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Fertility options for female cancer patients: facts and fiction
FERTILITY AND STERILITY威 VOL. 75, NO. 4, APRIL 2001 Copyright ©2001 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.
MODERN TRENDS Edward E. Wallach, M.D. Associate Editor
Fertility options for female cancer patients: facts and fiction M. Natalia Posada, M.D., Lisa Kolp, M.D., and Jairo E. Garcı´a, M.D. Division of Reproductive Endocrinology, Department of Gynecology and Obstetrics, The Johns Hopkins University School of Medicine, Baltimore, Maryland
Objective: To review the latest progress in the prevention of ovarian failure induced by chemo/radiotherapy, as well as the latest advances in culture technology and transplantation of frozen-thawed ovarian tissue. Design: The English-language literature was searched with PubMed and related references. Conclusion(s): The development of combination chemotherapy and radiotherapy has improved the long-term survival of young cancer patients who are then frequently faced with iatrogenic ovarian failure and its consequences. The use of prior and concomitant GnRH analogs with chemotherapy offers encouraging results in animal studies with regard to prevention of ovarian failure. Adequately controlled research projects are needed to define the utility of GnRHa cotreatment in women cancer patients exposed to prolonged chemotherapy. Ovarian tissue cryopreservation is the optimal procedure for follicle banking. Theoretic options include returning the banked tissue back to the original pedicle so that pregnancy could be achieved naturally. Alternatively, the tissue can be grafted to a heterotopic site, either as an autograft (i.e., rectus abdominis muscle sheath) or as a xenograft (i.e., immunodeficient mice). Follicles could also be grown in vitro. Until reliable ovarian culture technology becomes available, autologous transplantation offers the best prospect of using frozen-thawed ovarian tissue. A primary concern, however, is the issue of microscopic metastatic disease to the ovary and the possibility of tumor reimplantation. Areas of research should focus on optimizing the freeze/thaw procedure for ovarian tissue, minimizing the ischemia-reperfusion injury after transplantation, and detecting minimal residual disease in ovarian tissue grafts. (Fertil Steril威 2001;75:647–53. ©2001 by American Society for Reproductive Medicine.) Key Words: Cancer therapy, ovarian tissue, GnRH analog, cryopreservation, transplantation, culture
Received August 15, 2000; revised and accepted October 26, 2000. Reprint requests: M. Natalia Posada, Division of Reproductive Endocrinology and Infertility, Department of Gynecology and Obstetrics, 600 North Wolfe Street/Phipps 249, Baltimore, Maryland 21287-1247 (FAX: 410-614-9684; E-mail: natalia.posada@worldnet. att.net). 0015-0282/01/$20.00 PII S0015-0282(00)01781-7
The development of combination chemotherapy and radiotherapy has improved the long-term survival rate of young cancer patients. Many adolescent and childhood lymphomas and leukemias, as well as several solid tumors, can now be cured (1, 2). Combination chemotherapy regimens were designed with special care to avoid overlapping acute toxic effects, but the long-term consequences on reproductive potential were not anticipated (3–5). Cytotoxic chemotherapy used to treat malignances and some nonmalignant diseases commonly produces menstrual irregularities, immediate or subsequent ovarian failure, and associated infertility. As survival rates for young cancer patients continue to improve, protection against iatrogenic infertility caused by chemotherapy and/or radiotherapy assumes a greater priority. Drugs most frequently associated with ovarian failure are divided into three classes. The first group comprises those definitely associ-
ated with gonadal toxicity: cyclophosphamide, L-phenylalanine mustard, busulfan, and nitrogen mustard. The second group of cell-cycle– specific drugs are unlikely to cause gonadal toxicity: methotrexate, 5-fluorouracil, and 6-mercaptopurine. The third group involves drugs whose gonadal toxicity is unknown: doxorubicin, bleomycin, vinca alkaloids (vincristine and vinblastin), cisplatin, nitrosoureas, and cytosine arabinoside (2, 6). Total-body irradiation used in the preparative regimens for bone marrow transplantation (BMT) is deleterious to endocrine and gonadal function. Therefore, myeloablative therapy consisting of cyclophosphamide (Cy), busulfan (Bu), or melphalan may be used as an alternative to avoid the side effects of irradiation. However, BMT patients treated with Bu/Cy preparative regimens still have a significant increase in gonadal failure compared with that of patients who receive conventional chemotherapy (7, 8). 647
Ovarian failure is related to the patient’s age and total cumulative chemotherapy dose (9 –11). In a recent prospective study, it was found that 48% of patients with cancer had ovarian failure after treatment with alkylating agents (9). Ovarian failure was inversely related to the patient’s age. After BMT, 58 of 63 (92%) patients with cancer (mean age, 29 years) had ovarian failure as measured by gonadotropin levels and menstrual records (9). Younger age at transplantation may predict return of ovarian function in ⬃30% of patients 6 – 48 months after BMT (10). Whereas 10% of girls are likely to develop premature menopause after conventional chemotherapy for acute lymphoblastic leukemia (11), 12 out of 21 girls aged 11 to 21 years had clinical evidence of ovarian failure after high-dose chemotherapy and autologous BMT (7). Histologic studies examining the effects of chemotherapy on human ovarian tissue have shown that the end result is ovarian atrophy with marked loss of primordial follicles (PMF; [12]). A recent study in the mouse model suggests that follicular destruction occurs regardless of the alkylating drug dose (13). Mice, however, continued to ovulate and reproduce after a 54% reduction in the population of PMFs. Thus, immediate reproductive performance may not be a sensitive marker of cyclophosphamide (Cy)-induced PMFs destruction. In fact, clinically evident impairment of gonadal function is relatively rare after conventional chemotherapy for acute leukemia in pediatric patients or other cancer patients under the age of 30 years (9 –11). The clinical observation that older women appear to be more affected by exposure to chemotherapy can be explained by the fact that older women naturally have a smaller ovarian reserve to start with. Therefore, an apparent resumption of ovarian function after radiotherapy or chemotherapy doesn’t rule out a significant reduction in the ovarian reserve. These patients should not delay childbearing, given the real risk of premature ovarian failure (POF).
GNRH ANALOG COTREATMENT It would be interesting to know the mechanisms involved in chemotherapy-induced ovarian failure and its prevention. Currently, prior and concomitant treatment with GnRH analogs (GnRHa) appears to be the most promising approach to prevent ovarian failure induced by cancer therapy (2). Our current knowledge on the ovarian actions of GnRHa stems mainly from experiments in rats (14). In controlled studies examining the uptake of tritiated thymidine (3[HT]) by rat ovaries (primarily by the granulosa cells), there is a significant reduction in the degree of mitotic activity 13 days after starting the GnRHa administration (15). Treatment of the adult rat with GnRHa inhibits the process of recruitment from the pool of small follicles into the pool of larger follicles that undergo further development and atresia. Thus, it appears that GnRHa may prevent the follicles from reach648
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ing the chemotherapy-sensitive stage via suppression of the granulosa cells. The protective effect of GnRHa against Cy-induced ovarian follicular depletion has been documented in the rat model. When given in combination with Cy, GnRHa significantly increases the number of small follicles remaining after Cy treatment compared with the case of the Cy-only group (16 –18). Montz et al. (19) likewise documented a protective effect on the fertility of rats using GnRHa or progestin/Cy cotreatment. It is still uncertain, however, whether the same effects would occur in humans and other primates because of the questionable presence of GnRH receptors (GnRH-R) in primate ovaries (20). In addition, the protective effect of GnRHa may prove insufficient against the usually prolonged, high-dose chemotherapy regimens given to cancer patients in contrast to the 1-month course used in most animal models. Recently, specific GnRH agonistic and antagonistic binding was demonstrated in human luteinized granulosa cells. However, no high-affinity receptor binding was present in preovulatory follicles (21). The temporary presence of high-affinity GnRH agonistic and antagonistic binding sites at the time of ovulation would largely explain the conflicting data on the presence of the GnRH-R in the human ovary. Because the concentration of hypothalamic GnRH in the systemic circulation is considered to be too low to interact with the ovarian receptors, it has been proposed that GnRH or GnRH-like peptides produced in the ovary activate GnRH-R in an autocrine/paracrine manner (22). Expression of the GnRH-R gene in human follicles provides evidence that the ovary could be a target of extrapituitary GnRH action. However, to date there is no consensus on the ovarian actions of GnRH or its analogues (23). The protective effect of monthly injections of leuprolide acetate depot vs. placebo initiated 42 days before Cy treatment was documented in a group of six rhesus monkeys (24). The female rhesus monkeys receiving GnRHa cotreatment retained significantly more PMFs than did those in the Cyonly group. Two clinical studies have evaluated the effect of GnRHa cotreatment in chemotherapy-induced POF in cancer patients. A randomized controlled study failed to demonstrate efficacy for intranasal buserelin compared with placebo when the agonist was started 7 days before chemotherapy (MVPP) in an attempt to prevent POF. Doubts about achieving adequate ovarian suppression in this group of patients were raised by the investigators, however (25). Blumenfeld et al. (26) found a significant protective effect of D-Trp6-GnRHa (Decapeptyl) cotreatment against ovarian damage in young women with lymphoma undergoing chemotherapy. Of the patients who achieved remission, 15 of 16 (93.7%) patients in the GnRHa/chemotherapy cotreatment group had normal FSH levels and resumed ovarian cyclicity 3– 8 months after therapy. In contrast, only 7 of 18 (39%) patients receiving chemotherapy alone resumed cyclic ovarVol. 75, No. 4, April 2001
ian activity (P⬍0.05), and as a group, they had an abnormally high median FSH level of 31.5 IU/L (36 ⫾ 25.2; P⫽0.03), suggesting decreased ovarian function. In addition to the fact that GnRH seems to interact with ovarian granulosa cells, a large body of experimental evidence indicates that GnRH agonists and antagonists directly inhibit proliferation of ovarian cancer through GnRH receptors expressed by 80% of these tumors (27, 28). The presence of GnRH and GnRH receptor mRNA was recently demonstrated in human ovarian surface epithelial cells (23). In vitro studies show significant suppression of cell growth by exposure to cis-platinum in the presence of GnRHa in endometrial, breast, and ovarian cell lines with positive GnRH receptor mRNA expression (29). Although the addition of GnRHa treatment to standard platinum-based chemotherapy didn’t seem beneficial to patients with surgically treated advanced ovarian carcinoma (30), the effect of GnRHa cotreatment on patients with stage I or II ovarian carcinoma has not been addressed. Young patients with initial-stage ovarian cancer treated with unilateral oophorectomy, followed by chemotherapy, would benefit from strategies aimed at the preservation of the PMF population of the remaining ovary.
OTHER FERTILITY PRESERVATION OPTIONS Currently, female cancer patients have few options for fertility preservation. Although frozen embryo storage has been a standard practice in IVF centers since 1983 (31), it is far from satisfactory for preservation of reproductive potential. The fate of stored embryos should be considered at the outset, especially in cancer patients, because of the higher risk that the mothers will die. In addition, a harvest of viable embryos cannot be guaranteed, and there may not be enough time for a complete IVF cycle before cancer treatment commences. In addition, the procedure is inappropriate for children and is unacceptable to many women who do not have a partner and reject donor sperm as an alternative.
Oocyte Cryopreservation Some of these problems would be avoided, at least in adults with fertile ovaries, if oocytes could be collected for cryopreservation. The initial report by Chen in 1986 of the first births (one singleton and one twin) after human mature oocyte cryopreservation was highly encouraging (32). Until 1994, the combination data indicated that 383 mature oocytes had been thawed, resulting in the birth of four babies, in other words, a live birth rate of 1% per thawed oocyte. There were various reasons for this lack of success: low oocyte survival rates (25%– 40%), low fertilization rates after classical insemination, a high incidence of polyploidy, and poor developmental ability of the embryos. Simultaneously, extensive research done mostly on mouse oocytes evaluating the effects of various steps of the freeze/thaw FERTILITY & STERILITY威
protocol reported numerous induced abnormalities in the oocyte. These included damage and hardening of the zona pellucida, premature cortical reaction, depolymerization of the meiotic spindle during cellular cooling with the possibility of raising the incidence of aneuploidy, and disruption of cytoplasmic organelles (33). These concerns about mature oocyte cryopreservation prompted some teams to turn to immature germinal vesicle oocyte freezing (when they had reached full size and became meiotically competent but before they resumed maturation and progressed to the metaphase II stage; see ref. 34). Oocytes at this stage can be obtained from graafian follicles by the transvaginal route, although a modified needle and lower pressures than are used conventionally in IVF are needed to improve recovery rates. No advantage for immature oocyte freezing accrued from the results of various studies (35). Two main developments led to the revival of mature oocyte freezing. First, reappraisal of the effects of freezing on the oocyte structure gave a more optimistic outlook. At least 60% of surviving oocytes had normal spindles and chromosome configurations, with no evidence of an increased frequency of freezing-associated aneuploidy by fluorescence or cytogenetics (36, 37). The second major change was the introduction of ICSI to fertilize in vitro– cryopreserved oocytes with the same success rates as those obtained with control oocytes. In 1997, Porcu et al. reported the first birth after oocyte cryopreservation and ICSI (38). Recently, they reported nine additional births from 1,769 mature oocytes cryopreserved from 96 women who underwent 112 IVF cycles. The oocyte survival rate was 54%, the fertilization rate with ICSI was 56%, and the cleavage rate was 91.2% (39). Yang et al. (40) reported cryopreserved oocyte survival and pregnancy rates similar to those of frozen embryos with a modified oocyte cryopreservation regimen. Although the concerns about aneuploidy in human oocytes may be exaggerated, it is too soon to establish oocyte banking on the basis of the evidence to date. The freezing protocols are not yet reliable enough to translate into reproducible, clinically useful techniques.
PMF Banking After years of neglect, follicle banking is again being considered as a serious alternative to frozen oocyte storage (41). It has several advantages and could be the best and only strategy available for having children. Theoretic options include returning the banked tissue to the original pedicle so that pregnancy can be achieved naturally. Alternatively, the tissue can be grafted to a heterotopic site, either as an autograft (i.e., under the abdominal skin) or as an allogenic or xenogenic graft (i.e., in the SCID mouse; see refs. 42, 43). Follicles could also be isolated and grown in vitro. In the last three instances, IVF would be required.
Ovarian Tissue Culture PMFs can be considered the storage form of the oocyte in the ovary. Because ⬎90% of follicles are in the primordial 649
stage in the human ovary, they are a valuable source of oocytes that could be used for clinical purposes. Primordial oocytes are arrested in prophase of meiosis I. Oocytes in PMFs are far smaller than when fully ripened at metaphase II (approximately 1% by volume), and they are less differentiated, possessing fewer organelles and lacking a zona pellucida and cortical granules. Very little is known about the biology of PMFs and the events surrounding the initiation of growth and apoptosis (44, 45). PMFs can be stored frozen either after enzymatic or mechanical isolation or in thin slices of ovarian tissue. Eppig and O’Brien (46) were the first to report the development of primordial oocytes from newborn mouse ovaries to mature oocytes in vitro. The transfer of two-cell–stage embryos resulted in the live birth of a mouse pup. A two-step strategy was developed: first, the whole ovaries of newborn mice were cultured for 8 days to allow the development of secondary follicles within the ovarian tissue. The second step was to enzymatically isolate oocyte– granulosa cell complexes and culture them for an additional 14 days. This basic two-step strategy will probably serve as the essential framework for application to other species, for several reasons. First, it is difficult to isolate and culture intact PMFs. Second, the mechanism involved in the recruitment of PMFs into the pool of preantral follicles is unknown and may require the presence of ovarian factors external to the follicle itself. Third, properly timed isolation and culture of oocyte– granulosa cell complexes probably enhances the development and nutrition of larger antral follicles. Alternatively, final oocyte maturation could be achieved by a combination of in vivo transplantation of PMFs, followed by in vitro culture of growing follicles (47). Progress has been made toward ascertaining the hormonal support required to produce antral follicles after successful isolation and in vitro growth of human preantral follicles (ⱖ120 m). The rate of atresia between the preantral and early antral stages in vivo is very high. The histologic evaluation of freshly dissected and cultured early antral follicles revealed that only 10%–20% contained an oocyte (48). PMFs from human ovaries have been isolated successfully by a combination of a gentle enzymatic technique and manual dissection in both fresh and frozen-stored tissue blocks (in 1.5 M ethylene glycol [EG] using a slow freeze/ rapid thaw protocol). Overall, 71.6% ⫾ 2.4% of fresh and 71.5% ⫾ 4.7% of frozen-thawed PMFs were viable by live-dead staining (49). Efforts have been made to define the best culture conditions for the in vitro growth of human ovarian follicles (50, 51). Cryopreservation does not seem to affect the proportion of viable follicles (52). However, partially isolating PMFs increases the proportion of atretic follicles over 1–3 weeks with respect to nonisolated follicles. Human PMFs cultured for 4 weeks within slices of ovarian tissue reach secondary 650
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and, occasionally, early antral stages of development (53). The limited success with human follicular in vitro development is probably due to a lack of knowledge of the factors involved in primordial follicular development, as well as to the relative length of the human follicular growth cycle (44, 45).
Ovarian Tissue Transplantation Until reliable ovarian culture technology becomes available, autologous transplantation offers the best prospect of using frozen-banked human ovarian specimens for restoring fertility to patients. Advances in cryotechnology have improved the results and confirmed that frozen ovarian tissue can return to cyclical function after simple implantation without vascular anastomosis. Success of these models depends mostly on genetic homogeneity of the donor and host strains by using isografts or xenografts into immunodeficient hosts, as the ovary is not immunologically privileged. In 1960, Parrott reported successful pregnancies in mice after implantation of frozen-thawed ovarian grafts (54). In these early experiments, grafts were stored at ⫺79°C, and glycerol was used as a cryoprotectant. In 1993, Carroll and Gosden (55) reproduced these results, transplanting isolated mouse PMFs cryopreserved using dimethyl sulfoxide (DMSO). Cryopreserved marmoset ovarian tissue has also been transplanted under the kidney capsule of immunodeficient mice (56). Despite these successes, doubts remained as to whether the human ovary could be cryopreserved because it is bulkier and more fibrous and because the PMFs are more widely dispersed than in either rodents or marmoset monkeys. The restoration of fertility to oophorectomized sheep has been reassuring. Ovarian tissue slices (the cortex of each ovary was divided into four or five slices 1 mm thick) were preserved in liquid nitrogen using DMSO as a cryoprotectant (57). These animals became pregnant after transplantation of both fresh and thawed ovarian tissue. Recently, the first case of laparoscopic autotransplantation of frozen-thawed human ovarian tissue was reported in a 29-year-old woman. Ovarian cortical strips measuring 2 ⫻ 5 and 5 ⫻ 10 mm were cryopreserved in DMSO after bilateral salpingo-oophorectomy. One of the ovarian pieces was thawed, anchored to a Surgicell frame, and sutured to a pocket dissected on the pelvic wall. The patient ovulated 3 months later after ovarian stimulation with human menopausal gonadotropins (58).
Ovarian Cortex Biopsy Techniques A round biopter was recently designed as a safe and practical laparoscopic technique for obtaining ovarian cortical biopsy specimens (59). Given the fact that each square millimeter of ovarian surface in a 30-year-old woman contains an average of 35 PMFs, the combined area of six biopter specimens 5 mm in diameter would yield a total number of approximately 3,500 PMFs. However, for patients Vol. 75, No. 4, April 2001
who will almost certainly be sterilized (e.g., in those who will undergo BMT), unilateral oophorectomy for conservation of the entire cortex may be justifiable.
Ovarian Tissue Cryopreservation Despite these good results, it is important to optimize cryopreservation regimens for human ovarian tissue. The currently accepted theory states that the avoidance of large and numerous intracellular ice crystals (IIF) is a necessary condition for a cell to survive freezing (60, 61). Thus cooling rates should be fast enough to minimize the long exposure of cells to deleterious freezing conditions but slow enough to avoid the damaging effect of IIF. The assumption that dehydration conditions and the rate of cooling that are used for embryos will be suitable for the cells of the ovarian cortex, the follicular cells, and the oocyte may be untenable given the variation in the water content in each of these cells. Another essential part of the cryopreservation technique is permeation of the tissue with a cryoprotectant (CPA) to minimize ice formation. The permeation of isolated cells with CPAs is relatively fast, but diffusion into multicellular tissue systems is much slower, which means that exposure to the CPA may have to be prolonged or altered in order to reach adequate concentrations in the center of the tissue without overexposing the more peripheral cells. Several studies have started to address some of these issues. No difference in tissue necrosis was found after freezing thin pieces of ovarian cortex with cooled 1.5 M DMSO or 1.5 M 1,2 propanediol (PROH)/0.1M sucrose in a slow freeze–rapid thaw protocol (62). Newton et al. (63) evaluated the viability of follicles within 1-mm3 slices of ovarian tissue, donated by eight healthy patients, preserved by slow freezing in one of the following cryoprotectants: DMSO, EG, glycerol (GY), and PROH. Equilibration was done at 4°C for 30 minutes. Follicular viability was assessed by counting follicles in histological sections 18 days after grafting under the kidney capsules of severe combined immunodeficiency (SCID) mice, and the results were expressed as percentages of the numbers in comparable pieces of ungrafted tissue. Although only 10% of the control number of follicles was found in the GY group, there were no significant differences between the results with EG, DMSO, PROH, and fresh-grafted tissue. A total of 74% of the follicles survived in the grafted fresh tissue. Newton et al. (64) investigated the diffusion of these four cryoprotective agents into the human tissue at both 4°C and 37°C using proton nuclear magnetic resonance imaging analysis. After 30 minutes at 4°C, PROH and GY displayed a significantly lower equilibration rate than EG and DMSO (P⫽0.02). At 37°C, all CPAs penetrated at a faster rate, although there was no significant impact on their final tissue concentration, except for in the case of PROH, which achieved a significant rate of diffusion at this temperature (99.7% permeability). The significance of this finding in terms of follicular viability was not addressed. There was no FERTILITY & STERILITY威
further protection against cell damage by adding low concentrations of sucrose to DMSO. Gook et al. (65) evaluated the effect of varying dehydration conditions and cooling rates on the histological appearance of thin slices of human ovarian cortex after cryopreservation with 1.5 M PROH/0.1 M sucrose in phosphatebuffered saline with 10 mg/dL human serum albumin. Single-step dehydration for 90 minutes and slow cooling– rapid thaw resulted in the highest proportion of intact human primordial and primary follicles.
AREAS OF FUTURE RESEARCH The importance of understanding the molecular mechanisms of carcinogenesis cannot be overstated. This would be the basis for the development of tissue-specific or receptormediated chemotherapy. In the meantime, prospective randomized controlled studies are needed to define the protective role of GnRHa in cytotoxic-mediated loss of PMFs. It is important to optimize the dehydration conditions and cooling rates for ovarian tissue cryopreservation. Nonvascular implants are vulnerable to ischemia-reperfusion injury from reactive oxygen species. It will be important to minimize the period of ischemia because more follicles are lost at this stage than by freezing and thawing (42). Administering an antioxidant, vitamin E, seems to improve the survival of follicles in ovarian grafts by reducing ischemia-reperfusion injury (66). Although the physiologic viability of frozen-thawed transplanted ovarian tissue has been demonstrated, the eventual longevity of such transplants is probably limited. It is therefore important to consider timely transplantation of frozen-thawed ovarian cortical tissue to a cancer survivor preparing for conception. An additional concern is the issue of microscopic metastatic disease to the ovary and the possibility of tumor reimplantation in cancer patients (67– 69). Currently available standard techniques such as histologic evaluation cannot exclude microscopic tumor involvement (70). This risk would be avoided by either follicular growth in vitro or xenotransplantation of ovarian tissue (71, 72). In recent years, a number of characteristic chromosomal abnormalities in leukemia and lymphoma have been defined at the molecular level. Unfortunately, not all malignancies have such clear genetic markers (73). Detection of minimal residual disease in hematological malignancies would be helpful in terms of evaluating the efficacy of treatment, early detection of relapses, and quantitation of remaining tumor cells in bone marrow, peripheral blood cells, or even ovarian grafts before transplantation.
SUMMARY Given the promising preliminary results in human and animal studies, patients willing to preserve their fertility 651
should be offered GnRHa/chemotherapy cotreatment when at risk of POF, unless contraindicated. All patients, and especially women ⬎30 years of age and/or undergoing highdose chemotherapy and BMT, should be counseled about fertility preservation options. Frozen embryo storage is a standard practice in IVF centers and is, therefore, the cryopreservation technique most readily available. In this case, patients should consider issues such as the fate of stored embryos and the limitations in terms of harvesting an adequate amount of viable embryos. Ovarian tissue cryopreservation is the most efficient technique of oocyte banking and the only alternative for prepubertal women. Until reliable follicle culture technology becomes available, autologous ovarian tissue transplantation offers the best prospect of using frozen-banked ovarian cortex. Xenografts into immunodeficient hosts may avoid the risk of tumor reimplantation in cancer patients. Although there has been preliminary success with these models, their safety and clinical applicability needs further assessment. Ovarian tissue cryopreservation has brought hope to patients facing POF. References 1. Boring CC, Squires TS, Tong T. Cancer statistics 1991. CA Cancer J Clin 1991;41:19 –36. 2. Blumemfeld Z, Avivi I, Ritter M, Rowe JM. Preservation of fertility and ovarian function and minimizing chemotherapy-induced gonadotoxicity in young women. J Soc Gynecol Investig 1999;6:229 –39. 3. Chapman RM, Sutcliffe SB, Malpas JS. Cytotoxic-induced ovarian failure in women with Hodgkin’s disease. 1. Hormone function. JAMA 1979;242:1877– 81. 4. Clark ST, Radford JA, Crowther D, Swindell R, Shalet SM. Gonadal function following chemotherapy for Hodgkin’s disease: a comparative study of MVPP and a seven-drug hybrid regimen. J Clin Oncol 1995; 13:134 –9. 5. Horning SJ, Hoppe RT, Kaplan HS, Rosenberg SA. Female reproductive potential after treatment for Hodgkin’s disease. N Engl J Med 1981;304:1377– 82. 6. Reichman BS, Green KB. Breast cancer in young women: effect of chemotherapy on ovarian function, fertility, and birth defects. Natl Cancer Inst Mongr 1994;16:125–9. 7. Teinturer C, Harmann O, Valteau-Couanet D, Benhamou E, Bougneres PF. Ovarian function after autologous bone marrow transplantation in childhood: high-dose busulfan is a major cause of ovarian failure. Bone Marrow Transplant 1998;22:989 –94. 8. Leahy AM, Teunissen H, Friedman DL, Moshang T, Lange BJ, Meadows AT. Late effects of chemotherapy compared to bone marrow transplantation in the treatment of pediatric acute myeloid leukemia and myelodysplasia. Med Pediatr Oncol 1999;32:163–9. 9. Meirow D, Lewin A, Or R, Rachmilewitz E, Slavin S, Schenker JG, et al. Ovarian failure post-chemotherapy in young cancer patients—risk assessment indicate the need for intervention [abstract P-261]. In: 1997 Annual Meeting Program Supplement, Cincinnati, OH: American Society for Reproductive Medicine. Fertil Steril 1997:S218. 10. Schimmer AD, Quatermain M, Imrie K, Ali V, McCrae J, Stewart K, et al. Ovarian function after autologous bone marrow transplantation. J Clin Oncol 1998;16:2359 – 63. 11. Wallace W, Shalet SM, Tetlow LH, Morris-Jones PH. Ovarian function following the treatment of childhood acute lymphoblastic leukaemia. Med Pediatr Oncol 1993;21:333–9. 12. Familiari G, Caggiati A, Nottola SA, Ermini M, Di Benedetto MR, Motta PM. Ultrastructure of human ovarian primordial folllicles after combination chemotherapy for Hodgkin’s disease. Hum Reprod 1993; 8:2080 –7. 13. Meirow D, Lewis H, Nugent D, Epstein M. Subclinical depletion of primordial follicular reserve in mice treated with cyclophosphamide: clinical importance and proposed accurate investigative tool. Hum Reprod 1999;14:1903–7. 14. Hsueh AJ, Jones PB. Extrapituitary actions of gonadotropin-releasing hormone. Endocr Rev 1981;2:437– 61. 15. Ataya K, Moghissi K. Chemotherapy-induced premature ovarian failure: mechanisms and prevention. Steroids 1989;54:607–26.
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16. Ataya K, McKanna JA, Wintraub AM, Clark MR, LeMaire W. A luteinizing hormone-releasing hormone agonist for the prevention of chemotherapy-induced ovarian follicular loss in rats. Cancer Res 1985; 45:3651– 6. 17. Bokser L, Szende B, Schally AV. Protective effects of D-Trp6 luteinizing hormone-releasing hormone microcapsules against cyclophosphamide-induced gonadotoxicity in female rats. Br J Cancer 1990;61: 861–5. 18. Ataya KM, Ramahi-Ataya A. Reproductive performance of female rats treated with cyclophosphamide and/or LHRH agonist. Reprod Toxicol 1993;7:229 –35. 19. Montz FJ, Wolff AJ, Gambone JC. Gonadal protection and fecundity rates in cyclophosphamide-treated rats. Cancer Res 1991;51:2124 – 6. 20. Bramley TA, Menzies GS. Measurement of luteal and placental gonadotrophin-releasing hormone (GnRH) binding sites: role of inactivation of GnRH tracer. Mol Hum Reprod 1996;2:535–9. 21. Brus L, Lambalk CB, De Koning J, Helder MN, Janssens RMJ, Schoemaker J. Specific gonadotrophin-releasing hormone analogue binding predominantly in human luteinized follicular aspirates and not in human pre-ovulatory follicles. Hum Reprod 1997;12:769 –73. 22. Kang SK, Choi K, Cheng KW, Nathwani PS, Auersperg N, Leung PCK. Role of gonadotropin-releasing hormone as an autocrine growth factor in human ovarian surface epithelium. Endocrinology 2000;141: 72– 80. 23. Ortmann O, Diedrich K. Pituitary and extrapituitary actions of gonadotrophin-releasing hormone and its analogues. Hum Reprod 1999; 14(suppl 1):194 –206. 24. Ataya K, Rao LV, Lawrence E, Kimmel R. Luteinizing hormonereleasing hormone agonist inhibits cyclophosphamide-induced ovarian follicular depletion in rhesus monkeys. Biol Reprod 1995;52:365–72. 25. Waxman JH, Ahmed R, Smith D, Wrigley PFM, Gregory W, Shalet S, et al. Failure to preserve fertility in patients with Hodgkin’s disease. Cancer Chemother Pharmacol 1987;19:159 – 62. 26. Blumenfeld Z, Avivi I, Linn S, Epelbaum R, Ben-Shahar M, Haim N. Prevention of irreversible chemotherapy-induced ovarian damage in young women with lymphoma by a gonadotrophin-releasing hormone agonist in parallel to chemotherapy. Hum Reprod 1996;11:1620 – 6. 27. Emmons G, Schulz KD. Primary and salvage therapy with LH-RH analogues in ovarian cancer. Recent Results Cancer Res 2000;153:83– 94. 28. Srkalovic G, Schally AU, Wittliff JL, Day TG Jr, Jenison EL. Presence and characteristics of receptors for [D-Trp6] luteinizing hormone releasing hormone and epidermal growth factor in human ovarian cancer. Int J Oncol 1998;12:489 –98. 29. Ohta H, Sakamoto H, Satoh K. In vitro effects of gonadotropinreleasing hormone (GnRH) analogue on cancer cell sensitivity to cisplatinum. Cancer Lett 1998;134:111– 8. 30. Emmons G, Ortmann O, Feichert HM, Fassl H, Lors U, Kullander S, et al. Luteinizing hormone-releasing hormone agonist triptorelin in combination with cytotoxic chemotherapy in patients with advanced ovarian carcinoma. A prospective double blind randomized trial. Decapeptyl ovarian cancer study group. Cancer 1996;78:1452– 60. 31. Trounson A, Mohr L. Human pregnancy following cryopreservation, thawing and transfer of an eight cell embryo. Nature 1983;305:707–9. 32. Chen C. Pregnancy after human ovocyte cryopreservation. Lancet 1986;1:884 – 6. 33. Bernard A, Fuller BJ. Cryopreservation of human oocytes: a review of current problems and perspectives. Hum Reprod Update 1996;2:193– 07. 34. Baka SG, Toth TL, Veeck LL, Jones HW, Muasher SJ, Lazendorf SE. Evaluation of the spindle apparatus of in vitro matured human oocytes following cryopreservation. Hum Reprod 1995;7:1816 –20. 35. Mandelbaum J, Belaisch-Allart J, Juncda AM, Antonine JM, Plachot M, Alvarez S, et al. Cryopreservation in human assisted reproduction is now routine for embryos but remains a research procedure for oocytes. Hum Reprod 1998;13(suppl 3):161–74. 36. Gook DA, Osborn SM, Johnston WIH. Cryopreservation of mouse and human oocytes using 1,2-propanediol and the configuration of the meiotic spindle. Hum Reprod 1993;8:1101–9. 37. Gook DA, Osborn SM, Bourne H, Johnston WIH. Fertilization of human oocytes following cryopreservation; normal karyotypes and absence of stray chromosomes. Hum Reprod 1994;9:684 –91. 38. Porcu E, Fabbri R, Seracchioli R, Ciotti PM, Magrini O, Flamigni C. Birth of a healthy female after intracytoplasmic sperm injection of cryopreserved human oocytes. Fertil Steril 1997;68:724 – 6. 39. Porcu E, Fabbri R, Ciotti PM, Marsella T, Balicchia B, Damiano G, et al. Cycles of human oocyte cryopreservation and intracytoplasmic sperm injection: results of 112 cycles [abstract O-004]. In: 1999 Annual Meeting Program Supplement, Toronto, Canada: American Society for Reproductive Medicine and Canadian Fertility and Andrology Society. Fertil Steril 1999:S2. 40. Yang DS, Blohm PL, Cramer K, Nguyen K, Zhao YL, Winslow KL. A
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successful human oocyte cryopreservation regime: survival, implantation and pregnancy rates are comparable to that of cryopreserved embryos generated from sibling oocytes [abstract O-224]. In: 1999 Annual Meeting Program Supplement, Toronto, Canada: American Society for Reproductive Medicine and Canadian Fertility and Andrology Society. Fertil Steril 1999:S86. Oktay K, Newton H, Aubard Y, Salha O, Gosden RG. Cryopreservation of immature human oocytes and ovarian tissue: an emerging technology? Fertil Steril 1998;69:1–7. Aubard Y, Piver P, Cognie Y, Fermeaux V, Poulin N, Driancourt MA. Orthotopic and heterotopic autografts of frozen-thawed ovarian cortex in sheep. Hum Reprod 1999;14:2149 –54. Gunasena KT, Lakey JRT, Villiness PM, Critser ES, Critser JK. Allogeneic and xenogeneic transplantation of cryopreserved ovarian tissue to athymic mice. Biol Reprod 1997;57:226 –31. Gougeon A. Dynamics of follicular growth in the human: a model from preliminary results. Hum Reprod 1986;1:81–7. Gougeon A. Regulation of ovarian follicular development in primates: facts and hypothesis. Endocr Rev 1996;17:121–55. Eppig JJ, O’Brien MJ. Development in-vitro of mouse oocytes from primordial follicles. Biol Reprod 1996;54:197–207. Liu J, Van der Elst J, Van den Broecke R, Dumortier F, Dhont M. Maturation of mouse primordial follicles by combination of grafting and in vitro culture. Biol Reprod 2000;62:1218 –23. Abir R, Franks S, Mobberley MA, Moore PA, Margara RA, Winston RML. Mechanical isolation and in vitro growth of preantral and small antral human follicles. Fertil Steril 1997;68:682– 8. Oktay R, Nugent D, Newton H, Salha O, Chatterjee P, Gosden R. Isolation and characterization of primordial follicles from fresh and cryopreserved human ovarian tissue. Fertil Steril 1997;67:481– 6. Wandji SA, Srsen V, Nathanielsz PW, Eppig JJ, Fortune JE. Initiation of growth of baboon primordial follicles in vitro. Hum Reprod 1997; 12:1993– 01. Wright CS, Hovatta O, Margara R, Trew G, Winston RML, Franks S, et al. Effects of follicle-stimulating hormone and serum substitution on the in-vitro growth of human ovarian follicles. Hum Reprod 1999;14: 1555– 62. Hovatta O, Silye R, Abir R, Krausz T, Winston RML. Extracellular matrix improves survival of both stored and fresh human primordial and primary ovarian follicles in long-term culture. Hum Reprod 1997; 12:1032– 6. Hovatta O, Wright C, Krausz T, Hardy K, Winston RML. Human primordial, primary and secondary ovarian follicles in long term culture: effect of partial isolation. Hum Reprod 1999;14:2519 –24. Parrot DM. The fertility of mice with orthotopic ovarian grafts derived from frozen tissue. J Reprod Fertil 1960;1:230 – 41. Carroll J, Gosden RG. Transplantation of frozen-thawed mouse primordial follicles. Hum Reprod 1993;8:1163–7. Candy CJ, Wood MJ, Whittingham DG. Follicular development in cryopreserved marmoset ovarian tissue after transplantation. Hum Reprod 1995;10:2334 – 8. Gosden RG, Baird DT, Wade JC, Webb R. Restoration of fertility to oophorectomized sheep by ovarian autografts stored at ⫺196°C. Hum Reprod 1994;9:597– 603.
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58. Oktay K, Karlikaya G, Gosden R, Schwarz R. Ovarian function after autologous transplantation of frozen-banked human ovarian tissue [abstract O-054]. In: 1999 Annual Meeting Program Supplement, Toronto, Canada: American Society for Reproductive Medicine and Canadian Fertility and Andrology Society. Fertil Steril 1999:S21. 59. Meirow D, Fasouliotis SJ, Nugent D, Schenker JG, Gosden RG, Rutherford AJ. A laparoscopic technique for obtaining ovarian cortical biopsy specimens for fertility conservation in patients with cancer. Fertil Steril 1999;71:948 –51. 60. Mazur P. Freezing of living cells: mechanism and implications. Am J Physiol 1984;143:C125– 42. 61. Trad FS, Toner M, Biggers JD. Effects of cryoprotectants and iceseeding temperature on intracellular freezing and survival of human oocytes. Hum Reprod 1999;14:1569 –77. 62. Hovatta O, Silye R, Kransz T, Abir R, Margara R, Trew G, et al. Cryopreservation of human ovarian tissue using dimethylsulphoxide and propanediol-sucrose as cryoprotectants. Hum Reprod 1996;11: 1268 –72. 63. 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. 64. Newton H, Fisher J, Arnold JRP, Pegg DE, Faddy MJ, Gosden RG. Permeation of human ovarian tissue with cryoprotective agents in preparation for cryopreservation. Hum Reprod 1998:13:376 – 80. 65. 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. 66. Nugent D, Newton H, Gallivan L, Gosden RG. Protective effect of vitamin E on ischaemia-reperfusion injury in ovarian grafts. J Reprod Fertil 1998;114:341– 6. 67. Shaw JM, Bowles J, Koopman P, Wood EC, Trounson AO. Fresh and cryopreserved ovarian tissue samples from donors with lymphoma transmit the cancer to graft recipients. Hum Reprod 1996;11:1668 –73. 68. Gosden RG, Rutherford AJ, Norfolk DR. Transmission of malignant cells in ovarian grafts. Hum Reprod 1997;12:403. 69. Shaw J, Trounson A. Oncological implications in the replacement of ovarian tissue. Hum Reprod 1997;12:403–5. 70. Kim SS, Gosden RG, Radford JA, Harris M, Jox A, Rutherford AJ. A model to test the safety of human ovarian tissue transplantation after cryopreservation: xenografts of ovarian tissues from cancer patients into NOD/LtSz Scid mice [abstract O-003]. In: 1999 Annual Meeting Program Supplement, Toronto, Canada: American Society for Reproductive Medicine and Canadian Fertility and Andrology Society. Fertil Steril 1999:S1. 71. Moomjy M, Rosenwaks Z. Ovarian tissue cryopreservation: the time is now. Transplantation or in-vitro maturation: the time awaits. Fertil Steril 1998;69:999 –1000. 72. Meirow D, Yehuda DB, Prus D, Pollack A, Schenker JG, Rachmilewitz EA, et al. Ovarian tissue banking in patients with Hodgkin’s disease: is it safe? Fertil Steril 1998;69:996 – 8. 73. Kurokawa T, Kinoshita T, Ito T, Saito H, Hotta T. Detection of minimal residual disease in B cell lymphoma by PCR-mediated RNAse protection assay. Leukemia 1996;10:1222–31.
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MODERN TRENDS Edward E. Wallach, M.D. Associate Editor
Fertility options for female cancer patients: facts and fiction M. Natalia Posada, M.D., Lisa Kolp, M.D., and Jairo E. Garcı´a, M.D. Division of Reproductive Endocrinology, Department of Gynecology and Obstetrics, The Johns Hopkins University School of Medicine, Baltimore, Maryland
Objective: To review the latest progress in the prevention of ovarian failure induced by chemo/radiotherapy, as well as the latest advances in culture technology and transplantation of frozen-thawed ovarian tissue. Design: The English-language literature was searched with PubMed and related references. Conclusion(s): The development of combination chemotherapy and radiotherapy has improved the long-term survival of young cancer patients who are then frequently faced with iatrogenic ovarian failure and its consequences. The use of prior and concomitant GnRH analogs with chemotherapy offers encouraging results in animal studies with regard to prevention of ovarian failure. Adequately controlled research projects are needed to define the utility of GnRHa cotreatment in women cancer patients exposed to prolonged chemotherapy. Ovarian tissue cryopreservation is the optimal procedure for follicle banking. Theoretic options include returning the banked tissue back to the original pedicle so that pregnancy could be achieved naturally. Alternatively, the tissue can be grafted to a heterotopic site, either as an autograft (i.e., rectus abdominis muscle sheath) or as a xenograft (i.e., immunodeficient mice). Follicles could also be grown in vitro. Until reliable ovarian culture technology becomes available, autologous transplantation offers the best prospect of using frozen-thawed ovarian tissue. A primary concern, however, is the issue of microscopic metastatic disease to the ovary and the possibility of tumor reimplantation. Areas of research should focus on optimizing the freeze/thaw procedure for ovarian tissue, minimizing the ischemia-reperfusion injury after transplantation, and detecting minimal residual disease in ovarian tissue grafts. (Fertil Steril威 2001;75:647–53. ©2001 by American Society for Reproductive Medicine.) Key Words: Cancer therapy, ovarian tissue, GnRH analog, cryopreservation, transplantation, culture
Received August 15, 2000; revised and accepted October 26, 2000. Reprint requests: M. Natalia Posada, Division of Reproductive Endocrinology and Infertility, Department of Gynecology and Obstetrics, 600 North Wolfe Street/Phipps 249, Baltimore, Maryland 21287-1247 (FAX: 410-614-9684; E-mail: natalia.posada@worldnet. att.net). 0015-0282/01/$20.00 PII S0015-0282(00)01781-7
The development of combination chemotherapy and radiotherapy has improved the long-term survival rate of young cancer patients. Many adolescent and childhood lymphomas and leukemias, as well as several solid tumors, can now be cured (1, 2). Combination chemotherapy regimens were designed with special care to avoid overlapping acute toxic effects, but the long-term consequences on reproductive potential were not anticipated (3–5). Cytotoxic chemotherapy used to treat malignances and some nonmalignant diseases commonly produces menstrual irregularities, immediate or subsequent ovarian failure, and associated infertility. As survival rates for young cancer patients continue to improve, protection against iatrogenic infertility caused by chemotherapy and/or radiotherapy assumes a greater priority. Drugs most frequently associated with ovarian failure are divided into three classes. The first group comprises those definitely associ-
ated with gonadal toxicity: cyclophosphamide, L-phenylalanine mustard, busulfan, and nitrogen mustard. The second group of cell-cycle– specific drugs are unlikely to cause gonadal toxicity: methotrexate, 5-fluorouracil, and 6-mercaptopurine. The third group involves drugs whose gonadal toxicity is unknown: doxorubicin, bleomycin, vinca alkaloids (vincristine and vinblastin), cisplatin, nitrosoureas, and cytosine arabinoside (2, 6). Total-body irradiation used in the preparative regimens for bone marrow transplantation (BMT) is deleterious to endocrine and gonadal function. Therefore, myeloablative therapy consisting of cyclophosphamide (Cy), busulfan (Bu), or melphalan may be used as an alternative to avoid the side effects of irradiation. However, BMT patients treated with Bu/Cy preparative regimens still have a significant increase in gonadal failure compared with that of patients who receive conventional chemotherapy (7, 8). 647
Ovarian failure is related to the patient’s age and total cumulative chemotherapy dose (9 –11). In a recent prospective study, it was found that 48% of patients with cancer had ovarian failure after treatment with alkylating agents (9). Ovarian failure was inversely related to the patient’s age. After BMT, 58 of 63 (92%) patients with cancer (mean age, 29 years) had ovarian failure as measured by gonadotropin levels and menstrual records (9). Younger age at transplantation may predict return of ovarian function in ⬃30% of patients 6 – 48 months after BMT (10). Whereas 10% of girls are likely to develop premature menopause after conventional chemotherapy for acute lymphoblastic leukemia (11), 12 out of 21 girls aged 11 to 21 years had clinical evidence of ovarian failure after high-dose chemotherapy and autologous BMT (7). Histologic studies examining the effects of chemotherapy on human ovarian tissue have shown that the end result is ovarian atrophy with marked loss of primordial follicles (PMF; [12]). A recent study in the mouse model suggests that follicular destruction occurs regardless of the alkylating drug dose (13). Mice, however, continued to ovulate and reproduce after a 54% reduction in the population of PMFs. Thus, immediate reproductive performance may not be a sensitive marker of cyclophosphamide (Cy)-induced PMFs destruction. In fact, clinically evident impairment of gonadal function is relatively rare after conventional chemotherapy for acute leukemia in pediatric patients or other cancer patients under the age of 30 years (9 –11). The clinical observation that older women appear to be more affected by exposure to chemotherapy can be explained by the fact that older women naturally have a smaller ovarian reserve to start with. Therefore, an apparent resumption of ovarian function after radiotherapy or chemotherapy doesn’t rule out a significant reduction in the ovarian reserve. These patients should not delay childbearing, given the real risk of premature ovarian failure (POF).
GNRH ANALOG COTREATMENT It would be interesting to know the mechanisms involved in chemotherapy-induced ovarian failure and its prevention. Currently, prior and concomitant treatment with GnRH analogs (GnRHa) appears to be the most promising approach to prevent ovarian failure induced by cancer therapy (2). Our current knowledge on the ovarian actions of GnRHa stems mainly from experiments in rats (14). In controlled studies examining the uptake of tritiated thymidine (3[HT]) by rat ovaries (primarily by the granulosa cells), there is a significant reduction in the degree of mitotic activity 13 days after starting the GnRHa administration (15). Treatment of the adult rat with GnRHa inhibits the process of recruitment from the pool of small follicles into the pool of larger follicles that undergo further development and atresia. Thus, it appears that GnRHa may prevent the follicles from reach648
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ing the chemotherapy-sensitive stage via suppression of the granulosa cells. The protective effect of GnRHa against Cy-induced ovarian follicular depletion has been documented in the rat model. When given in combination with Cy, GnRHa significantly increases the number of small follicles remaining after Cy treatment compared with the case of the Cy-only group (16 –18). Montz et al. (19) likewise documented a protective effect on the fertility of rats using GnRHa or progestin/Cy cotreatment. It is still uncertain, however, whether the same effects would occur in humans and other primates because of the questionable presence of GnRH receptors (GnRH-R) in primate ovaries (20). In addition, the protective effect of GnRHa may prove insufficient against the usually prolonged, high-dose chemotherapy regimens given to cancer patients in contrast to the 1-month course used in most animal models. Recently, specific GnRH agonistic and antagonistic binding was demonstrated in human luteinized granulosa cells. However, no high-affinity receptor binding was present in preovulatory follicles (21). The temporary presence of high-affinity GnRH agonistic and antagonistic binding sites at the time of ovulation would largely explain the conflicting data on the presence of the GnRH-R in the human ovary. Because the concentration of hypothalamic GnRH in the systemic circulation is considered to be too low to interact with the ovarian receptors, it has been proposed that GnRH or GnRH-like peptides produced in the ovary activate GnRH-R in an autocrine/paracrine manner (22). Expression of the GnRH-R gene in human follicles provides evidence that the ovary could be a target of extrapituitary GnRH action. However, to date there is no consensus on the ovarian actions of GnRH or its analogues (23). The protective effect of monthly injections of leuprolide acetate depot vs. placebo initiated 42 days before Cy treatment was documented in a group of six rhesus monkeys (24). The female rhesus monkeys receiving GnRHa cotreatment retained significantly more PMFs than did those in the Cyonly group. Two clinical studies have evaluated the effect of GnRHa cotreatment in chemotherapy-induced POF in cancer patients. A randomized controlled study failed to demonstrate efficacy for intranasal buserelin compared with placebo when the agonist was started 7 days before chemotherapy (MVPP) in an attempt to prevent POF. Doubts about achieving adequate ovarian suppression in this group of patients were raised by the investigators, however (25). Blumenfeld et al. (26) found a significant protective effect of D-Trp6-GnRHa (Decapeptyl) cotreatment against ovarian damage in young women with lymphoma undergoing chemotherapy. Of the patients who achieved remission, 15 of 16 (93.7%) patients in the GnRHa/chemotherapy cotreatment group had normal FSH levels and resumed ovarian cyclicity 3– 8 months after therapy. In contrast, only 7 of 18 (39%) patients receiving chemotherapy alone resumed cyclic ovarVol. 75, No. 4, April 2001
ian activity (P⬍0.05), and as a group, they had an abnormally high median FSH level of 31.5 IU/L (36 ⫾ 25.2; P⫽0.03), suggesting decreased ovarian function. In addition to the fact that GnRH seems to interact with ovarian granulosa cells, a large body of experimental evidence indicates that GnRH agonists and antagonists directly inhibit proliferation of ovarian cancer through GnRH receptors expressed by 80% of these tumors (27, 28). The presence of GnRH and GnRH receptor mRNA was recently demonstrated in human ovarian surface epithelial cells (23). In vitro studies show significant suppression of cell growth by exposure to cis-platinum in the presence of GnRHa in endometrial, breast, and ovarian cell lines with positive GnRH receptor mRNA expression (29). Although the addition of GnRHa treatment to standard platinum-based chemotherapy didn’t seem beneficial to patients with surgically treated advanced ovarian carcinoma (30), the effect of GnRHa cotreatment on patients with stage I or II ovarian carcinoma has not been addressed. Young patients with initial-stage ovarian cancer treated with unilateral oophorectomy, followed by chemotherapy, would benefit from strategies aimed at the preservation of the PMF population of the remaining ovary.
OTHER FERTILITY PRESERVATION OPTIONS Currently, female cancer patients have few options for fertility preservation. Although frozen embryo storage has been a standard practice in IVF centers since 1983 (31), it is far from satisfactory for preservation of reproductive potential. The fate of stored embryos should be considered at the outset, especially in cancer patients, because of the higher risk that the mothers will die. In addition, a harvest of viable embryos cannot be guaranteed, and there may not be enough time for a complete IVF cycle before cancer treatment commences. In addition, the procedure is inappropriate for children and is unacceptable to many women who do not have a partner and reject donor sperm as an alternative.
Oocyte Cryopreservation Some of these problems would be avoided, at least in adults with fertile ovaries, if oocytes could be collected for cryopreservation. The initial report by Chen in 1986 of the first births (one singleton and one twin) after human mature oocyte cryopreservation was highly encouraging (32). Until 1994, the combination data indicated that 383 mature oocytes had been thawed, resulting in the birth of four babies, in other words, a live birth rate of 1% per thawed oocyte. There were various reasons for this lack of success: low oocyte survival rates (25%– 40%), low fertilization rates after classical insemination, a high incidence of polyploidy, and poor developmental ability of the embryos. Simultaneously, extensive research done mostly on mouse oocytes evaluating the effects of various steps of the freeze/thaw FERTILITY & STERILITY威
protocol reported numerous induced abnormalities in the oocyte. These included damage and hardening of the zona pellucida, premature cortical reaction, depolymerization of the meiotic spindle during cellular cooling with the possibility of raising the incidence of aneuploidy, and disruption of cytoplasmic organelles (33). These concerns about mature oocyte cryopreservation prompted some teams to turn to immature germinal vesicle oocyte freezing (when they had reached full size and became meiotically competent but before they resumed maturation and progressed to the metaphase II stage; see ref. 34). Oocytes at this stage can be obtained from graafian follicles by the transvaginal route, although a modified needle and lower pressures than are used conventionally in IVF are needed to improve recovery rates. No advantage for immature oocyte freezing accrued from the results of various studies (35). Two main developments led to the revival of mature oocyte freezing. First, reappraisal of the effects of freezing on the oocyte structure gave a more optimistic outlook. At least 60% of surviving oocytes had normal spindles and chromosome configurations, with no evidence of an increased frequency of freezing-associated aneuploidy by fluorescence or cytogenetics (36, 37). The second major change was the introduction of ICSI to fertilize in vitro– cryopreserved oocytes with the same success rates as those obtained with control oocytes. In 1997, Porcu et al. reported the first birth after oocyte cryopreservation and ICSI (38). Recently, they reported nine additional births from 1,769 mature oocytes cryopreserved from 96 women who underwent 112 IVF cycles. The oocyte survival rate was 54%, the fertilization rate with ICSI was 56%, and the cleavage rate was 91.2% (39). Yang et al. (40) reported cryopreserved oocyte survival and pregnancy rates similar to those of frozen embryos with a modified oocyte cryopreservation regimen. Although the concerns about aneuploidy in human oocytes may be exaggerated, it is too soon to establish oocyte banking on the basis of the evidence to date. The freezing protocols are not yet reliable enough to translate into reproducible, clinically useful techniques.
PMF Banking After years of neglect, follicle banking is again being considered as a serious alternative to frozen oocyte storage (41). It has several advantages and could be the best and only strategy available for having children. Theoretic options include returning the banked tissue to the original pedicle so that pregnancy can be achieved naturally. Alternatively, the tissue can be grafted to a heterotopic site, either as an autograft (i.e., under the abdominal skin) or as an allogenic or xenogenic graft (i.e., in the SCID mouse; see refs. 42, 43). Follicles could also be isolated and grown in vitro. In the last three instances, IVF would be required.
Ovarian Tissue Culture PMFs can be considered the storage form of the oocyte in the ovary. Because ⬎90% of follicles are in the primordial 649
stage in the human ovary, they are a valuable source of oocytes that could be used for clinical purposes. Primordial oocytes are arrested in prophase of meiosis I. Oocytes in PMFs are far smaller than when fully ripened at metaphase II (approximately 1% by volume), and they are less differentiated, possessing fewer organelles and lacking a zona pellucida and cortical granules. Very little is known about the biology of PMFs and the events surrounding the initiation of growth and apoptosis (44, 45). PMFs can be stored frozen either after enzymatic or mechanical isolation or in thin slices of ovarian tissue. Eppig and O’Brien (46) were the first to report the development of primordial oocytes from newborn mouse ovaries to mature oocytes in vitro. The transfer of two-cell–stage embryos resulted in the live birth of a mouse pup. A two-step strategy was developed: first, the whole ovaries of newborn mice were cultured for 8 days to allow the development of secondary follicles within the ovarian tissue. The second step was to enzymatically isolate oocyte– granulosa cell complexes and culture them for an additional 14 days. This basic two-step strategy will probably serve as the essential framework for application to other species, for several reasons. First, it is difficult to isolate and culture intact PMFs. Second, the mechanism involved in the recruitment of PMFs into the pool of preantral follicles is unknown and may require the presence of ovarian factors external to the follicle itself. Third, properly timed isolation and culture of oocyte– granulosa cell complexes probably enhances the development and nutrition of larger antral follicles. Alternatively, final oocyte maturation could be achieved by a combination of in vivo transplantation of PMFs, followed by in vitro culture of growing follicles (47). Progress has been made toward ascertaining the hormonal support required to produce antral follicles after successful isolation and in vitro growth of human preantral follicles (ⱖ120 m). The rate of atresia between the preantral and early antral stages in vivo is very high. The histologic evaluation of freshly dissected and cultured early antral follicles revealed that only 10%–20% contained an oocyte (48). PMFs from human ovaries have been isolated successfully by a combination of a gentle enzymatic technique and manual dissection in both fresh and frozen-stored tissue blocks (in 1.5 M ethylene glycol [EG] using a slow freeze/ rapid thaw protocol). Overall, 71.6% ⫾ 2.4% of fresh and 71.5% ⫾ 4.7% of frozen-thawed PMFs were viable by live-dead staining (49). Efforts have been made to define the best culture conditions for the in vitro growth of human ovarian follicles (50, 51). Cryopreservation does not seem to affect the proportion of viable follicles (52). However, partially isolating PMFs increases the proportion of atretic follicles over 1–3 weeks with respect to nonisolated follicles. Human PMFs cultured for 4 weeks within slices of ovarian tissue reach secondary 650
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and, occasionally, early antral stages of development (53). The limited success with human follicular in vitro development is probably due to a lack of knowledge of the factors involved in primordial follicular development, as well as to the relative length of the human follicular growth cycle (44, 45).
Ovarian Tissue Transplantation Until reliable ovarian culture technology becomes available, autologous transplantation offers the best prospect of using frozen-banked human ovarian specimens for restoring fertility to patients. Advances in cryotechnology have improved the results and confirmed that frozen ovarian tissue can return to cyclical function after simple implantation without vascular anastomosis. Success of these models depends mostly on genetic homogeneity of the donor and host strains by using isografts or xenografts into immunodeficient hosts, as the ovary is not immunologically privileged. In 1960, Parrott reported successful pregnancies in mice after implantation of frozen-thawed ovarian grafts (54). In these early experiments, grafts were stored at ⫺79°C, and glycerol was used as a cryoprotectant. In 1993, Carroll and Gosden (55) reproduced these results, transplanting isolated mouse PMFs cryopreserved using dimethyl sulfoxide (DMSO). Cryopreserved marmoset ovarian tissue has also been transplanted under the kidney capsule of immunodeficient mice (56). Despite these successes, doubts remained as to whether the human ovary could be cryopreserved because it is bulkier and more fibrous and because the PMFs are more widely dispersed than in either rodents or marmoset monkeys. The restoration of fertility to oophorectomized sheep has been reassuring. Ovarian tissue slices (the cortex of each ovary was divided into four or five slices 1 mm thick) were preserved in liquid nitrogen using DMSO as a cryoprotectant (57). These animals became pregnant after transplantation of both fresh and thawed ovarian tissue. Recently, the first case of laparoscopic autotransplantation of frozen-thawed human ovarian tissue was reported in a 29-year-old woman. Ovarian cortical strips measuring 2 ⫻ 5 and 5 ⫻ 10 mm were cryopreserved in DMSO after bilateral salpingo-oophorectomy. One of the ovarian pieces was thawed, anchored to a Surgicell frame, and sutured to a pocket dissected on the pelvic wall. The patient ovulated 3 months later after ovarian stimulation with human menopausal gonadotropins (58).
Ovarian Cortex Biopsy Techniques A round biopter was recently designed as a safe and practical laparoscopic technique for obtaining ovarian cortical biopsy specimens (59). Given the fact that each square millimeter of ovarian surface in a 30-year-old woman contains an average of 35 PMFs, the combined area of six biopter specimens 5 mm in diameter would yield a total number of approximately 3,500 PMFs. However, for patients Vol. 75, No. 4, April 2001
who will almost certainly be sterilized (e.g., in those who will undergo BMT), unilateral oophorectomy for conservation of the entire cortex may be justifiable.
Ovarian Tissue Cryopreservation Despite these good results, it is important to optimize cryopreservation regimens for human ovarian tissue. The currently accepted theory states that the avoidance of large and numerous intracellular ice crystals (IIF) is a necessary condition for a cell to survive freezing (60, 61). Thus cooling rates should be fast enough to minimize the long exposure of cells to deleterious freezing conditions but slow enough to avoid the damaging effect of IIF. The assumption that dehydration conditions and the rate of cooling that are used for embryos will be suitable for the cells of the ovarian cortex, the follicular cells, and the oocyte may be untenable given the variation in the water content in each of these cells. Another essential part of the cryopreservation technique is permeation of the tissue with a cryoprotectant (CPA) to minimize ice formation. The permeation of isolated cells with CPAs is relatively fast, but diffusion into multicellular tissue systems is much slower, which means that exposure to the CPA may have to be prolonged or altered in order to reach adequate concentrations in the center of the tissue without overexposing the more peripheral cells. Several studies have started to address some of these issues. No difference in tissue necrosis was found after freezing thin pieces of ovarian cortex with cooled 1.5 M DMSO or 1.5 M 1,2 propanediol (PROH)/0.1M sucrose in a slow freeze–rapid thaw protocol (62). Newton et al. (63) evaluated the viability of follicles within 1-mm3 slices of ovarian tissue, donated by eight healthy patients, preserved by slow freezing in one of the following cryoprotectants: DMSO, EG, glycerol (GY), and PROH. Equilibration was done at 4°C for 30 minutes. Follicular viability was assessed by counting follicles in histological sections 18 days after grafting under the kidney capsules of severe combined immunodeficiency (SCID) mice, and the results were expressed as percentages of the numbers in comparable pieces of ungrafted tissue. Although only 10% of the control number of follicles was found in the GY group, there were no significant differences between the results with EG, DMSO, PROH, and fresh-grafted tissue. A total of 74% of the follicles survived in the grafted fresh tissue. Newton et al. (64) investigated the diffusion of these four cryoprotective agents into the human tissue at both 4°C and 37°C using proton nuclear magnetic resonance imaging analysis. After 30 minutes at 4°C, PROH and GY displayed a significantly lower equilibration rate than EG and DMSO (P⫽0.02). At 37°C, all CPAs penetrated at a faster rate, although there was no significant impact on their final tissue concentration, except for in the case of PROH, which achieved a significant rate of diffusion at this temperature (99.7% permeability). The significance of this finding in terms of follicular viability was not addressed. There was no FERTILITY & STERILITY威
further protection against cell damage by adding low concentrations of sucrose to DMSO. Gook et al. (65) evaluated the effect of varying dehydration conditions and cooling rates on the histological appearance of thin slices of human ovarian cortex after cryopreservation with 1.5 M PROH/0.1 M sucrose in phosphatebuffered saline with 10 mg/dL human serum albumin. Single-step dehydration for 90 minutes and slow cooling– rapid thaw resulted in the highest proportion of intact human primordial and primary follicles.
AREAS OF FUTURE RESEARCH The importance of understanding the molecular mechanisms of carcinogenesis cannot be overstated. This would be the basis for the development of tissue-specific or receptormediated chemotherapy. In the meantime, prospective randomized controlled studies are needed to define the protective role of GnRHa in cytotoxic-mediated loss of PMFs. It is important to optimize the dehydration conditions and cooling rates for ovarian tissue cryopreservation. Nonvascular implants are vulnerable to ischemia-reperfusion injury from reactive oxygen species. It will be important to minimize the period of ischemia because more follicles are lost at this stage than by freezing and thawing (42). Administering an antioxidant, vitamin E, seems to improve the survival of follicles in ovarian grafts by reducing ischemia-reperfusion injury (66). Although the physiologic viability of frozen-thawed transplanted ovarian tissue has been demonstrated, the eventual longevity of such transplants is probably limited. It is therefore important to consider timely transplantation of frozen-thawed ovarian cortical tissue to a cancer survivor preparing for conception. An additional concern is the issue of microscopic metastatic disease to the ovary and the possibility of tumor reimplantation in cancer patients (67– 69). Currently available standard techniques such as histologic evaluation cannot exclude microscopic tumor involvement (70). This risk would be avoided by either follicular growth in vitro or xenotransplantation of ovarian tissue (71, 72). In recent years, a number of characteristic chromosomal abnormalities in leukemia and lymphoma have been defined at the molecular level. Unfortunately, not all malignancies have such clear genetic markers (73). Detection of minimal residual disease in hematological malignancies would be helpful in terms of evaluating the efficacy of treatment, early detection of relapses, and quantitation of remaining tumor cells in bone marrow, peripheral blood cells, or even ovarian grafts before transplantation.
SUMMARY Given the promising preliminary results in human and animal studies, patients willing to preserve their fertility 651
should be offered GnRHa/chemotherapy cotreatment when at risk of POF, unless contraindicated. All patients, and especially women ⬎30 years of age and/or undergoing highdose chemotherapy and BMT, should be counseled about fertility preservation options. Frozen embryo storage is a standard practice in IVF centers and is, therefore, the cryopreservation technique most readily available. In this case, patients should consider issues such as the fate of stored embryos and the limitations in terms of harvesting an adequate amount of viable embryos. Ovarian tissue cryopreservation is the most efficient technique of oocyte banking and the only alternative for prepubertal women. Until reliable follicle culture technology becomes available, autologous ovarian tissue transplantation offers the best prospect of using frozen-banked ovarian cortex. Xenografts into immunodeficient hosts may avoid the risk of tumor reimplantation in cancer patients. Although there has been preliminary success with these models, their safety and clinical applicability needs further assessment. Ovarian tissue cryopreservation has brought hope to patients facing POF. References 1. Boring CC, Squires TS, Tong T. Cancer statistics 1991. CA Cancer J Clin 1991;41:19 –36. 2. Blumemfeld Z, Avivi I, Ritter M, Rowe JM. Preservation of fertility and ovarian function and minimizing chemotherapy-induced gonadotoxicity in young women. J Soc Gynecol Investig 1999;6:229 –39. 3. Chapman RM, Sutcliffe SB, Malpas JS. Cytotoxic-induced ovarian failure in women with Hodgkin’s disease. 1. Hormone function. JAMA 1979;242:1877– 81. 4. Clark ST, Radford JA, Crowther D, Swindell R, Shalet SM. Gonadal function following chemotherapy for Hodgkin’s disease: a comparative study of MVPP and a seven-drug hybrid regimen. J Clin Oncol 1995; 13:134 –9. 5. Horning SJ, Hoppe RT, Kaplan HS, Rosenberg SA. Female reproductive potential after treatment for Hodgkin’s disease. N Engl J Med 1981;304:1377– 82. 6. Reichman BS, Green KB. Breast cancer in young women: effect of chemotherapy on ovarian function, fertility, and birth defects. Natl Cancer Inst Mongr 1994;16:125–9. 7. Teinturer C, Harmann O, Valteau-Couanet D, Benhamou E, Bougneres PF. Ovarian function after autologous bone marrow transplantation in childhood: high-dose busulfan is a major cause of ovarian failure. Bone Marrow Transplant 1998;22:989 –94. 8. Leahy AM, Teunissen H, Friedman DL, Moshang T, Lange BJ, Meadows AT. Late effects of chemotherapy compared to bone marrow transplantation in the treatment of pediatric acute myeloid leukemia and myelodysplasia. Med Pediatr Oncol 1999;32:163–9. 9. Meirow D, Lewin A, Or R, Rachmilewitz E, Slavin S, Schenker JG, et al. Ovarian failure post-chemotherapy in young cancer patients—risk assessment indicate the need for intervention [abstract P-261]. In: 1997 Annual Meeting Program Supplement, Cincinnati, OH: American Society for Reproductive Medicine. Fertil Steril 1997:S218. 10. Schimmer AD, Quatermain M, Imrie K, Ali V, McCrae J, Stewart K, et al. Ovarian function after autologous bone marrow transplantation. J Clin Oncol 1998;16:2359 – 63. 11. Wallace W, Shalet SM, Tetlow LH, Morris-Jones PH. Ovarian function following the treatment of childhood acute lymphoblastic leukaemia. Med Pediatr Oncol 1993;21:333–9. 12. Familiari G, Caggiati A, Nottola SA, Ermini M, Di Benedetto MR, Motta PM. Ultrastructure of human ovarian primordial folllicles after combination chemotherapy for Hodgkin’s disease. Hum Reprod 1993; 8:2080 –7. 13. Meirow D, Lewis H, Nugent D, Epstein M. Subclinical depletion of primordial follicular reserve in mice treated with cyclophosphamide: clinical importance and proposed accurate investigative tool. Hum Reprod 1999;14:1903–7. 14. Hsueh AJ, Jones PB. Extrapituitary actions of gonadotropin-releasing hormone. Endocr Rev 1981;2:437– 61. 15. Ataya K, Moghissi K. Chemotherapy-induced premature ovarian failure: mechanisms and prevention. Steroids 1989;54:607–26.
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