—may not correlate with success as defined by live offspring.¹⁴ In a study using heterotopic and orthotopic autografts of frozen-then-thawed ovarian cortex in sheep, "successfully" transplanted ewes could not produce live lambs despite ART. Other investigators have also reported transplant "successes" in animals that failed to produce offspring.¹⁵

Of course, surgical reimplantation of previously cryopreserved ovarian tissue is also more challenging than using "fresh" tissues, but the former has met with success in animals, with subsequent pregnancies. In 1994, Gosden and colleagues—using thin cortical sections, rather than whole-organ vascular transplants—reported pregnancies after transplanting fresh and frozen ewe ovaries. In this small study, no difference between fresh and frozen transplanted ovaries was detectable. Technically, the operation was a success, in that seemingly normal baby sheep were born following normal mating. On the downside, though, the transplanted ovaries lost a large number of follicles, probably dooming them to early ovarian failure.¹⁶ Greater success with transplantation in mice, compared to sheep, illustrates the significance of species-specific differences in ovarian physiology. In one study, 100% of the transplanted mice ovaries functioned!¹⁷

Animal researchers don't need to transplant an entire ovary, however. Small fragments of ovary grafted at different orthotopic or heterotopic locations are sufficient to retain function, perhaps because of the relatively small size of the ovary with regard to surface to volume ratio. However, to improve long-term ovarian function and follicle reserve, some researchers advocate a vascular whole-organ transplant. Building on pioneering work by James Scott and others, investigators have successfully re-implanted orthotopic and heterotopic fresh whole organ animal ovaries.^12,18 Others have achieved an en bloc syngeneic transplantation of cryopreserved rat ovaries, fallopian tubes, and the upper segment of the uterus.¹³

Researchers have also applied these techniques to humans using sections of cryopreserved ovaries.¹⁹ No pregnancies have yet resulted but they've achieved preliminary success, at least as measured by transient ovarian function, including apparent folliculogenesis and hormone production.²⁰

Temporary xenografting. The use of a temporary animal host for human ovaries may be an alternative to cryopreservation because it avoids the loss of follicle reserve that currently is inevitable with cryopreservation. Temporary xenografting offers several other theoretical advantages, including the ability to continually monitor the status of the graft.²¹ Successful animal xenograft recipients (i.e., the temporary hosts), are followed daily for estrogen production, gonadotropin levels, and other parameters of ovarian function and with sonography. Temporary xenografts could theoretically also be used to screen cancer patients for occult disease in the transplanted ovary. If the severely immunosuppressed animal host fails to develop metastatic cancer after the transplant, it is possible that the transplant contains no potentially malignant cells that might be reintroduced into the donor.²² Of course, concerns about cross-species retroviral infections will need further investigation.

An alternative to frozen embryos? Investigators have pursued ovarian tissue transplantation because of its theoretical advantages over freezing embryos. With transplantation, for instance, a woman need not designate a male gamete source to retain future fertility, as is required for embryo cryopreservation. In addition, the ovary's hormonal function is preserved. However these advantages may not be enough to balance the theoretical risk of occult metastatic disease in the preserved whole ovary in cases of primary or metastatic ovarian cancer. As with other organ recipients, the effects of long-term immunosuppression on the patient and her child are of concern.²³ A final answer awaits many more years of follow-up of patients and their exposed children.²⁴

Clearly, we must find some method to preserve ovarian function while a patient is undergoing gonadal-toxic therapy. Less clear is whether that technique will be transplantation, ovary cortical cryopreservation, xenografting, or some other gonadal protecting option. For these reasons, some patients will always want to consider gamete cryopreservation.

Female gamete cryopreservation lags behind frozen sperm technology

Male gamete cryopreservation has been a reality since the early 1970s, when cryopreserved sperm first came into routine use.²⁵ Advances since then include intracytoplasmic injection using cryopreserved sperm.²⁶

The progress on preserving female gametes, or metaphase II oocytes, has been far slower. Oocytes are more difficult to preserve because they're the largest cells in the body and contain a complex intracellular architecture that is critical to biologic function. Research continues, however, because oocyte freezing is an attractive alternative to embryo preservation. Preserving eggs raises fewer ethical and legal red flags than does use of residual frozen embryos. This alternative is especially crucial for patients at risk of premature ovarian failure due to antineoplastic treatments during childhood.

Human oocytes are particularly fragile during the cryopreservation process because of their large size, high water content, and susceptibility to chromosomal rearrangements.²⁷ Techniques such as slow freezing and rapid thawing can minimize intracellular ice formation and thus subsequent structural damage. Selecting the right cryoprotectant is also critical and requires variation of the freezing protocol.²⁸ To circumvent the difficulties encountered in oocyte cryopreservation, some research facilities have incorporated vitrification. This process—performed before cryopreservation—uses high concentrations of carbohydrate-containing cryoprotectants to solidify the cell in a glass-like state and minimize ice damage. Superior results in animals have been achieved with vitrified cryopreserved oocytes, and although rare, there have been some human births.^29-31

Oocyte cryopreservation without vitrification has been performed since the 1980s, but with limited success.³² Intracytoplasmic sperm injection (ICSI) seemed to have partly obviated initial problems with fertilization.³³ However, the first report of a live birth using cryopreserved oocytes and ICSI did not appear until late in the 1990s.³⁴ Although ICSI may improve the success rate with cryopreserved oocyte fertilization, it may also add another reason for concern over the long-term health consequences in the children conceived.^35,36 By the end of last year, fewer than 50 babies had been delivered following fertilization of previously cryopreserved oocytes, a testament to its challenges (Debra Gook, oral communication, October 2002).

Perhaps these concerns will give way to completely new alternatives to cryopreservation as gamete reconstruction research produces astonishing alternatives and hopefully, results. For instance, the reconstitution of oocytes from bone marrow-derived stem cells may soon be an option.^37-39 Another alternative includes first activating the oocytes to allow them to complete the final meiotic division and then freezing the haploid zygotes as we have been freezing embryos. This obviates the large cytoplasm and temperature-sensitive spindles that make oocyte freezing difficult. However, drawbacks with this technique include the need for artificial activation before freezing. Second, fertilization requires nuclear transfer techniques that currently are not routine in any IVF lab. However, using this approach live births in mice have been achieved in our lab (H. Liu, MD; J.J.Z.; J.A.G., unpublished data 2003).

Conclusions

Despite reassuring data on the questionable risk of cancer associated with ART, women and clinicians continue to be apprehensive about fertility issues in cancer patients. For this reason, most patients and primary care physicians hesitate to use ART in cancer patients. We recommend that women with cancer seek institutions that are supportive of the difficult decisions (reproductive and otherwise) that they face. Ob/gyns can refer these patients to tertiary centers that use a formal program of counseling and close collaboration between oncologists, perinatologists, and reproductive endocrinologists. These specialists can educate and inform patients, enabling them to choose wisely among every possible option to achieve their family planning objectives. As with other new developments in ART, we'll need to confront the ethical debates that will surely ensue.^40,41

In the best of circumstances, pursuing the goal of having a family demands difficult decision-making. A regimen of fertility impairing-cancer therapy adds an extra measure of anxiety to family planning for all concerned. Fortunately, there are some new options and others on the horizon for preserving fertility. Maintaining her fertility potential and keeping her options open may greatly comfort a cancer patient and her family. At the same time, trying to deal with two of life's greatest challenges—family planning and the life-and-death struggle of cancer—can elicit tremendous anxiety in a woman, her family, and clinician. We as physicians should urge family planning and raise fertility issues with our patients who have—or have had cancer.

REFERENCES

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2. Del Priore G, Grifo JA, Zhang JJ, et al. Exploring a cancer patient's reproductive options. Contemporary OB-GYN. 2002;47(1):53-66.

3. Del Priore G, Smith JR, Boyle DC, et al. Uterine transplantation, abdominal trachelectomy, and other reproductive options for cancer patients. Ann N Y Acad Sci. 2001;943:287-295.

4. Gosden RG. Gonadal tissue cryopreservation and transplantation. Reprod Biomed Online. 2002;4(suppl 1):64-67.

5. Scott JR, Hendrickson M, Lash S, et al. Pregnancy after tubo-ovarian transplantation. Obstet Gynecol. 1987;70:229-234.

6. Fageeh W, Raffa H, Jabbad H, et al. Transplantation of the human uterus. Int J Gynaecol Obstet. 2002;76:245-251.

7. Keith LG, Del Priore G. Uterine transplantation in humans: a new frontier. Int J Gynaecol Obstet. 2002;76:243-244.

8. Oktay K, Economos K, Rucinski J, et al. Endocrine function and oocyte retrieval after autologous transplantation of ovarian cortical pieces to the forearm. JAMA. 2001;286:1490-1493.

9. Oktay K, Karlikaya G. Ovarian function after autologous transplantation of frozen, banked human ovarian tissue. N Engl J Med. 2000;342:1919.

10. Eraslan S, Hamernik RJ, Hardy JD. Replantation of uterus and ovaries in dogs, with successful pregnancy. Arch Surg. 1966;92:9-12.

11. Del Priore G, Diflo T, Silber S, et al. Ovary and uterine transplant: a successful rat model. J Soc Gynecol Investig. 2002;9(1s):274a.

12. Jeremias E, Bedaiwy MA, Gurunluoglu R, et al. Heterotopic autotransplantation of the ovary with microvascular anastomosis: a novel surgical technique. Fertil Steril. 2002;77:1278-1282.

13. Wang X, Chen H, Yin H, et al. Fertility after intact ovary transplantation. Nature. 2002;415(6870):385.

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

15. Zakaria F, Boyle D, Del Priore G, et al. Uterine transplant: a successful porcine model. Fertil Steril. 2001;76(3 S):S106. Abstract 0-279.

16. Gosden RG, Baird DT, Wade JC, et al. Restoration of fertility to oophorectomized sheep by ovarian autografts stored at 2196 degrees C. Hum Reprod. 1994;9:597-603.

17. Harp R, Leibach J, Black J, et al. Cryopreservation of murine ovarian tissue. Cryobiology. 1994;31:336-343.

18. Scott JR, Keye WR, Poulson AM, et al. Microsurgical ovarian transplantation in the primate. Fertil Steril. 1981;36:512-515.

19. Oktay K, Aydin B, Karlikaya G. A technique for laparoscopic transplantation of frozen-banked ovarian tissue. Fertil Steril. 2001;75:1212-1216.

20. Radford JA, Lieberman BA, Brison DR, et al. Orthotopic reimplantation of cryopreserved ovarian cortical strips after high-dose chemotherapy for Hodgkin's lymphoma. Lancet. 2001;357:1172-1175.

21. Gunasena KT, Lakey JR, Villines PM, et al. Antral follicles develop in xenografted cryopreserved African elephant (Loxodonta africana) ovarian tissue. Anim Reprod Sci. 1998;53:265-275.

22. Kim SS, Radford J, Harris M, et al. Ovarian tissue harvested from lymphoma patients to preserve fertility may be safe for autotransplantation. Hum Reprod. 2001;16:2056-2060.

23. Kyllonen L, Salmela K, Pukkala E. Cancer incidence in a kidney-transplanted population. Transpl Int. 2000;13(suppl 1):S394-S398.

24. Scott JR, Branch DW, Holman J. Autoimmune and pregnancy complications in the daughter of a kidney transplant patient. Transplantation. 2002;73:815-816.

25. Khorram O, Patrizio P, Wang C, et al. Reproductive technologies for male infertility. J Clin Endocrinol Metab. 2001;86:2373-2379.

26. Tournaye H, Merdad T, Silber S, et al. No differences in outcome after intracytoplasmic sperm injection with fresh or with frozen-thawed epididymal spermatozoa. Hum Reprod. 1999;14:90-95.

27. Oktay K, Newton H, Aubard Y, et al. Cryopreservation of immature human oocytes and ovarian tissue: an emerging technology? Fertil Steril. 1998;69:1-7.

28. Gook DA, Edgar DH. Cryopreservation of the human female gamete: current and future issues. Hum Reprod. 1999;14):2938-2940.

29. Lane M, Gardner DK. Live births following vitrification of mouse oocytes using the VitroLoop. Fertil Steril. 2000; 74(3 suppl):S47. Abstract 0-122.

30. Yoon TK, Chung HM, Lim JM, et al. Pregnancy and delivery of healthy infants developed from vitrified oocytes in a stimulated in vitro fertilization-embryo transfer program. Fertil Steril. 2000;74:180-181.

31. Kuleshova L, Gianaroli L, Magli C, et al. Birth following vitrification of a small number of human oocytes: case report. Hum Reprod. 1999;14:3077-3079.

32. Chen C. Pregnancy after human oocyte cryopreservation. Lancet. 1986;1:884-886.

33. Porcu E, Fabbri R, Petracchi S, et al. Ongoing pregnancy after intracytoplasmic injection of testicular spermatozoa into cryopreserved human oocytes. Am J Obstet Gynecol. 1999;180:1044-1045.

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

35. Fabbri R, Porcu E, Marsella T, et al. Human oocyte cryopreservation: new perspectives regarding oocyte survival. Hum Reprod. 2001;16:411-416.

36. Men H, Monson RL, Parrish JJ, et al. Detection of DNA damage in bovine metaphase II oocytes resulting from cryopreservation. Mol Reprod Dev. 2003;64:245-250.

37. Hubner K, Fuhrmann G, Christenson LK, et al. Derivation of oocytes from mouse embryonic stem cells. Science. 2003;23:1251-1256.

38. Gook DA, Osborn SM, Johnston WI. Parthenogenetic activation of human oocytes following cryopreservation using 1,2-propanediol. Hum Reprod. 1995;10:654-658.

39. Liu H, Zhang J, Krey LC, et al. In-vitro development of mouse zygotes following reconstruction by sequential transfer of germinal vesicles and haploid pronuclei. Hum Reprod. 2000;15:1997-2002.

40. Robertson JA. Ethical issues in ovarian transplantation and donation. Fertil Steril. 2000;73:443-446.

41. Minkoff H, Santoro N. Ethical considerations in the treatment of infertility in women with human immunodeficiency virus infection. N Engl J Med. 2000;342: 1748-1750.

Dr. Del Priore is Director of Gynecologic Oncology at the Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, N.Y. Dr. Zhang is a Fellow and Dr. Grifo is Director, Division of Reproductive Endocrinology and Infertility, at the NYU School of Medicine, New York, N.Y.

Key points

• New options for preserving a cancer survivor's fertility—and others on the horizon—may greatly comfort an anxious patient and her family, but unfortunately, all transplanted ovarian tissue to date has functioned for only a limited time, which will likely be the fate of the first human transplant.

• Clearly we must find some method to preserve ovarian function while a patient is undergoing gonadal-toxic therapy. But will that technique be transplantation, ovary cortical cryopreservation, xenografting, or some other option?

• Xenografting—using a temporary animal host for human ovaries—may be an attractive alternative to cryopreservation because it avoids the loss of follicle reserve that is inevitable with cryopreservation.

• Vitrification overcomes some of the difficulties inherent in the fragile process of oocyte cryopreservation by solidifying the cell in a glass-like state, minimizing ice damage. Although rare, there have been some human births from vitrified cryopreserved oocytes.

 

Giuseppe DelPriore, Jamie Grifo. Gamete cryopreservation and ovarian tissue transplantation. Contemporary Ob/Gyn Aug. 1, 2003;48:85-91.