Monitoring the ovaries after autotransplantation of cryopreserved ovarian tissue: endocrine studies, in vitro fertilization cycles, and live birth

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1 Monitoring the ovaries after autotransplantation of cryopreserved ovarian tissue: endocrine studies, in vitro fertilization cycles, and live birth Dror Meirow, M.D., a Jacob Levron, M.D., a Talia Eldar-Geva, Ph.D., M.D., b Izhar Hardan, M.D., c Eduard Fridman, M.D., d Ziva Yemini, M.Sc., a and Jehoshua Dor, M.D. a a IVF Unit, Department of Obstetrics and Gynecology, Sheba Medical Center, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel; b Department of Obstetrics and Gynecology, Shaare Zedek Medical Center, Ben Gurion University of the Negev, Jerusalem, Israel; c Department of Hematology, and d Department of Pathology, Sheba Medical Center, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel Objective: To investigate fertility potential of ovarian tissue harvested after chemotherapy, to monitor ovarian recovery after transplantation, and to compare with in vitro fertilization (IVF) cycles. Design: Clinical and endocrine study. Setting: IVF unit and hematology department in a tertiary university hospital. Patient(s): A 28-year-old patient suffering from non-hodgkin s lymphoma had some of her ovarian tissue cryopreserved shortly after conventional chemotherapy and failure to respond to ovarian stimulation but before sterilizing treatment. Intervention(s): Transplantation of cryopreserved ovarian tissue; four IVF cycles. Main Outcome Measure(s): Gonadotropins, ovarian steroids, anti-mullerian hormone (AMH), inhibin B, ovarian histology, sonography, and outcome of IVF cycles. Result(s): Large number of primordial follicles were present in the harvested tissue. During the first months after transplantation, gonadotropins were high, AMH and inhibin B were low, and in three IVF cycles, eggs were not found. After recovery of endocrine activity 9 months after transplantation, a mature oocyte was retrieved. Embryo transfer resulted in a normal pregnancy and delivery of a healthy baby. Although spontaneous menstruation resumed after delivery, endocrine profile 22 months after transplantation indicated low reserve. Conclusion(s): The recovery of endocrine function after transplantation correlated with the result of oocyte recovery. Fertility preservation using ovarian tissue is effective also in cases when the ovaries are injured after chemotherapy. However, transplant life span is limited. (Fertil Steril 2007;87:418.e by American Society for Reproductive Medicine.) Key Words: Fertility preservation, ovarian tissue, chemotherapy, ovarian reserve, IVF, inhibin B, anti-mullerian hormone Advances in high-dose chemotherapy and radiotherapy treatments have significantly improved the cure rates of many young patients with some hematologic malignancies and solid tumors, but unfortunately, sterilization and early menopause are common long-term side effects of treatment (1, 2). Therefore, as an integral part of treatment, procedures to preserve fertility are commonly practiced. Various strategies of fertility preservation are used depending on the risks and probabilities of gonadal failure, the patient s general health at diagnosis, and the partner status (3, 4). These include IVF and embryo cryopreservation, which are standard established procedures with predictable and well-documented outcomes in terms of pregnancies and childbirth rates (5). Harvesting and cryopreservation of ovarian tissue before commencing sterilizing chemotherapy has been practiced Received November 23, 2005; revised April 15, 2005; accepted May 14, Reprint requests: Dror Meirow, M.D., Department of Obstetrics and Gynecology, Chaim Sheba Medical Center, Tel Hashomer 52621, Israel (FAX: ; meirow@post.tau.ac.il). during the past decade. With ovarian cryobanking, abundant primordial follicles, containing small, less differentiated oocytes are stored (6). Ovarian tissue banking is an experimental procedure (7), and optimal conditions of freezingthawing and grafting are yet to be determined (8). This technique requires surgical grafting procedures once cancer treatment is completed and the patient is sterilized. Spontaneous pregnancy and delivery have been reported after fresh ovarian tissue donated from a fertile woman was successfully transplanted into her sterile monozygotic twin (9). Fertility preservation procedures are preferably performed before administration of chemotherapy. However, in some cases, attempts are initiated only after the patient has already been exposed to chemotherapy treatments. Recently, we have reported a pregnancy and delivery after transplantation of cryopreserved thawed ovarian cortical tissue into the ovaries of a young patient who was treated for non-hodgkin s lymphoma (NHL) and suffered from ovarian failure (10). Ovarian tissue was harvested shortly after exposure to chemotherapy, but prior to high-dose chemother /07/$32.00 Fertility and Sterility Vol. 87, No. 2, February 2007 doi: /j.fertnstert Copyright 2007 American Society for Reproductive Medicine, Published by Elsevier Inc. 418.e7

2 apy. To follow short- and long-term function of the transplant, endocrine studies were performed and correlated with the outcome of ovarian stimulation and IVF cycles. Ovarian histology was used to assess follicular density, and transvaginal sonography was used to monitor the ovaries. MATERIALS AND METHODS Patient Treatment A 28-year-old woman, married with one child, was diagnosed with a primary mediastinal B cell non-hodgkin s lymphoma. A first-line chemotherapy protocol was administered weekly (induction chemotherapy) for 12 weeks (Fig. 1). A relapse occurred 6 months after completion of therapy. Therefore, two courses of a second-line protocol (salvage chemotherapy) (11) were administered, followed by high-dose chemotherapy with autologous stem cell support 7 weeks later. Since then, the patient has been in complete remission. Prior to undergoing chemotherapy, the patient had had regular cycles, and 1.5 years earlier had a normal pregnancy and delivery. After completion of the first chemotherapy protocol, the patient had monthly regular cycles until the second chemotherapy course, and then oral contraceptives were administered for 1 month. Due to the high risk of sterilization with high-dose chemotherapy and stem cell support, attempts were made to preserve fertility in the 7 weeks available after the second chemotherapy course (Fig. 1). Initially, the ovaries were stimulated with the intention of storing embryos after IVF. However, after 14 days of intense ovarian stimulation (up to 525 IU/day FSH), the ovaries did not respond (E 2 20 pg/ml) and no follicles were seen on vaginal sonography. Therefore, it was decided to cryopreserve ovarian tissue in an attempt to preserve fertility. At laparoscopy, both ovaries appeared normal, 10 cm 3 each. Approximately two thirds of the right ovarian cortex was removed, and seven pieces of ovarian cortex were cryopreserved by methods described in detail below; in addition, a small fragment was stored for future reference. Histologic examination revealed a high number of primordial follicles ( 150 mm 3 ) with normal morphology (Fig. 2A), a few secondary follicles ( 30 mm 3 ), but no antral follicles. No malignant cells were identified in the tissue sent for evaluation. After high-dose chemotherapy, the patient suffered from 2 years of persistent amenorrhea, and repeated endocrine studies indicated ovarian failure. Being fully aware of her disease status and prognosis, and the evidence of persistent ovarian failure, the patient asked to autotransplant the stored ovarian tissue in an attempt to restore fertility. Institutional review board (IRB) approval was obtained. Initially, a small fragment of tissue was thawed, which contained primordial follicles with normal architecture (Fig. 2B). Four of the seven stored pieces of ovarian tissue were then thawed and used for the current transplantation. In order to transplant the ovarian tissue into the ovaries, a laparotomy was performed concurrently with tissue preparation. Both ovaries had fibrotic white capsules; the right ovary was significantly smaller (1.7 cm 3 ) and there were no adhesions in the pelvis. The thawed ovarian tissue was transplanted as previously described (10). Briefly, in the left ovary, three pairs of 5-mm transverse incisions were made through the tunica albuginea, and using blunt dissection, cavities were formed beneath the cortex, into which each of the three strips was gently placed. The incisions were sutured with 4/0 Vicryl to avoid adhesions. In the right ovary, the ovarian FIGURE 1 Timescale (months) from diagnosis to high-dose chemotherapy and stem cell support, showing chemotherapy protocols, menstrual cycles, and fertility preservation procedures. OTCP, ovarian tissue cryopreservation; OCP, oral contraception pills; SCT, high-dose chemotherapy and stem cell support. First-line chemotherapy protocol (VACOP-B): etoposide, adriamycin, cyclophosphamide, vincristine, bleomycin, and steroids. Second-line chemotherapy protocol (MINE/ESHAP): mesna, ifosfamide, mitoxantrone, etoposide, Ara-C, cis-platinum, and steroids (11). High-dose chemotherapy (BEAM): BCNU 300 mg/sm, etoposide 1600 mg/sm, Ara-C 1600 mg/sm, and melphalan 140 mg/sm. 418.e8 Meirow et al. Birth after ovarian tissue transplantation Vol. 87, No. 2, February 2007

3 FIGURE 2 Ovarian histology. (A) Ovarian tissue harvested for cryopreservation 5 weeks after second chemotherapy course. A large number of primordial follicles ( 150 mm 3 ) with normal architecture are present. Original magnification, 100. (B) Two primordial follicles after freezing-thawing procedure showing normal morphology. Original magnification, 400. (C) Samples of ovarian tissue 2 years after high-dose chemotherapy, removed during the surgery to transplant ovarian tissue, show cortical atrophy. No follicles are present. Original magnification, 20. fragments were injected below the cortex. A good vascular bed was present in both ovaries. Samples were taken from the ovaries to evaluate the histology of the tissue that had been exposed to high-dose chemotherapy. Sonography, serum hormone measurements of gonadotrophins, estradiol (E 2 ), anti-mullerian hormone (AMH), and inhibin B were measured repeatedly to detect and assess the recovery and function of the ovaries. During the 9 months after transplantation, four attempts were made (at 2, 3, 8, and 9 months) to aspirate mature eggs from the transplant. Ovarian Tissue Handling Ovarian tissue cryopreservation The harvested cortex was promptly transferred to the laboratory adjacent to the operating theater in oocyte wash buffer (IVF oocyte wash buffer; Cook, Sydney, Australia) at room temperature. (It is important to emphasize that when laparoscopy is performed in locations remote from the laboratory, as a few previous cases we had, the harvested tissue is transported on ice.) While working under laminar flow hood in medium, the specimen was gently grasped with an atraumatic grasper. Using a number 10 blade, the cortex was cleaned from the medulla. The cortex was then cut into ten 5-mm pieces 1 2 mm thick. A small fragment of cortex was evaluated for follicle density and for possible presence of lymphoma cells. In order to assess freezing and storage conditions for this patient prior to tissue thawing and transplantation procedure, additional smaller cortical tissue mm was cryopreserved and kept in a separate vial. As a freezing condition, slow freezing method modified from Gosden s protocol was used (12). The sliced cortex (1 2 mm thick) was equilibrated with the cryoprotective medium for 30 minutes in an automatic roller (1 Hz) using 1.5 M dimethylsulfoxide (DMSO) (D2650; Sigma, St. Louis, MO) in an oocyte wash buffer, 15% synthetic serum substitute supplement (Irvine Scientific, Santa Ana, CA), and 0.1 M sucrose. The tissue was then transferred to freezing vials (no , Nunc freezing vials; Nuclon, Roskilde, Denmark), which were filled to 1.8 ml with freezing solution and placed into a programmable freezer. A slow freezing protocol was employed: cooling of 1 C/min to 9 C, manual seeding, further cooling at a rate of 0.3 C/min to 36 C, followed by faster reduction of 5 C/min to 140 C. The tissue was then transferred to liquid nitrogen. Ovarian tissue thawing and preparation The vials were removed from the liquid nitrogen, held in air at room temperature for one-half minute, then placed in a water bath 37 C and stirred for 2 minutes to melt the medium in the tube. The content of the vials (medium and tissue) was washed in a Petri dish containing first-step thawing solution: oocyte wash buffer (IVF oocyte wash buffer; Cook), synthetic serum substitute supplement 15% (Irvine Scientific), 1.0 M DMSO (D2650; Sigma) 0.1 M sucrose. The tissue was Fertility and Sterility 418.e9

4 transferred to washing vials using prepared thawing solutions for graded dilution of the cryoprotectant. The first stage was carried out by rolling the tissue (1 Hz) in a large volume of oocyte wash buffer (50-mL vials), 0.1 M sucrose, and 1.0 M DMSO for 5 minutes. The procedure and solutions for the following second dilution stage (0.5 M DMSO) and third dilution stage (without DMSO) were identical to the first stage except DMSO concentration; 5 minutes for each stage. By using large volume, we avoided the dilution effect of fluid coming out of the large ovarian strips and kept the desired concentrations stable. All stages were at room temperature. One of the cortical strips was dissected into very small fragments, which were immersed in a small volume of medium and placed in a syringe with a 17-gauge needle. The tissue was immediately transplanted as laparotomy was already performed at the operating theater adjacent to the laboratory. Hormone Measurements, Histologic Analysis, and Pelvic Sonography Hormone levels of E 2, progesterone, FSH, and LH were measured by a chemiluminescent immunometric method (Immulite 2000; Diagnostic Products Corporation, Los Angeles, CA). The sensitivities of E 2 and progesterone were 20 pg/ml and 0.2 ng/ml; the interassay coefficients of variation (CV) were 11% and 15%, respectively. The sensitivities of FSH and LH were 0.1 IU/L and 0.05 IU/L and the CVs were 6% and 7%, respectively. Inhibin B concentrations were measured using two-site ELISA (Serotec, Oxford, UK). Assay sensitivity was 7.8 pg/ml. Inter- and intraassay CVs were 15% and 7%, respectively. The concentrations of AMH were measured using an ultrasensitive two-site ELISA (Diagnostic Systems Laboratories, Inc., Webster, TX). The assay sensitivity was 0.02 ng/ml. Inter- and intraassay CVs were 8.0% and 4.6%, respectively. All serum samples were assayed in duplicate. In-house high- and low-quality control samples in each assay were used, with interassay variability of 12% and 9% for inhibin B and AMH, respectively. Sera from 30 individuals showing results within the expected range were assayed with the patient s serum. Ovarian cortex was sampled at random. The tissue was serially sectioned into 5- m slices, stained with hematoxylineosin, and the numbers of ovarian follicles counted in every fifth section. Ultrasound scans were performed using Logic 9 (General Electric, Milwaukee, WI) with a transvaginal transducer of 4 8 mhz. Ovarian and follicular flow were measured by Doppler. Modified natural IVF cycle After menstruation, spontaneous follicle development was monitored by sonography and estrogen-progesterone measurements. When follicle size reached 15 mm, GnRH antagonist (GnRH-a) 0.25 mg (Cetrorelix; ASTA Medica AG, Frankfurt, Germany) and 225 IU human menopausal gonadotropins (Menogon Ferring, Malmo, Sweden) were added daily. When follicle size reached mm, HCG (Chorigon; TEVA, Petach- Tikva, Israel) was added and follicle aspiration was performed hours later (13). RESULTS Recovery of Ovarian Function Before ovarian tissue transplantation, repeated measurements of FSH were high ( IU/l), and AMH and inhibin B levels were undetectable. Histologic evaluation of the patient s ovary sampled during the transplantation surgery indicated that the ovaries were atrophic; no follicles were observed in the tissue (Fig. 2C). These findings, as well as 2 years of amenorrhea, clearly indicated that the patient had ovarian failure. Ovarian response was monitored continuously during the immediate post-transplantation months (Fig. 3). When large follicles developed, HCG was administered and follicles were aspirated in an attempt to recruit eggs and restore fertility (Table 1). During the first post-transplantation month, estrogen-progesterone tablets were administered. In the second cycle, a follicle developed (28 mm), associated with a rise of E 2 serum levels up to 124 pg/ml. However, levels of inhibin B and AMH were undetectable. In the third cycle, when the ovaries were stimulated with gonadotropins, a single follicle developed associated with minor elevations of both inhibin B up to 35 pg/ml and E 2 serum levels up to 328 pg/ml. However, basal FSH was 28 IU/L and AMH levels undetectable, and no egg was found at follicle aspiration. Throughout the forth and fifth post-transplantation months, the ovaries were inactive: E 2, inhibin B, and AMH were low, and the ovaries did not respond to stimulation. AMH levels that were undetectable during the first five post-transplantation months started to rise in the sixth month, and high AMH levels (5.3 ng/ml) were measured in the eighth month. In the eighth month, spontaneous menstruation occurred, followed by growth of a single follicle (19 mm), concomitant with a minor elevation of inhibin B up to 28 pg/ml and rise of E 2 levels to 236 pg/ml. After HCG administration, a rise in progesterone levels (8.0 ng/ml) indicated luteinization. However, no egg was found at follicle aspiration. In all cycles monitored, ovarian activity and follicle growth were detected only on the left side, where ovarian strips had been transplanted, and Doppler measurements at the periphery of these follicles revealed normal flow (Fig. 4A). No activity was documented from the right ovary, where the small fragments had been injected. Egg Retrieval, Pregnancy, and Live Birth Basal hormone measurements on day 4 of the ninth posttransplantation month showed FSH 7.9 IU/L, LH 6.8 IU/L, E pg/ml, and progesterone 0.5 ng/ml. Therefore, a modified natural cycle was used for stimulation. Inhibin B levels during the follicular phase were high (up to e10 Meirow et al. Birth after ovarian tissue transplantation Vol. 87, No. 2, February 2007

5 FIGURE 3 Estradiol (E 2 ) levels, inhibin B levels, and follicle diameter in the second month (A), third month (B), eighth month (C), and ninth month (D) after ovarian tissue transplantation. Inhibin B is presented in squares ( ) and points connected in dashed lines. Levels of E 2 are presented in triangles (Œ) and solid lines connect the points during follicular phase. At the eighth and ninth months (C, D), hormone levels are also presented during luteal phase. In the third and the ninth months (B, D), the ovaries were stimulated. Follicles with diameter of 19 to 22 mm developed in all cycles; however, inhibin B levels were low during the second, third, and eighth months (A, B, C) and did not correlate with follicular size. Only in the ninth month were inhibin B levels high and showed normal pattern of rise and fall during follicle growth. In this cycle, an egg was retrieved (D). pg/ml) with a normal pattern of initial rise and fall (Fig. 3). On day 11, follicle size was 20 mm, endometrial thickness was 7 mm, and E 2 level was 253 pg/ml. HCG 7500 IU was administered and 35 hours later a single mature egg with a large cumulus was retrieved. The egg was fertilized with the husband s sperm in vitro without intracytoplasmic sperm injection (ICSI), and on day 2 postfertilization, a four-cell embryo (Fig. 4B) was transferred to the uterus. After embryo transfer, progesterone supplementation was administered using vaginal tablets (Utrogestan; Besins Iscovesco, Paris, France) 200 mg two tablets three times daily, and 12 days later the pregnancy test was positive. Repeated ultrasonography during the pregnancy showed normal fetal growth and development (Fig. 4C). At 38 weeks 5 days of gestation, a healthy-appearing female infant weighing 3,000 g was delivered by cesarean section. The left ovary was small (3.75 mm 3 ) and the right almost undetectable. At the age Fertility and Sterility 418.e11

6 TABLE 1 Ovarian response and IVF cycles after ovarian cortical tissue transplantation. Hormone basal levels Months posttransplantation Vaginal bleeding Ovarian stimulation FSH IU/L AMH ng/ml Inhibin B pg/ml Follicle (mm) Eggs 2 Post-pill a Post-pill a FSH Post-pill a GnRH-a FSH Post-pill a Spontaneous b Spontaneous b Modified natural (13) Note: Although follicles were present from the second cycle, an egg was aspirated only at the ninth cycle when hormone levels were normal. a Bleeding ascending stimulation resulted from estrogen progesterone administration (pills). b Spontaneous menstruation. of 3 months, the baby was examined and showed normal growth and normal responses to neurodevelopmental tests. Three to 7 months after delivery, the patient had four spontaneous menses. However, at 5 months after delivery, hormone profile was FSH 83 IU/L, LH 61 IU/L, and E 2 41 pg/ml. DISCUSSION This work demonstrates recovery of a cryopreserved-thawed ovarian transplant, as monitored by endocrine studies and IVF cycles. The successful IVF cycle and pregnancy 9 months after transplantation attest to the good quality and fertility potential of the cryopreserved primordial follicles, despite the fact that ovarian tissue was cryopreserved a short time after chemotherapy. It also indicates that the ovary is a good location for transplantation, as conventional fertility procedures can be used. The ovarian failure rate after conventional chemotherapy for non-hodgkin s lymphoma is 10% 44% (14). However, FIGURE 4 (A) Transvaginal ultrasonography 8 months after transplantation demonstrating the left ovary, measuring mm with a follicle of 18 mm in diameter. Doppler flow in the periphery of the follicle revealed normal vascularization, Resistance Index (RI) (B) Four-cell embryo on day 2 after fertilization, prior to embryo transfer. (C) Transvaginal ultrasound at 10 weeks gestation, demonstrating a single fetus with a Crown to Rump Length (CRL) of 35 mm. 418.e12 Meirow et al. Birth after ovarian tissue transplantation Vol. 87, No. 2, February 2007

7 the risk of sterilization by high-dose chemotherapy is very high (80% 100%) (15). Thus, it is highly important to make every possible effort to preserve future fertility prior to high-dose chemotherapy and bone marrow support. The first line of chemotherapy treatment in this patient did not result in ovarian failure. Thus, after relapse and second-line chemotherapy treatment, the patient requested that embryos be stored for future use, in spite of the possible hazards resulting from recent exposure to chemotherapy, i.e., high abortion and malformation rates, as shown in animal studies (16). The advantages of embryo freezing for fertility preservation are that it is a well-established procedure, the patient s general health was good, there was sufficient time to conduct the procedure, and the woman had a partner. However, the ovaries did not respond after 2 weeks of intense ovarian stimulation. It was possible that the patient already had ovarian failure after the second-line chemotherapy but it is also possible that the ovaries were merely inactive and the patient was not sterile. A short period of amenorrhea, followed by recovery of the ovary, occurs frequently after exposure to nonsterilizing chemotherapy. Chemotherapy destroys follicles differentially: large maturing follicles that respond to gonadotropin stimulation, and which are recruited for IVF, are more vulnerable than are primordial follicles representing ovarian reserve (17). Ovarian tissue taken at laparoscopy confirmed that numerous primordial follicles were present (Fig. 2A). At that stage, the potential for future fertility with the frozen ovarian tissue and the quality of the preserved primordial follicles were unknown. After high-dose chemotherapy, the patient experienced 2 years of ovarian failure, as indicated by amenorrhea, ovarian histology, and endocrine studies. Hormone levels of AMH and inhibin B were undetectable. These two members of the TGF- superfamily are produced exclusively in granulosa cells from early stage ovarian follicles (18, 19). Serum AMH is an indicator of the pool of FSH-independent follicles, and levels of both AMH and inhibin B are good markers of ovarian reserve and decline gradually with age (20, 21). Ovarian histology complementary to clinical and hormonal evaluation is a reliable tool in evaluating premature ovarian failure (22). Orthotopic and heterotopic sites for graft transplantation that have previously been reported include the arm, abdominal wall, pelvis, and ovary (9, 23 27). In this patient, both ovaries were present and blood flow was preserved. Therefore, we assumed that the ovary would be a suitable transplantation site. Follicle growth in the ovary enables the patient to be managed in a conventional IVF setting and it allows future spontaneous conception. Two different transplantation techniques were used (10): large cortical pieces were implanted under the cortex of the left ovary, while small fragments were injected under the cortex of the right ovary, a technique that has been shown to be successful in the rabbit model (28). The latter technique has the advantage of surgical simplicity, but at present, only the large cortical strips transplanted into the left ovary have resumed function. During the second and third months after transplantation, low ovarian activity was detected, in spite of high gonadotropins and undetectable levels of AMH (Fig. 3; Table 1). This may have resulted from a few growing follicles in the transplanted tissue that survived the freezing, thawing, and transplantation procedures. The discrepancy between follicle growth and hormone secretion could be attributed to granulose cell damage. However, there were no eggs in the follicles. In the eighth month after ovarian tissue transplantation, spontaneous menstruation occurred, AMH levels were high (5.3 ng/ml), and the left ovary was active. The conditions used for freezing ovarian tissue are effective for storing primordial follicles (29) but are not effective in preserving the small number of growing follicles that exist in the tissue. The time needed for primordial follicles to grow, mature, and ovulate is of the order 6 months (30). When cryopreserved thawed human ovarian cortex was grafted into immunodeficient mice, multiple growing follicles were observed after 6 months (31). Hence, it was not entirely unexpected that the ovarian transplant responded 8 months postoperation. There have been a few reports of ovarian recovery and spontaneous pregnancies after high-dose chemotherapy (32) and in a patient after ovarian tissue freezing (33). Theoretically, the patient may have ovulated from a few follicles that remained in the ovary and not merely from the transplanted cortical strips. However, by means of repeated measurements of basal AMH and inhibin B, semiquantitative changes in ovarian endocrine function were evaluated (Table 1). The indications of ovarian failure after the high-dose chemotherapy, and the high AMH levels, indicating high activity of early stage growing follicles after transplantation, provided strong evidence for recovery of the transplant rather than the activity of a few residual follicles. These were followed by a significant rise in inhibin B levels that were as high as normal levels in ovulatory women (34). Ovarian cryobanking as a strategy to preserve fertility was perceived as a novel option after the first pregnancies in the sheep model (12). Successful IVF and pregnancy after egg collection from freshly transplanted ovarian tissue was reported in monkeys (35). Heterotopic autotransplantation of ovarian tissue followed by oocyte retrieval and IVF in a sterilized breast cancer patient resulted in embryo development but no pregnancy (26). A spontaneous pregnancy and delivery of a healthy baby has been reported recently after transplantation of frozen thawed ovarian tissue in a woman previously exposed to conventional dose chemotherapy treatments (27). In that report, however, the data provided did not necessarily indicate that the patient suffered from ovarian failure (36). We were able to restore fertility by transplantation of cryopreserved thawed ovarian tissue into the ovaries of a sterilized patient followed by oocyte retrieval and IVF. Large follicles were aspirated from the left ovary; however, Fertility and Sterility 418.e13

8 an egg was found only at the fourth attempt (Table 1). During the first two cycles, follicle growth and E 2 secretion were monitored. However, the ovaries were not functioning adequately, as indicated by high FSH and low AMH and inhibin B levels. No eggs were retrieved in aspirated follicles ( empty follicles ) as frequently found in patients with low ovarian reserve and elevated FSH. In spite of high AMH levels in the eighth month, FSH was still high, inhibin B levels low, and no egg was found at follicle aspiration. Only in the ninth month, when all endocrine functions were normal, was a mature egg aspirated. A prospective study has clearly shown excellent correlation of serum AMH levels measured within 3 months preceding IVF cycle with ovarian response in IVF (21). The IVF allowed inspection of the egg and assessment of fertilization and early embryo development. After conception, normal development and adequate fetal growth were reassuring. The healthy baby, as evaluated by the neonatologists and the normal responses to neurodevelopmental tests, is an indicator that the procedure is safe. However, confirmation of this observation will have to await other future pregnancies and long-term follow-up. Ovarian tissue cryopreservation in cancer patients carries the risk of grafting microscopic undetected malignant cells that could remain within the cryopreserved-thawed grafts. In order to reduce the risk, it is highly recommended to use the best available methods to detect minimal disease (37 39). The lymphoma cells in this patient did not have markers that could identify small numbers of malignant cells in a tissue sample. However, the tissue was harvested for cryopreservation after induction of remission, at which time there was no evidence of active disease, and conventional pathology did not disclose cancer cells in the tissue taken for cryopreservation. At this stage, we cannot know if the transplant will continue to function. The spontaneous menstruation 3 to 7 months after delivery (21 25 months after transplantation of ovarian tissue) and the high levels of AMH and inhibin B prior to pregnancy are indications of good ovarian reserve. However, high gonadotropin levels 5 months after delivery indicate that the ovaries are at least inactive at this stage or in failure. The number of primordial follicles in the strips of ovarian cortex transplanted is limited and a significant number of follicles are lost during the ischemic period after transplantation (40). Furthermore, the ovaries were exposed to the destructive effect of chemotherapy before the tissue was cryopreserved. Thus, a limited period of graft survival could be expected. This was the reason why we started with IVF cycles early and did not await natural conception. In the future, if the woman and her husband ask for another child and the transplant will not function, we can thaw the remaining ovarian tissue strips and perform an additional transplantation. As this case demonstrates, the optimal approach for fertility preservation varies and should be individualized. We believe that in young patients, fertility preservation should be an integral part of cancer treatment. Acknowledgments: The authors thank Prof. Irving Spitz, Institute of Hormone Research, Shaare-Zedek Medical Center, Jerusalem, for discussions and critical review of the data and manuscript. REFERENCES 1. McVie JG. Cancer treatment: the last 25 years. Cancer Treat Rev 1999;25: Larsen EC, Muller J, Schmiegelow K, Rechnitzer C, Andersen AN. Reduced ovarian function in long-term survivors of radiation- and chemotherapy-treated childhood cancer. J Clin Endocrinol Metab 2003; 88: Meirow D. 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