Autotransplantation of cryopreserved ovarian tissue to the right forearm 4½ years after autologous stem cell transplantation

Abstract

Premature ovarian failure (POF) may occur as a consequence of chemotherapy and of irradiation to the ovaries (1). Alkylating agents such as cyclophosphamide and chlorambucil are particularly prone to cause ovarian damage (2). The human oocytes are extremely sensitive to radiation. Recently, it was estimated that the radiation required to destroy 50% of the oocytes in human ovaries is less than 2 Grey (3). Besides cryopreservation of embryos and of unfertilized oocytes, cryopreservation of ovarian tissue harvested prior to gonadotoxic treatment is one approach to try to preserve a patient's fertility. Thus, investigators have reported autotransplantation of fresh ovarian tissue to the forearm (4) and of cryopreserved ovarian tissue heterotopically to the retroperitoneum (5), the rectus abdominis muscle (6) or orthotopically to the remaining ovary (7,8). Recently, Byskov et al. transplanted cryopreserved ovarian tissue to the remaining ovary and could later obtain mature oocytes from the transplant (8). Oktay et al. transplanted cryopreserved ovarian tissue beneath the skin of a woman's abdomen and succeeded in fertilizing a mature oocyte obtained from the transplant (9). Similarly, Lee et al. obtained mature oocytes from cryopreserved ovarian tissue transplanted to subcutaneous pockets of macaque monkeys. One monkey delivered a healthy female infant at the end of 2003 following in vitro fertilization (IVF) of an oocyte obtained from an abdominal pocket (10). The present report describes the first autotransplantation of cryopreserved ovarian tissue to the forearm of a woman. A 30-year-old, nulliparous, woman was diagnosed with primary Sjogren's syndrome in 1991. In 1993, she was diagnosed with pure red cell aplasia (PRCA), requiring numerous blood transfusions. From 1995, attempts to treat the PRCA were made with antithymocyte globulin (ATG), anti-CD52 and anti-CD4 antibodies, desferrioxamine (Desferal®, Novartis AG, Basel, Switzerland), azatioprin (Imurel®, Glaxo SmithKline, UK), cyclosporine, extracorporal photophoresis, and immunoglobuline infusions (Gammagard®, Baxter international Inc., Deerfield, IL, USA). In February 1998, stem cell mobilization was successfully attempted with cyclophosphamide and granulocyte colony-stimulating factor (G-CSF). Later in 1998, autologous stem cell transplantation was performed. Prior to treatment, conservation of her fertility was taken into consideration. IVF was not possible as she had no partner. Thus, cryopreservation of ovarian tissue was decided on and one ovary was removed during laparoscopy. Immersed in Gamete-100 medium (Vitrolife Sweden AB, Gothenburg, Sweden) at 37 °C, the ovarian cortex was separated from the stroma and cut into small pieces. The cortical pieces were immediately transported in 37 °C Gamete-100 medium to the laboratory at IVF-Öresund, Malmö. The tissue was further cut into 1–2 mm × 1–2 mm × 1 mm pieces and transferred to a cryosolution containing 1·5 m propanediol and 0·1 m sucrose in PBS (Cryosolution no. 2; Vitrolife Sweden AB) at ambient temperature. Fifteen straws, prefilled with cryosolution, were loaded with the pieces of ovarian tissue and immediately put in a programmable freezing device, CTE 920 (Cryo-Technik-Erlangen GmbH, Erlangen, Germany), for cryopreservation. This device has a self-seeding system, which results in crystallization at an optimal temperature. A slow freezing protocol was used and the straws were moved for further storage in liquid N2. After laparoscopy, the patient received a combination of cyclophosphamide and fractionated total body irradiation (Cy-fTBI) with a total of 8400 mg of cyclophosphamide, and 8 Grey of total body irradiation. Then she was successfully infused with 4·0 × 106/kg body weight of human CD34+ hematopoietic progenitor cells. A few months later, she had climacteric symptoms and amenorrhea with a follicle-stimulating hormone (FSH) level of 31 IU/l, luteinizing hormone (LH) level of 16 IU/l, and estradiol level of <70 pmol/l, suggesting primary ovarian failure (POF). She then received hormone replacement therapy (HRT). Early in 2003, she attended for infertility. HRT treatment was intermittently discontinued, rendering the patient climacteric with hot flushes and an FSH level of 25 IE 2 weeks later suggesting continuing POF. She gave informed consent for autotransplantation of her cryopreserved ovarian tissue. On April 29, 2003, containers with cryopreserved ovarian tissue were transported in liquid N2 from IVF-Öresund in Malmö to the University Hospital of Lund. The thawing procedure was similar to the one described by Oktay and Karlikaya (5). In short, two straws containing 10 ovarian pieces were thawed in room temperature for 30 seconds, then placed in a 37 °C water bath for 2 min. The contents of each straw were expelled in a solution containing 1·5 m propanediol, 0·1 m sucrose and 20% of fresh autologous serum in phenolred-free minimal essential medium (with l-glutamine, ribonucleosides and deoxyribonucleosides; Gibco, cat. no. 41061-029, Invitrogen Inc., Carlsbad, CA, USA). After washing stepwise in decreasing concentrations of cryoprotectant, the tissue was finally transferred to and kept in a transport medium consisting of 10 µg/ml cefuroxim, 0·26 IU/ml of FSH (Serono S.A., Geneva, Switzerland) and 20% fresh autologous serum in phenolred-free minimal essential medium for 5 min before transplantation. All dishes were kept on ice and after completion of the procedure the temperature of the media was 8 °C. The transplantation was largely performed according to Oktay et al. (11). On the right arm, a 12 mm transverse incision was made approximately 8 cm distally of the antecubital fossa above the brachioradial muscle. Using blunt dissection, a pocket 2 cm in depth, was created distally between the subcutaneous tissue and the muscle fascia. Lifting the skin above the pocket with small hooks to keep it expanded, the 10 pieces of tissue were put into the pocket and distributed with a micro pick-up. The pieces were of uneven sizes and shapes, i.e. 1–2 mm × 1–2 mm × 0·5–1 mm. The incision was sutured with a continuous intradermal 4-0 polyglycolic suture. The forearm was loosely bandaged and put in a mitella. The patient was asked to use the mitella for the next 4 days. For the next 7 days, 75 IU/day of FSH was injected and spread out into the subcutaneous tissue above the pocket to improve follicular growth (12). Hormone treatment by an estradiol 100 µg/24 h patch was continued. Power Doppler ultrasound, using an 8 MHz crystal, was performed regularly during the following weeks to monitor vascularization and follicle development. Follicle development was first observed after 18 weeks. The estradiol patch treatment was then discontinued. A week later, estradiol levels had increased to a maximum of 6861 pmol/l (Fig. 1). To further stimulate follicle development, doses of 50 IU of FSH increasing to 100 IU were given subcutaneously every day from day 135 to day 145. As shown in Fig. 1, estradiol levels declined during FSH treatment, remaining on lower levels. After 7 months, estradiol levels remained at levels of 70 pmol/l or lower. The follicle initially observed increased slowly in size to a maximum average diameter of 12·6 mm (Fig. 2). A second follicle was noted on day 162, reaching a maximum average diameter of 6·7 mm on day 216. A new attempt to stimulate the ovarian tissue with FSH 8 months after transplantation failed, suggesting that the tissue was exhausted. Graph showing plasma estradiol levels (red bars) and follicle diameters (lines) after autotransplantation of cryopreserved ovarian tissue to a subcutaneous pocket on the right forearm. Ultrasound examination of the right forearm at the time the largest follicle reached its maximum diameter, using an 8 MHz crystal. Follicle marked with two ‘x’. In this case, the tissue did not survive for longer than approximately 7 months. The two observed follicles increased slowly in size over several weeks and were regarded as abnormal. Thus, oocyte harvesting was not attempted. Several issues must first be addressed to further develop the method. Autotransplantation has two main goals: to restore hormone production as well as follicular development in order to achieve pregnancy. The levels and duration of estradiol production are probably intimately connected to the total number of intact follicles in the tissue at the time of adequate revascularization. At the outset, this number will be dependent on the age of the patient and the amount of tissue transplanted. However, follicles are lost during the different procedures the tissue is exposed to from oophorectomy to complete revascularization. Different cryoprotectants, for instance, are associated with more or less follicle damage (13). In addition, exposure to the cryoprotectant before freezing may be critical for follicle survival (13). Ischemia is another important factor that might cause follicle damage. Ischemia starts as soon as the ovary is removed. Even when the number of follicles is not reduced during the first 30 min after removal (14), apoptotic processes might start before the tissue is cryopreserved. After the replacing of tissue, ischemia will persist until revascularization, causing major follicle loss. Baird et al. showed that 65% of the follicular population was damaged after transplantation of fresh ovarian tissue from sheep under the renal capsule of immunodeficient virgin female SCID mice, whereas only 7% was damaged by the cryopreservation and thawing procedures (15). To reduce follicular loss during revascularization, it is necessary to find ways of speeding up the revascularization process. Dissen et al. showed that vascular endothelial growth factor (VEGF) is a potentially important endogenous angiogenic stimulus for subsequent revascularization of autotransplanted ovaries in young rats (16). Vascular corrosion casting followed by scanning electron microscopy revealed that the transplanted ovary became profusely revascularized within 48 hr after transplantation. Vascular in-growth was accompanied by a 40–60-fold increase in expression of the genes encoding VEGF and transforming growth factor-beta 1 (TGF-(β1)), and it seemed that FSH secretion contributed to this upregulation (16). For IVF, metaphase-II oocytes have to be obtained from the autotransplanted ovarian tissue. To optimize this procedure, several questions need to be addressed, including when and how to stimulate the growing follicles, and when to harvest oocytes. The reason we did not harvest the small follicles observed in the forearm was our expectation of further growth beyond the 12 mm. However, Lee et al. in their experiment with macaques, aspirated 4-mm follicles achieving six metaphase-II oocytes from 15 follicles in ovarian tissue transplanted to the abdominal wall (10). Another issue is at what site to place the frozen-thawed ovarian tissue at autotransplantation. There are several principles to pay attention to: revascularization should occur rapidly, the transplantation should be possible with minimal surgery, and the graft should be easily accessible to examination and follicle aspiration. Whether revascularization is site-dependent is unknown. A risk of transplant exposure to suboptimal temperatures or to mechanical stress would depend on transplantation site. Tissue transplanted under the skin of the forearm, as in the present case, probably will be exposed to both higher and lower temperatures than ovaries in their normal location. Thus, the possible thermal injury the oocytes are exposed to, might exceed the freezing and thawing procedures. Finally, convenience and aesthetics should not be forgotten. Thus, transplantation to the side of the throat, as was recently suggested (17), seems less attractive. Lee et al. achieved the largest numbers of metaphase-II oocytes from transplants on the abdomen (10), a site Oktay et al. also used for the first successful fertilization of an oocyte from cryopreserved human ovarian tissue (9). In conclusion, autotransplantation of cryopreserved ovarian tissue to the forearm is feasible. Further research is needed to elucidate how to increase the number of surviving follicles, prolong tissue survival, and achieve intact, mature oocytes to be fertilized.