- Email: [email protected]
Long-term ovarian function and fertility after heterotopic autotransplantation of cryobanked human ovarian tissue: 8-year experience in cancer patients
Long-term ovarian function and fertility after heterotopic autotransplantation of cryobanked human ovarian tissue: 8-year experience in cancer patients S. Samuel Kim, M.D.,a Woo S. Lee, M.D.,b Mi K. Chung, Ph.D.,b Hoi Chang Lee, M.Sc.,c Hyang H. Lee, M.Sc.,c and David Hill, Ph.D.d a Reproductive Endocrinology & Infertility, University of Kansas, Kansas City, Kansas; b CHA Fertility Center, Seoul, South Korea; c Eulji University School of Medicine, Seoul, South Korea; and d ART Reproductive Center, Beverly Hills, California
Objective: To assess the long-term ovarian function and fertility after heterotopic autotransplantion of frozen– thawed ovarian tissue in cancer patients. Design: Prospective clinical case series. Setting: Academic medical center Patient(s): Four young cancer patients who completed cancer treatment. Intervention(s): Cryopreserved ovarian tissue (2000–2002) was thawed and transplanted to the heterotopic site (between the rectus muscle and fascia) between 2002 and 2005. Main Outcome Measure(s): [1] Serial blood tests (FSH, LH, estradiol, progesterone, testosterone) and ultrasound examinations. [2] Oocyte retrieval and in vitro fertilization. Result(s): The hormonal profiles were consistent with the postmenopausal level before transplantation. The return of the ovarian function was evidenced by hormonal profiles between 12 and 20 weeks after transplantation. Three patients underwent a second transplantation, as restored ovarian function lasted only 3 to 5 months. After the second transplantation, long-term ovarian function (lasting for 15–41 months) was established in all three patients. Six oocytes (one GV, four MI, one MII) were retrieved from the grafts. Three MI oocytes were developed to full maturity in vitro. Four MII oocytes were fertilized and developed to the cleavage stage embryos (up to six-cell). Conclusion(s): Autotransplantation of frozen–thawed ovarian tissue to a heterotopic site restored long-term ovarian function (for >40 months), and showed a potential to restore fertility in cancer patients. (Fertil Steril 2009;91:2349–54. 2009 by American Society for Reproductive Medicine.) Key Words: Heterotopic transplantation, fertility preservation, ovarian tissue, autotransplantation, cryopreservation, ovarian function, cancer, embryo
The interest and desire for robust technologies to preserve fertility in women with cancer have grown rapidly, as the number of long-term cancer survivors has increased markedly the last 10 years. Indeed, >10 million people living in the United States are cancer survivors, and approximately 1 in 715 adults in the United Kingdom will be childhood cancer survivors by 2010 (1). Currently, the 5-year survival rate for all cancers combined in women is over 64%. In particular, the 5-year survival rate for breast cancer is approaching 90% (2). Although there are a few options for fertility preservation in female cancer patients, most of the options except embryo cryopreservation are investigational at the moment (3). An emerging technology, involving autotransplantaion of ovarian tissue banked at low temperature, offers the possibility
Received February 15, 2008; revised and accepted April 9, 2008; published online August 4, 2008. Presented at the 2007 ASRM annual meeting (received the SRS Prize Paper Award). S.S.C. has nothing to disclose. W.S.L. has nothing to disclose. M.K.C. has nothing to disclose. H.C.L. has nothing to disclose. H.H.L. has nothing to disclose. D.H. has nothing to disclose. Reprint requests: S. Samuel Kim, Reproductive Endocrinology & Infertility, University of Kansas, 3901 Rainbow Boulevard, Kansas City, KS 66160 (FAX: 913-588-6271; E-mail: [email protected]).
0015-0282/09/$36.00 doi:10.1016/j.fertnstert.2008.04.019
of restoring ovarian function and fertility after highly gonadotoxic cancer treatment. The feasibility of clinical applications of this technique was not entertained until 1994, when successful restoration of fertility after transplantation of frozen–thawed ovarian tissue was reported for the first time in an ewe (4). Since then, there has been a steady increase in the interest in the clinical application of human ovarian transplantation worldwide. Although the number of cases is small, the successful human ovarian autotransplantation (orthotopic and heterotopic) on an experimental basis have been reported since 2000 (5–7). To date, four healthy babies were born after orthotopic autotransplantation of cryobanked human ovarian tissue (8–10). In addition, Silber et al. (11) demonstrated successful restoration of ovarian function in eight women with premature ovarian failure after transplantation of fresh donor ovarian tissue from their identical twin sisters. Autotransplantation of human ovarian tissue can be done either orthotopically or heterotopically (12). The pregnancies and live births reported so far were resulted from transplantation to the orthotopic sites. To date, there is no report of viable pregnancy after ovarian transplantation to the heterotopic sites. Does that mean that heterotopic transplantation is not a practical technology to restore fertility? Because the experience with heterotopic ovarian
Fertility and Sterility Vol. 91, No. 6, June 2009 Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc.
2349
transplantation is very limited in humans, it will be difficult to make a final verdict on this issue without further investigation.
medium (Sigma, St. Louis, MO), and processed for freezing in <1 hour after recovery.
Theoretically, orthotopic autotransplantation can restore normal reproductive function, which leads to natural conception. Although the follicular loss from freezing and thawing of ovarian tissue appears to be minimal (13, 14), tissue ischemia after grafting can cause a significant loss of follicles (15, 16). The expected relatively short life span of ovarian tissue after transplantation may not be sufficient to restore fertility, and repeated transplantation will be required to extend the ovarian function. Where few follicles remain and early graft exhaustion is expected, it may be reasonable to use heterotopic sites such as subcutaneous tissue to avoid multiple invasive procedures.
Ovarian cortex was processed into thin slices of 1 to 2 mm thickness at room temperature (25 C) and cut into small sections (between 5 5 and 10 10 mm in size). Ovarian cortical sections were transferred into 1.8-mL cryogenic vials (Nunc Intermed, Kamstrup, Denmark) containing 1.5 M dimethyl sulfoxide (DMSO) (Sigma) with 1% human serum albumin (Sigma) and 0.1 M sucrose (Sigma) in Leibovitz L-15 medium (two sections per vial). The vials were gently shaken for 30 minutes at 4 C to promote equilibration, cooled in a programmable freezer according to our ovarian freezing program (cooled at 2 C/min to 7 C, seeding manually at 7 C, 0.3 C/min to 40 C, 10 C/min to 120 C), and plunged into liquid nitrogen (196 C) for storage.
There are many unknowns with human ovarian transplantation that need to be investigated such as the optimal graft site, the method to improve follicle survival, the efficacy of transplantation, and quality of eggs and embryos from the grafted ovarian tissue. Moreover, it is important to know how long the restored ovarian function can be maintained. In our clinical series of heterotopic ovarian transplantation between 2000 and 2007 we were able to assess the long-term ovarian function after transplantation. In addition, we investigated fertility restoration through the steps of ovarian transplantation to the heterotopic site, oocyte retrieval from ovarian grafts, IVF of the retrieved oocytes, and embryo culture. MATERIALS AND METHODS Study Patients The study was approved by the institutional review board, and informed consent was obtained from each patient before transplantation. Four study patients (three with cervical cancer, one with breast cancer) were identified. The age range of the patients was 29 to 38 years old. The main purpose of ovarian tissue cryopreservation was fertility preservation, except one patient who was more interested in restoration of endocrine function. Of three cervical cancer patients, two patients with stage IIb squamous cell carcinoma of cervix underwent radiation therapy and chemotherapy (cisplatin), the third one with stage Ib cancer was treated with radical hysterectomy, pelvic lymphadenectomy followed by radiotherapy. The ovary was collected by laparotomy or laparoscopy before chemotherapy and radiotherapy in all cases. The patient with stage IIa invasive intraductal adenocarcinoma of the breast underwent modified radical mastectomy followed by chemotherapy (four cycles of adriamycin and cytoxan). The ovary was collected by laparoscopy before the initiation of chemotherapy. Cryopreservation of Ovarian Tissue Before chemotherapy/radiotherapy we collected a whole ovary to secure the sufficient follicular pool for the future use. The small ovarian biopsy was sent to pathology. There was no histologic evidence of ovarian metastasis of cancer cells in ovarian biopsy. The recovered ovaries were transported immediately to the laboratory in Leibovitz L-15 2350
Kim et al.
Transplantation of Thawed Ovarian Tissue For transplantation, cryopreserved ovarian tissue were thawed rapidly (100 C/min) by agitating in a warm water bath (35 C) for 2 to 3 minutes, and washed in a stepwise manner to minimize osmotic damages (1.0 M DMSO þ 0.1 M sucrose, 0.5 M DMSO þ 0.1 M sucrose, and 0.1 M sucrose). Each washing step took 3 minutes at room temperature. Thawed ovarian sections were incubated in Leibovitz L-15 medium containing 500 IU/mL penicillin G (Sigma) and 382 IU/mL streptomycin (Sigma) and 100 mg/mL ascorbic acid for 30 minutes at 37 C in the incubator (5% CO2 in air) before transplantation. Eight to 10 thawed cortical sections were threaded onto 3-0 vicryl sutures (Ethicon, Edinburgh, UK). After making a skin incision (1–2 cm) in the abdomen, the space for transplantation was created between the rectus muscle and the rectus sheath by blunt and sharp dissection. Threaded ovarian tissue was carefully placed into the space between the rectus sheath and the rectus muscle using a pull-through method (Fig 1). After the closure of the skin, compression dressing was applied to the transplant site. Monitoring Blood samples for the sequential evaluation of the serum concentrations of FSH, LH, estradiol (E2), progesterone (P), and testosterone were collected monthly after transplantation. Serum hormones were measured by a solid-phase chemiluminiscent assay using an ADVIA Automated Analyzer (Bayer Corporation, Tarrytown, NY). The intra- and interassay coefficients of variation were 6.5% and 9.2% for E2, 3.9% and 4.2% for FSH, 4.9% and 5.5% for LH, and 6.7% and 7.9% for P, respectively. Ultrasonographic examination was performed using 7.0-MHz abdominal transducer (HDI 3000: ATL, Bothell, WA) monthly on the same day of blood sampling. When restoration of ovarian function was confirmed, blood was collected weekly. Ovarian Stimulation and Oocyte Retrieval The ovarian graft was stimulated with daily administration of 300 IU of rFSH until the size of a dominant follicle reached at
Heterotopic transplantation of human ovarian tissue
Vol. 91, No. 6, June 2009
FIGURE 1 The process of heterotopic transplantation of frozen–thawed ovarian tissue to the space between the rectus muscle and the rectus sheath.
Kim. Heterotopic transplantation of human ovarian tissue. Fertil Steril 2009.
14 to 16 mm in diameter. The oocyte retrieval was performed under the ultrasound guidance 36 hours after administration of rhCG (Ovidrel 250 mg, Organon, Roseland, NJ). In Vitro Maturation, Fertilization, and Embryo Culture The immature oocytes (GV, MI) were incubated for 24 hours in an organ culture dish containing 2 mL of maturation medium: G2 medium (series III, Vitrolife, Sweden) supplemented with 20% (vol/vol) human follicular fluid, 75 mIU/ mL recombinant FSH, 0.5 IU/mL hCG, and 1mg/mL E2 at 37 C with 5% CO2 in humidified air. After the culture, oocytes were denuded by hyaluronidase solution (80 IU/mL, Sigma), and their maturity was determined by the presence of the first polar body. The mature oocytes (MII) were fertilized by husband’s sperm by intracytoplasmic sperm injection (ICSI), and showed two distinct pronuclei at 16 hours after ICSI. These pronuclear zygotes were further cultured in 2 mL culture medium (P-1 medium, Irvine Scientific, Santa Ana, CA) for 48 hours. RESULTS The hormonal profiles of all four patients were consistent with the postmenopausal level before transplantation (FSH 80–100 mIU/mL; E2 undetectable). The return of the ovarian function was evidenced by the elevation of serum E2 levels and by the decrease of FSH levels below 20 mIU/mL between 12 and 20 weeks after transplantation (Fig. 2). However, restored ovarian function lasted only 3 to 5 months. The option of repeated transplantation was discussed, and all three patients (except one with relapsed disease) agreed to undergo second transplantation. The return of ovarian function after the second transplantation was faster in all three patients (between 2 and 4 months) (Fig. 3). In contrast to the first transplantation, we observed the establishment of long-term ovarian function (lasting for 15–36 months) after the second transplantation. All three patients maintained the FSH levels below 15 mIU/mL during this follow-up period. In addition, restoration of the cyclic FSH/LH secretion was evidenced by the weekly hormonal profiles (Fig. 4). Of note, the interval of the most cycles during the monitoring period Fertility and Sterility
was approximately 6 weeks. During the weekly monitoring period, the occasional elevation of the P level above 3.0 ng/mL was observed, suggesting the occurrence of spontaneous ovulation in the ovarian transplant. As an example, the LH surge (69.8 IU/L) followed by the elevation of the P concentration (9.6 ng/mL) was noticed in patient A. The total testosterone concentrations were in the range of 8 to 91 ng/dL while ovarian grafts were functioning. The ultrasound revealed dominant follicles in the heterotopic site. In fact, all three patients reported a palpable growing lump, when the size of the follicle reached approximately 1 cm in diameter.
FIGURE 2 Monthly FSH and E2 levels after the first transplantation in three cancer patients (a–c). The FSH levels were postmenopausal at the time of transplantation. Three months after transplantation the serum E2 levels increased above 40 pg/mL in all patients. The serum FSH levels returned to below 20 IU/L in all patients 5 months posttransplantation.
Kim. Heterotopic transplantation of human ovarian tissue. Fertil Steril 2009.
2351
FIGURE 3
FIGURE 4
Monthly FSH and E2 levels after the second transplantation in three cancer patients (A–C). Estradiol production (above 40 pg/mL) from the ovarian grafts was noticed in <2 months after the second transplantation in all three patients. However, the serum FSH levels were still elevated for 4 months after transplantation except the patient B.
Restoration of the cyclic FSH/LH secretion was evidenced by weekly hormonal profiles.
Kim. Heterotopic transplantation of human ovarian tissue. Fertil Steril 2009.
18). Although the current freezing technique is relatively satisfactory, there are uncertainties about the optimal future use of stored ovarian tissue because the most follicles survived through the freezing and thawing process are primordial or primary follicles. Autotransplantation can be a practical method to restore ovarian function and to mature oocytes in stored ovarian tissue. However, our clinical experience and knowledge with this technology is very limited, especially with heterotopic transplantation. Kim. Heterotopic transplantation of human ovarian tissue. Fertil Steril 2009.
We were able to retrieve six oocytes (GV 1, MI 4, MII 1) from the ovarian grafts in two patients between August 2003 and November 2005. Three MI oocytes were developed to full maturity in vitro (Fig. 5). All four MII oocytes were fertilized and cultured in vitro for 2 to 3 days, which resulted in four embryos at the six-cell, three- cell, two-cell, and pronuclear stage (Fig. 5). All embryos were cryopreserved. In the near future, the stored embryos will be thawed and transferred to the surrogate. DISCUSSION Because germ cells are susceptible to cytotoxic treatment (especially alkylating agents and/or radiation), premature ovarian failure is a common sequel to aggressive cancer treatment. As sterility can impact on the quality of life for many young cancer survivors, there has been growing awareness of the importance of fertility conservation for young cancer patients. Cryopreservation of semen can be offered for adult males as a safeguard for fertility. Although cryopreservation of embryos or oocytes can be a strategy to preserve fertility in women, lengthy ovarian stimulation and follicle aspiration can delay cancer treatment and limit its application for women with cancer (12). An emerging strategy, involving autotransplantation of ovarian tissue banked at low temperature, offers the possibility of restoring ovarian function in women and children after highly gonadotoxic cancer treatment. The follicular survival in the stored ovarian tissue after freezing and thawing is approximately 70% to 80% (17, 2352
Kim et al.
There are many unresolved issues involved with heterotopic transplantation of ovarian tissue in humans. One of them is the optimal site for heterotopic transplantation. In theory, the optimal site should be rich in vascularity, as rapid revascularization of the graft is crucial for the survival of ovarian follicles. In addition, the optimal heterotopic site should be easily accessible without an invasive procedure, because the repeated ovarian transplantation and/or egg retrieval may be necessary if early graft exhaustion is expected (19). The subcutaneous tissue or the superficial muscle appears to be a practical heterotopic site for human ovarian transplantation. Indeed, the return of ovarian function was demonstrated when ovarian cortical strips were transplanted subcutaneously to the forearm (20). In our previous study (unpublished), different heterotopic sites such as breast tissue, subcutaneous tissue of the hip, and pre-rectus space were tested. These sites were selected after considering multiple factors including vascularity, cosmetic aspects, convenience, easiness for grafting, and accessibility for egg retrievals. For us, the abdominal site (between the rectus sheath and the rectus muscle) appeared to be the best heterotopic site for human ovarian transplantation. Another concern related to transplanting ovarian tissue to the heterotopic site is the environmental factors that can affect the follicular development. Obviously, the environment of the heterotopic site we selected is different from that of orthotopic site in the pelvic cavity. The environmental factors that can be altered with heterotopic transplantation include (but not limited to) temperature, pressure, space for follicular growth, peritoneal fluid, cytokines, angiogenic factors, and
Heterotopic transplantation of human ovarian tissue
Vol. 91, No. 6, June 2009
FIGURE 5 Two MI oocytes retrieved from the ovarian graft were matured to MII oocytes in vitro. These MII oocytes were fertilized with the husband’s sperm by ICSI and cultured for 3 days to the six-cell and three-cell stage embryos.
Kim. Heterotopic transplantation of human ovarian tissue. Fertil Steril 2009.
hormonal milieu. The follicles cannot grow and mature normally without an optimal nurturing environment. Hence, it needs to be explored if the environment of the heterotopic site can be suitable for growing follicles. Previously, we investigated the quality of human oocytes developed in the subcutaneous space using a severe combined immunodeficient mouse xenograft model (21). The result was alarming, as the microtubule and chromatin patterns of human oocytes developed in the subcutaneous space of the host animal were abnormal in most cases. Although not proven, it could be caused by the suboptimal environmental condition of the graft site. On the other hand, the results of the present study are encouraging, because the most oocytes retrieved from the heterotopic ovarian graft were fertilized normally (four of six). Of four fertilized oocytes, three embryos developed to the cleavage stage. The quality and developmental rate of embryos, however, appeared to be below average. There are many factors besides the graft site that can affect the quality and development of the oocytes and embryos. Thus, it is too premature to draw any conclusions about the relations between the quality of oocyte/embryo and heterotopic graft site. By 3 to 5 months posttransplantation, reestablishment of ovarian function was clinically evidenced: the patient no longer experienced hot flushes, and the follicular development was noticed by palpation as well as by ultrasound. Interestingly, all patients felt a growing lump accompanied by slight discomfort in the graft site, and one patient experienced an itching sensation, which coincided with the timing of follicular enlargement. The hormonal profiles also indicated sex steroid hormone production (E2, testosterone) from the ovarian graft, followed by the return of the FSH level below 20 mIU/mL. The latent periods of ovarian function after transplantation were shorter than expected. The data from orthotopic autotranslantation in patients with Hodgkin’s lymphoma indicated that the return of ovarian function took about 6 Fertility and Sterility
months (7, 9). It has been our knowledge that entire follicles except primordial follicles (and some primary follicles) in human ovarian tissue are lost in the process of freezing, thawing, and transplantation (19, 22). Because the growth of a primordial follicle to the large antral stage in humans takes >6 months (23), the return of endocrine function in 3 months after heterotopic transplantation is an unexpected finding. We can speculate that either some growing follicles in ovarian tissue survived through freezing, thawing, and grafting, or unknown factors associated with heterotopic autotransplantation accelerated the growth phase of primordial follicles. Of note, an acceleration of follicular growth has been observed with in vitro follicular culture (24). In the present study, we occasionally noticed the spontaneous LH surge followed by the elevation of P level above the ovulatory value (3 ng/mL) after heterotopic autotransplantation with frozen– thawed human ovarian tissue, which strongly suggested the occurrence of spontaneous ovulation in the ovarian graft. The duration of ovarian function correlates with the number of surviving follicles in the graft, which is influenced by many factors such as the age of the patient. The single most important factor determining the graft longevity is the degree of ischemia that causes a significant follicular loss after transplantation (12, 16). In fact, <30% of primordial follicles survived after grafting in studies using a sheep model (15, 22). Nevertheless, relatively long-term ovarian function (22 months) after autotransplantation of frozen–thawed ovarian tissue in ewe was demonstrated (15). In humans, there was a report that ovarian function persisted >2 years after heterotopic transplantation of fresh ovarian tissue to the forearm (25), whereas other studies showed that ovarian function lasted only for 2 to 5 months after autografting of frozen– thawed human ovarian tissue (5, 7, 26). In our present study, we noticed that the restored ovarian function after transplantation lasted for 3 to 5 months initially, but with the second transplantation long-term ovarian function (lasting for 1–3 years) has been established in all three patients. Of note, the return of ovarian function after 2353
the second transplantation was faster (taking only 2-4 months). This could be explained by the residual effect of the first transplantation. In addition, we can speculate possible synergistic effects of growth factors and hormones. In summary, cryopreserved human ovarian tissue can be used to restore long-term ovarian function by repeated autotransplantation to a heterotopic site. It appears that heterotopic transplantation to a space between the rectus sheath and the rectus muscle is not only convenient but effective. In addition, restoration of fertility can be achieved by heterotopic transplantation, as we have demonstrated that successful retrieval and fertilization of the eggs from the ovarian graft led to the embryo development up to the six-cell stage with in vitro culture. To our knowledge, this is the first longterm follow-up study (accumulating data for 8 years between 2000 and 2007) that investigated restoration of ovarian function as well as fertility after heterotopic autotransplantation of frozen–thawed human ovarian tissue. REFERENCES 1. Wallace WH, Anderson RA, Irvine DS. Fertility preservation for young patients with cancer: who is at risk and what can be offered? Lancet Oncol 2005;6:209–18. 2. Jemal A, Clegg LX, Ward E, Ries LA, Wu X, Jamison PM, et al. Annual report to the nation on the status of cancer, 1975–2001, with a special feature regarding survival. Cancer 2004;101:3–27. 3. Kim SS. Fertility preservation in female cancer patients: current developments and future directions. Fertil Steril 2006;85:1–11. 4. Gosden RG, Baird DT, Wade JC, Webb R. Restoration of fertility to oophorectomized sheep by ovarian autografts stored at 196 degrees C. Hum Reprod 1994;9:597–603. 5. Kim SS, Hwang IT, Lee HC. Heterotopic autotransplantation of cryobanked human ovarian tissue as a strategy to restore ovarian function. Fertil Steril 2004;82:930–2. 6. Oktay K, Karlikaya G. Ovarian function after transplantation of frozen, banked autologous ovarian tissue. N Engl J Med 2000;342:1919. 7. Radford JA, Lieberman BA, Brison DR, Smith AR, Critchlow JD, Russell SA, et al. Orthotopic reimplantation of cryopreserved ovarian cortical strips after high-dose chemotherapy for Hodgkin’s lymphoma. Lancet 2001;357:1172–5. 8. Demeestere I, Simon P, Emiliani S, Delbaere A, Englert Y. Fertility preservation: successful transplantation of cryopreserved ovarian tissue in a young patient previously treated for Hodgkin’s disease. Oncologist 2007;12:1437–42. 9. Donnez J, Dolmans MM, Demylle D, Jadoul P, Pirard C, Squifflet J, et al. Livebirth after orthotopic transplantation of cryopreserved ovarian tissue. Lancet 2004;364:1405–10.
2354
Kim et al.
10. Meirow D, Levron J, Eldar-Geva T, Hardan I, Fridman E, Zalel Y, et al. Pregnancy after transplantation of cryopreserved ovarian tissue in a patient with ovarian failure after chemotherapy. N Engl J Med 2005;353: 318–21. 11. Silber SJ, Gosden RG. Ovarian transplantation in a series of monozygotic twins discordant for ovarian failure. N Engl J Med 2007;356: 1382–4. 12. Kim SS, Battaglia DE, Soules MR. The future of human ovarian cryopreservation and transplantation: fertility and beyond. Fertil Steril 2001;75:1049–56. 13. Candy CJ, Wood MJ, Whittingham DG. Restoration of a normal reproductive lifespan after grafting of cryopreserved mouse ovaries. Hum Reprod 2000;15:1300–4. 14. Candy CJ, Wood MJ, Whittingham DG. Effect of cryoprotectants on the survival of follicles in frozen mouse ovaries. J Reprod Fertil 1997;110: 11–9. 15. Baird DT, Webb R, Campbell BK, Harkness LM, Gosden RG. Long-term ovarian function in sheep after ovariectomy and transplantation of autografts stored at 196 C. Endocrinology 1999;140:462–71. 16. Kim SS, Battaglia DE, Soules MR. The future of human ovarian cryopreservation and transplantation: fertility and beyond. Fertil Steril 2001;75:1049–56. 17. Gook DA, Edgar DH, Stern C. Effect of cooling rate and dehydration regimen on the histological appearance of human ovarian cortex following cryopreservation in 1, 2-propanediol. Hum Reprod 1999;14:2061–8. 18. Newton H, Aubard Y, Rutherford A, Sharma V, Gosden R. Low temperature storage and grafting of human ovarian tissue. Hum Reprod 1996;11:1487–91. 19. Kim SS, Yin H, Gosden RG. Cryobanking of ovarian and testicular tissue for children and young adults. In: Tulandi T, Gosden RG, eds. Preservation of fertility. London: Taylor & Francis, 2004:157–75. 20. Oktay K, Economos K, Kan M, Rucinski J, Veeck L, Rosenwaks Z. Endocrine function and oocyte retrieval after autologous transplantation of ovarian cortical strips to the forearm. JAMA 2001;286:1490–3. 21. Kim SS, Kang HG, Kim NH, Lee HC, Lee HH. Assessment of the integrity of human oocytes retrieved from cryopreserved ovarian tissue after xenotransplantation. Hum Reprod 2005;20:2502–8. 22. Aubard Y, Piver P, Cogni Y, Fermeaux V, Poulin N, Driancourt MA. Orthotopic and heterotopic autografts of frozen-thawed ovarian cortex in sheep. Hum Reprod 1999;14:2149–54. 23. Gougeon A. Dynamics of follicular growth in the human: a model from preliminary results. Hum Reprod 1986;1:81–7. 24. Thomas FH, Armstrong DG, Telfer EE. Activin promotes oocyte development in ovine preantral follicles in vitro. Reprod Biol Endocrinol 2003;1:76. 25. Oktay K, Buyuk E, Rosenwaks Z, Rucinski J. A technique for transplantation of ovarian cortical strips to the forearm. Fertil Steril 2003;80: 193–8. 26. Callejo J, Salvador C, Miralles A, Vilaseca S, Lailla JM, Balasch J. Longterm ovarian function evaluation after autografting by implantation with fresh and frozen-thawed human ovarian tissue. J Clin Endocrinol Metab 2001;86:4489–94.
Heterotopic transplantation of human ovarian tissue
Vol. 91, No. 6, June 2009
Objective: To assess the long-term ovarian function and fertility after heterotopic autotransplantion of frozen– thawed ovarian tissue in cancer patients. Design: Prospective clinical case series. Setting: Academic medical center Patient(s): Four young cancer patients who completed cancer treatment. Intervention(s): Cryopreserved ovarian tissue (2000–2002) was thawed and transplanted to the heterotopic site (between the rectus muscle and fascia) between 2002 and 2005. Main Outcome Measure(s): [1] Serial blood tests (FSH, LH, estradiol, progesterone, testosterone) and ultrasound examinations. [2] Oocyte retrieval and in vitro fertilization. Result(s): The hormonal profiles were consistent with the postmenopausal level before transplantation. The return of the ovarian function was evidenced by hormonal profiles between 12 and 20 weeks after transplantation. Three patients underwent a second transplantation, as restored ovarian function lasted only 3 to 5 months. After the second transplantation, long-term ovarian function (lasting for 15–41 months) was established in all three patients. Six oocytes (one GV, four MI, one MII) were retrieved from the grafts. Three MI oocytes were developed to full maturity in vitro. Four MII oocytes were fertilized and developed to the cleavage stage embryos (up to six-cell). Conclusion(s): Autotransplantation of frozen–thawed ovarian tissue to a heterotopic site restored long-term ovarian function (for >40 months), and showed a potential to restore fertility in cancer patients. (Fertil Steril 2009;91:2349–54. 2009 by American Society for Reproductive Medicine.) Key Words: Heterotopic transplantation, fertility preservation, ovarian tissue, autotransplantation, cryopreservation, ovarian function, cancer, embryo
The interest and desire for robust technologies to preserve fertility in women with cancer have grown rapidly, as the number of long-term cancer survivors has increased markedly the last 10 years. Indeed, >10 million people living in the United States are cancer survivors, and approximately 1 in 715 adults in the United Kingdom will be childhood cancer survivors by 2010 (1). Currently, the 5-year survival rate for all cancers combined in women is over 64%. In particular, the 5-year survival rate for breast cancer is approaching 90% (2). Although there are a few options for fertility preservation in female cancer patients, most of the options except embryo cryopreservation are investigational at the moment (3). An emerging technology, involving autotransplantaion of ovarian tissue banked at low temperature, offers the possibility
Received February 15, 2008; revised and accepted April 9, 2008; published online August 4, 2008. Presented at the 2007 ASRM annual meeting (received the SRS Prize Paper Award). S.S.C. has nothing to disclose. W.S.L. has nothing to disclose. M.K.C. has nothing to disclose. H.C.L. has nothing to disclose. H.H.L. has nothing to disclose. D.H. has nothing to disclose. Reprint requests: S. Samuel Kim, Reproductive Endocrinology & Infertility, University of Kansas, 3901 Rainbow Boulevard, Kansas City, KS 66160 (FAX: 913-588-6271; E-mail: [email protected]).
0015-0282/09/$36.00 doi:10.1016/j.fertnstert.2008.04.019
of restoring ovarian function and fertility after highly gonadotoxic cancer treatment. The feasibility of clinical applications of this technique was not entertained until 1994, when successful restoration of fertility after transplantation of frozen–thawed ovarian tissue was reported for the first time in an ewe (4). Since then, there has been a steady increase in the interest in the clinical application of human ovarian transplantation worldwide. Although the number of cases is small, the successful human ovarian autotransplantation (orthotopic and heterotopic) on an experimental basis have been reported since 2000 (5–7). To date, four healthy babies were born after orthotopic autotransplantation of cryobanked human ovarian tissue (8–10). In addition, Silber et al. (11) demonstrated successful restoration of ovarian function in eight women with premature ovarian failure after transplantation of fresh donor ovarian tissue from their identical twin sisters. Autotransplantation of human ovarian tissue can be done either orthotopically or heterotopically (12). The pregnancies and live births reported so far were resulted from transplantation to the orthotopic sites. To date, there is no report of viable pregnancy after ovarian transplantation to the heterotopic sites. Does that mean that heterotopic transplantation is not a practical technology to restore fertility? Because the experience with heterotopic ovarian
Fertility and Sterility Vol. 91, No. 6, June 2009 Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc.
2349
transplantation is very limited in humans, it will be difficult to make a final verdict on this issue without further investigation.
medium (Sigma, St. Louis, MO), and processed for freezing in <1 hour after recovery.
Theoretically, orthotopic autotransplantation can restore normal reproductive function, which leads to natural conception. Although the follicular loss from freezing and thawing of ovarian tissue appears to be minimal (13, 14), tissue ischemia after grafting can cause a significant loss of follicles (15, 16). The expected relatively short life span of ovarian tissue after transplantation may not be sufficient to restore fertility, and repeated transplantation will be required to extend the ovarian function. Where few follicles remain and early graft exhaustion is expected, it may be reasonable to use heterotopic sites such as subcutaneous tissue to avoid multiple invasive procedures.
Ovarian cortex was processed into thin slices of 1 to 2 mm thickness at room temperature (25 C) and cut into small sections (between 5 5 and 10 10 mm in size). Ovarian cortical sections were transferred into 1.8-mL cryogenic vials (Nunc Intermed, Kamstrup, Denmark) containing 1.5 M dimethyl sulfoxide (DMSO) (Sigma) with 1% human serum albumin (Sigma) and 0.1 M sucrose (Sigma) in Leibovitz L-15 medium (two sections per vial). The vials were gently shaken for 30 minutes at 4 C to promote equilibration, cooled in a programmable freezer according to our ovarian freezing program (cooled at 2 C/min to 7 C, seeding manually at 7 C, 0.3 C/min to 40 C, 10 C/min to 120 C), and plunged into liquid nitrogen (196 C) for storage.
There are many unknowns with human ovarian transplantation that need to be investigated such as the optimal graft site, the method to improve follicle survival, the efficacy of transplantation, and quality of eggs and embryos from the grafted ovarian tissue. Moreover, it is important to know how long the restored ovarian function can be maintained. In our clinical series of heterotopic ovarian transplantation between 2000 and 2007 we were able to assess the long-term ovarian function after transplantation. In addition, we investigated fertility restoration through the steps of ovarian transplantation to the heterotopic site, oocyte retrieval from ovarian grafts, IVF of the retrieved oocytes, and embryo culture. MATERIALS AND METHODS Study Patients The study was approved by the institutional review board, and informed consent was obtained from each patient before transplantation. Four study patients (three with cervical cancer, one with breast cancer) were identified. The age range of the patients was 29 to 38 years old. The main purpose of ovarian tissue cryopreservation was fertility preservation, except one patient who was more interested in restoration of endocrine function. Of three cervical cancer patients, two patients with stage IIb squamous cell carcinoma of cervix underwent radiation therapy and chemotherapy (cisplatin), the third one with stage Ib cancer was treated with radical hysterectomy, pelvic lymphadenectomy followed by radiotherapy. The ovary was collected by laparotomy or laparoscopy before chemotherapy and radiotherapy in all cases. The patient with stage IIa invasive intraductal adenocarcinoma of the breast underwent modified radical mastectomy followed by chemotherapy (four cycles of adriamycin and cytoxan). The ovary was collected by laparoscopy before the initiation of chemotherapy. Cryopreservation of Ovarian Tissue Before chemotherapy/radiotherapy we collected a whole ovary to secure the sufficient follicular pool for the future use. The small ovarian biopsy was sent to pathology. There was no histologic evidence of ovarian metastasis of cancer cells in ovarian biopsy. The recovered ovaries were transported immediately to the laboratory in Leibovitz L-15 2350
Kim et al.
Transplantation of Thawed Ovarian Tissue For transplantation, cryopreserved ovarian tissue were thawed rapidly (100 C/min) by agitating in a warm water bath (35 C) for 2 to 3 minutes, and washed in a stepwise manner to minimize osmotic damages (1.0 M DMSO þ 0.1 M sucrose, 0.5 M DMSO þ 0.1 M sucrose, and 0.1 M sucrose). Each washing step took 3 minutes at room temperature. Thawed ovarian sections were incubated in Leibovitz L-15 medium containing 500 IU/mL penicillin G (Sigma) and 382 IU/mL streptomycin (Sigma) and 100 mg/mL ascorbic acid for 30 minutes at 37 C in the incubator (5% CO2 in air) before transplantation. Eight to 10 thawed cortical sections were threaded onto 3-0 vicryl sutures (Ethicon, Edinburgh, UK). After making a skin incision (1–2 cm) in the abdomen, the space for transplantation was created between the rectus muscle and the rectus sheath by blunt and sharp dissection. Threaded ovarian tissue was carefully placed into the space between the rectus sheath and the rectus muscle using a pull-through method (Fig 1). After the closure of the skin, compression dressing was applied to the transplant site. Monitoring Blood samples for the sequential evaluation of the serum concentrations of FSH, LH, estradiol (E2), progesterone (P), and testosterone were collected monthly after transplantation. Serum hormones were measured by a solid-phase chemiluminiscent assay using an ADVIA Automated Analyzer (Bayer Corporation, Tarrytown, NY). The intra- and interassay coefficients of variation were 6.5% and 9.2% for E2, 3.9% and 4.2% for FSH, 4.9% and 5.5% for LH, and 6.7% and 7.9% for P, respectively. Ultrasonographic examination was performed using 7.0-MHz abdominal transducer (HDI 3000: ATL, Bothell, WA) monthly on the same day of blood sampling. When restoration of ovarian function was confirmed, blood was collected weekly. Ovarian Stimulation and Oocyte Retrieval The ovarian graft was stimulated with daily administration of 300 IU of rFSH until the size of a dominant follicle reached at
Heterotopic transplantation of human ovarian tissue
Vol. 91, No. 6, June 2009
FIGURE 1 The process of heterotopic transplantation of frozen–thawed ovarian tissue to the space between the rectus muscle and the rectus sheath.
Kim. Heterotopic transplantation of human ovarian tissue. Fertil Steril 2009.
14 to 16 mm in diameter. The oocyte retrieval was performed under the ultrasound guidance 36 hours after administration of rhCG (Ovidrel 250 mg, Organon, Roseland, NJ). In Vitro Maturation, Fertilization, and Embryo Culture The immature oocytes (GV, MI) were incubated for 24 hours in an organ culture dish containing 2 mL of maturation medium: G2 medium (series III, Vitrolife, Sweden) supplemented with 20% (vol/vol) human follicular fluid, 75 mIU/ mL recombinant FSH, 0.5 IU/mL hCG, and 1mg/mL E2 at 37 C with 5% CO2 in humidified air. After the culture, oocytes were denuded by hyaluronidase solution (80 IU/mL, Sigma), and their maturity was determined by the presence of the first polar body. The mature oocytes (MII) were fertilized by husband’s sperm by intracytoplasmic sperm injection (ICSI), and showed two distinct pronuclei at 16 hours after ICSI. These pronuclear zygotes were further cultured in 2 mL culture medium (P-1 medium, Irvine Scientific, Santa Ana, CA) for 48 hours. RESULTS The hormonal profiles of all four patients were consistent with the postmenopausal level before transplantation (FSH 80–100 mIU/mL; E2 undetectable). The return of the ovarian function was evidenced by the elevation of serum E2 levels and by the decrease of FSH levels below 20 mIU/mL between 12 and 20 weeks after transplantation (Fig. 2). However, restored ovarian function lasted only 3 to 5 months. The option of repeated transplantation was discussed, and all three patients (except one with relapsed disease) agreed to undergo second transplantation. The return of ovarian function after the second transplantation was faster in all three patients (between 2 and 4 months) (Fig. 3). In contrast to the first transplantation, we observed the establishment of long-term ovarian function (lasting for 15–36 months) after the second transplantation. All three patients maintained the FSH levels below 15 mIU/mL during this follow-up period. In addition, restoration of the cyclic FSH/LH secretion was evidenced by the weekly hormonal profiles (Fig. 4). Of note, the interval of the most cycles during the monitoring period Fertility and Sterility
was approximately 6 weeks. During the weekly monitoring period, the occasional elevation of the P level above 3.0 ng/mL was observed, suggesting the occurrence of spontaneous ovulation in the ovarian transplant. As an example, the LH surge (69.8 IU/L) followed by the elevation of the P concentration (9.6 ng/mL) was noticed in patient A. The total testosterone concentrations were in the range of 8 to 91 ng/dL while ovarian grafts were functioning. The ultrasound revealed dominant follicles in the heterotopic site. In fact, all three patients reported a palpable growing lump, when the size of the follicle reached approximately 1 cm in diameter.
FIGURE 2 Monthly FSH and E2 levels after the first transplantation in three cancer patients (a–c). The FSH levels were postmenopausal at the time of transplantation. Three months after transplantation the serum E2 levels increased above 40 pg/mL in all patients. The serum FSH levels returned to below 20 IU/L in all patients 5 months posttransplantation.
Kim. Heterotopic transplantation of human ovarian tissue. Fertil Steril 2009.
2351
FIGURE 3
FIGURE 4
Monthly FSH and E2 levels after the second transplantation in three cancer patients (A–C). Estradiol production (above 40 pg/mL) from the ovarian grafts was noticed in <2 months after the second transplantation in all three patients. However, the serum FSH levels were still elevated for 4 months after transplantation except the patient B.
Restoration of the cyclic FSH/LH secretion was evidenced by weekly hormonal profiles.
Kim. Heterotopic transplantation of human ovarian tissue. Fertil Steril 2009.
18). Although the current freezing technique is relatively satisfactory, there are uncertainties about the optimal future use of stored ovarian tissue because the most follicles survived through the freezing and thawing process are primordial or primary follicles. Autotransplantation can be a practical method to restore ovarian function and to mature oocytes in stored ovarian tissue. However, our clinical experience and knowledge with this technology is very limited, especially with heterotopic transplantation. Kim. Heterotopic transplantation of human ovarian tissue. Fertil Steril 2009.
We were able to retrieve six oocytes (GV 1, MI 4, MII 1) from the ovarian grafts in two patients between August 2003 and November 2005. Three MI oocytes were developed to full maturity in vitro (Fig. 5). All four MII oocytes were fertilized and cultured in vitro for 2 to 3 days, which resulted in four embryos at the six-cell, three- cell, two-cell, and pronuclear stage (Fig. 5). All embryos were cryopreserved. In the near future, the stored embryos will be thawed and transferred to the surrogate. DISCUSSION Because germ cells are susceptible to cytotoxic treatment (especially alkylating agents and/or radiation), premature ovarian failure is a common sequel to aggressive cancer treatment. As sterility can impact on the quality of life for many young cancer survivors, there has been growing awareness of the importance of fertility conservation for young cancer patients. Cryopreservation of semen can be offered for adult males as a safeguard for fertility. Although cryopreservation of embryos or oocytes can be a strategy to preserve fertility in women, lengthy ovarian stimulation and follicle aspiration can delay cancer treatment and limit its application for women with cancer (12). An emerging strategy, involving autotransplantation of ovarian tissue banked at low temperature, offers the possibility of restoring ovarian function in women and children after highly gonadotoxic cancer treatment. The follicular survival in the stored ovarian tissue after freezing and thawing is approximately 70% to 80% (17, 2352
Kim et al.
There are many unresolved issues involved with heterotopic transplantation of ovarian tissue in humans. One of them is the optimal site for heterotopic transplantation. In theory, the optimal site should be rich in vascularity, as rapid revascularization of the graft is crucial for the survival of ovarian follicles. In addition, the optimal heterotopic site should be easily accessible without an invasive procedure, because the repeated ovarian transplantation and/or egg retrieval may be necessary if early graft exhaustion is expected (19). The subcutaneous tissue or the superficial muscle appears to be a practical heterotopic site for human ovarian transplantation. Indeed, the return of ovarian function was demonstrated when ovarian cortical strips were transplanted subcutaneously to the forearm (20). In our previous study (unpublished), different heterotopic sites such as breast tissue, subcutaneous tissue of the hip, and pre-rectus space were tested. These sites were selected after considering multiple factors including vascularity, cosmetic aspects, convenience, easiness for grafting, and accessibility for egg retrievals. For us, the abdominal site (between the rectus sheath and the rectus muscle) appeared to be the best heterotopic site for human ovarian transplantation. Another concern related to transplanting ovarian tissue to the heterotopic site is the environmental factors that can affect the follicular development. Obviously, the environment of the heterotopic site we selected is different from that of orthotopic site in the pelvic cavity. The environmental factors that can be altered with heterotopic transplantation include (but not limited to) temperature, pressure, space for follicular growth, peritoneal fluid, cytokines, angiogenic factors, and
Heterotopic transplantation of human ovarian tissue
Vol. 91, No. 6, June 2009
FIGURE 5 Two MI oocytes retrieved from the ovarian graft were matured to MII oocytes in vitro. These MII oocytes were fertilized with the husband’s sperm by ICSI and cultured for 3 days to the six-cell and three-cell stage embryos.
Kim. Heterotopic transplantation of human ovarian tissue. Fertil Steril 2009.
hormonal milieu. The follicles cannot grow and mature normally without an optimal nurturing environment. Hence, it needs to be explored if the environment of the heterotopic site can be suitable for growing follicles. Previously, we investigated the quality of human oocytes developed in the subcutaneous space using a severe combined immunodeficient mouse xenograft model (21). The result was alarming, as the microtubule and chromatin patterns of human oocytes developed in the subcutaneous space of the host animal were abnormal in most cases. Although not proven, it could be caused by the suboptimal environmental condition of the graft site. On the other hand, the results of the present study are encouraging, because the most oocytes retrieved from the heterotopic ovarian graft were fertilized normally (four of six). Of four fertilized oocytes, three embryos developed to the cleavage stage. The quality and developmental rate of embryos, however, appeared to be below average. There are many factors besides the graft site that can affect the quality and development of the oocytes and embryos. Thus, it is too premature to draw any conclusions about the relations between the quality of oocyte/embryo and heterotopic graft site. By 3 to 5 months posttransplantation, reestablishment of ovarian function was clinically evidenced: the patient no longer experienced hot flushes, and the follicular development was noticed by palpation as well as by ultrasound. Interestingly, all patients felt a growing lump accompanied by slight discomfort in the graft site, and one patient experienced an itching sensation, which coincided with the timing of follicular enlargement. The hormonal profiles also indicated sex steroid hormone production (E2, testosterone) from the ovarian graft, followed by the return of the FSH level below 20 mIU/mL. The latent periods of ovarian function after transplantation were shorter than expected. The data from orthotopic autotranslantation in patients with Hodgkin’s lymphoma indicated that the return of ovarian function took about 6 Fertility and Sterility
months (7, 9). It has been our knowledge that entire follicles except primordial follicles (and some primary follicles) in human ovarian tissue are lost in the process of freezing, thawing, and transplantation (19, 22). Because the growth of a primordial follicle to the large antral stage in humans takes >6 months (23), the return of endocrine function in 3 months after heterotopic transplantation is an unexpected finding. We can speculate that either some growing follicles in ovarian tissue survived through freezing, thawing, and grafting, or unknown factors associated with heterotopic autotransplantation accelerated the growth phase of primordial follicles. Of note, an acceleration of follicular growth has been observed with in vitro follicular culture (24). In the present study, we occasionally noticed the spontaneous LH surge followed by the elevation of P level above the ovulatory value (3 ng/mL) after heterotopic autotransplantation with frozen– thawed human ovarian tissue, which strongly suggested the occurrence of spontaneous ovulation in the ovarian graft. The duration of ovarian function correlates with the number of surviving follicles in the graft, which is influenced by many factors such as the age of the patient. The single most important factor determining the graft longevity is the degree of ischemia that causes a significant follicular loss after transplantation (12, 16). In fact, <30% of primordial follicles survived after grafting in studies using a sheep model (15, 22). Nevertheless, relatively long-term ovarian function (22 months) after autotransplantation of frozen–thawed ovarian tissue in ewe was demonstrated (15). In humans, there was a report that ovarian function persisted >2 years after heterotopic transplantation of fresh ovarian tissue to the forearm (25), whereas other studies showed that ovarian function lasted only for 2 to 5 months after autografting of frozen– thawed human ovarian tissue (5, 7, 26). In our present study, we noticed that the restored ovarian function after transplantation lasted for 3 to 5 months initially, but with the second transplantation long-term ovarian function (lasting for 1–3 years) has been established in all three patients. Of note, the return of ovarian function after 2353
the second transplantation was faster (taking only 2-4 months). This could be explained by the residual effect of the first transplantation. In addition, we can speculate possible synergistic effects of growth factors and hormones. In summary, cryopreserved human ovarian tissue can be used to restore long-term ovarian function by repeated autotransplantation to a heterotopic site. It appears that heterotopic transplantation to a space between the rectus sheath and the rectus muscle is not only convenient but effective. In addition, restoration of fertility can be achieved by heterotopic transplantation, as we have demonstrated that successful retrieval and fertilization of the eggs from the ovarian graft led to the embryo development up to the six-cell stage with in vitro culture. To our knowledge, this is the first longterm follow-up study (accumulating data for 8 years between 2000 and 2007) that investigated restoration of ovarian function as well as fertility after heterotopic autotransplantation of frozen–thawed human ovarian tissue. REFERENCES 1. Wallace WH, Anderson RA, Irvine DS. Fertility preservation for young patients with cancer: who is at risk and what can be offered? Lancet Oncol 2005;6:209–18. 2. Jemal A, Clegg LX, Ward E, Ries LA, Wu X, Jamison PM, et al. Annual report to the nation on the status of cancer, 1975–2001, with a special feature regarding survival. Cancer 2004;101:3–27. 3. Kim SS. Fertility preservation in female cancer patients: current developments and future directions. Fertil Steril 2006;85:1–11. 4. Gosden RG, Baird DT, Wade JC, Webb R. Restoration of fertility to oophorectomized sheep by ovarian autografts stored at 196 degrees C. Hum Reprod 1994;9:597–603. 5. Kim SS, Hwang IT, Lee HC. Heterotopic autotransplantation of cryobanked human ovarian tissue as a strategy to restore ovarian function. Fertil Steril 2004;82:930–2. 6. Oktay K, Karlikaya G. Ovarian function after transplantation of frozen, banked autologous ovarian tissue. N Engl J Med 2000;342:1919. 7. Radford JA, Lieberman BA, Brison DR, Smith AR, Critchlow JD, Russell SA, et al. Orthotopic reimplantation of cryopreserved ovarian cortical strips after high-dose chemotherapy for Hodgkin’s lymphoma. Lancet 2001;357:1172–5. 8. Demeestere I, Simon P, Emiliani S, Delbaere A, Englert Y. Fertility preservation: successful transplantation of cryopreserved ovarian tissue in a young patient previously treated for Hodgkin’s disease. Oncologist 2007;12:1437–42. 9. Donnez J, Dolmans MM, Demylle D, Jadoul P, Pirard C, Squifflet J, et al. Livebirth after orthotopic transplantation of cryopreserved ovarian tissue. Lancet 2004;364:1405–10.
2354
Kim et al.
10. Meirow D, Levron J, Eldar-Geva T, Hardan I, Fridman E, Zalel Y, et al. Pregnancy after transplantation of cryopreserved ovarian tissue in a patient with ovarian failure after chemotherapy. N Engl J Med 2005;353: 318–21. 11. Silber SJ, Gosden RG. Ovarian transplantation in a series of monozygotic twins discordant for ovarian failure. N Engl J Med 2007;356: 1382–4. 12. Kim SS, Battaglia DE, Soules MR. The future of human ovarian cryopreservation and transplantation: fertility and beyond. Fertil Steril 2001;75:1049–56. 13. Candy CJ, Wood MJ, Whittingham DG. Restoration of a normal reproductive lifespan after grafting of cryopreserved mouse ovaries. Hum Reprod 2000;15:1300–4. 14. Candy CJ, Wood MJ, Whittingham DG. Effect of cryoprotectants on the survival of follicles in frozen mouse ovaries. J Reprod Fertil 1997;110: 11–9. 15. Baird DT, Webb R, Campbell BK, Harkness LM, Gosden RG. Long-term ovarian function in sheep after ovariectomy and transplantation of autografts stored at 196 C. Endocrinology 1999;140:462–71. 16. Kim SS, Battaglia DE, Soules MR. The future of human ovarian cryopreservation and transplantation: fertility and beyond. Fertil Steril 2001;75:1049–56. 17. Gook DA, Edgar DH, Stern C. Effect of cooling rate and dehydration regimen on the histological appearance of human ovarian cortex following cryopreservation in 1, 2-propanediol. Hum Reprod 1999;14:2061–8. 18. Newton H, Aubard Y, Rutherford A, Sharma V, Gosden R. Low temperature storage and grafting of human ovarian tissue. Hum Reprod 1996;11:1487–91. 19. Kim SS, Yin H, Gosden RG. Cryobanking of ovarian and testicular tissue for children and young adults. In: Tulandi T, Gosden RG, eds. Preservation of fertility. London: Taylor & Francis, 2004:157–75. 20. Oktay K, Economos K, Kan M, Rucinski J, Veeck L, Rosenwaks Z. Endocrine function and oocyte retrieval after autologous transplantation of ovarian cortical strips to the forearm. JAMA 2001;286:1490–3. 21. Kim SS, Kang HG, Kim NH, Lee HC, Lee HH. Assessment of the integrity of human oocytes retrieved from cryopreserved ovarian tissue after xenotransplantation. Hum Reprod 2005;20:2502–8. 22. Aubard Y, Piver P, Cogni Y, Fermeaux V, Poulin N, Driancourt MA. Orthotopic and heterotopic autografts of frozen-thawed ovarian cortex in sheep. Hum Reprod 1999;14:2149–54. 23. Gougeon A. Dynamics of follicular growth in the human: a model from preliminary results. Hum Reprod 1986;1:81–7. 24. Thomas FH, Armstrong DG, Telfer EE. Activin promotes oocyte development in ovine preantral follicles in vitro. Reprod Biol Endocrinol 2003;1:76. 25. Oktay K, Buyuk E, Rosenwaks Z, Rucinski J. A technique for transplantation of ovarian cortical strips to the forearm. Fertil Steril 2003;80: 193–8. 26. Callejo J, Salvador C, Miralles A, Vilaseca S, Lailla JM, Balasch J. Longterm ovarian function evaluation after autografting by implantation with fresh and frozen-thawed human ovarian tissue. J Clin Endocrinol Metab 2001;86:4489–94.
Heterotopic transplantation of human ovarian tissue
Vol. 91, No. 6, June 2009