Hum. Reprod. Advance Access originally published online on May 9, 2006
Human Reproduction 2006 21(8):2006-2009; doi:10.1093/humrep/del140
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Biochemical pregnancy after fertilization of an oocyte aspirated from a heterotopic autotransplant of cryopreserved ovarian tissue: Case Report
1 The Fertility Clinic 2 Laboratory of Reproductive Biology and 3 The Department of Gynecology, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark
4 To whom correspondence should be addressed at: The Fertility Clinic, Copenhagen University Hospital, Rigshospitalet, Blegdamsvej 9, 2100 Copenhagen, Denmark. E-mail: mikkel.rosendahl{at}rh.dk
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Abstract |
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Autotransplantation of frozen/thawed ovarian tissue in women undergoing cancer therapy has so far led to the birth of two healthy babies. In both cases, it can be discussed whether the fertilized oocyte originated from the transplant or from the native ovary. We now present a biochemical pregnancy achieved after heterotopical autotransplantation of cryopreserved ovarian cortical tissue and hence the unquestionable proof that pregnancy can occur after transplantation of cryopreserved ovarian tissue. A woman diagnosed with Hodgkin’s lymphoma had ovarian tissue cryopreserved at the age of 28, before receiving chemotherapy and radiation therapy that rendered her amenorrhoeic. After complete remission, she had autotransplantation of ovarian tissue to the remaining ovary, to the right pelvic wall and to a midline subperitoneal pocket on the lower abdominal wall. The transplanted tissue resumed hormone secretion and follicles developed in all three locations. Three times during 8 months, when follicles could not be visualized in other locations, oocytes were aspirated from the subperitoneal autotransplanted tissue on the lower abdominal wall. Twice, an oocyte was retrieved, fertilized by intracytoplasmatic sperm injection (ICSI) and transferred to the woman’s uterus. One of the treatments resulted in a positive pregnancy test 14 days after transfer. Clinical pregnancy, however, was not achieved. In conclusion, heterotopic autotransplantation of cryopreserved ovarian tissue can sustain follicle development. The oocytes of aspirated mature follicles are capable of fertilization after ICSI, and the resulting embryo is competent of producing hCG at detectable levels.
Key words: biochemical pregnancy/cancer/cryopreservation/heterotopic autotransplantation/ovarian tissue
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Introduction |
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In female patients, premature ovarian failure (POF) is a potential long-term side effect following treatment of a malignant disease with chemotherapy and/or irradiation (McVie, 1999
). The consequences of POF are loss of primordial follicles, decreased or ceased sex hormone secretion and infertility (Meirow and Nugent, 2001).
One option for preservation of fertility in these patients is cryopreservation of ovarian tissue prior to cancer treatment (Meirow et al., 1999; Radford et al., 2001; Radford, 2003; Oktay and Buyuk, 2004).
In our clinic, we have frozen ovarian biopsies or entire ovaries from a total of 176 women, and in cooperation with Aarhus University Hospital, Skejby, Denmark, five autotransplantations of ovarian tissue have been made (Schmidt et al., 2005). At present, two children have been born worldwide as a result of autotransplantation – one after spontaneous ovulation and intercourse (Donnez et al., 2004) and the other after IVF (Meirow et al., 2005). The two pregnancies resulted from tissue orthotopically transplanted to the remaining ovary or to a peritoneal window just beneath the ovarian hilus. We now present data from a patient who achieved a biochemical pregnancy after oocyte retrieval from a subperitoneal, heterotopic transplant located in the midline below the lower abdominal wall.
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Case report |
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A 28-year-old woman was diagnosed with Hodgkin’s disease, clinical stage IIB. She had the entire right ovary removed prior to receiving gonadotoxic chemotherapy with alkylating agents, radiation therapy towards the neck, supra- and infraclavicular regions, both axillaries and the mediastinum and autologous stem cell transplantation (see Schmidt et al., 2005
; patient 9 for details). The cortex of the retrieved ovary was dissected into 29 pieces (5 x 5 mm) and cryopreserved according to the method previously described (Tryde Schmidt et al., 2004). During treatment, the patient became amenorrhoeic and developed vasomotor symptoms.
Almost 2 years after onset of treatment, the patient requested autotransplantation of the cryopreserved tissue. Complete remission had been obtained and her haematologist approved autotransplantation. At this time, vaginal sonography revealed a small remaining ovary with a volume of 1 cm3 without antral follicles. High levels of follicle-stimulating hormone (FSH) (62–68 IU/l) and luteinizing hormone (33–42 IU/l) combined with undetectable estradiol levels (<0.04 nmol/l) confirmed ovarian failure. By a combined laparoscopy/mini-laparotomy, twelve pieces of frozen/thawed ovarian cortex were transplanted to three locations: (i) in the remaining ovary, two subcortical pockets were made and two pieces of cortex were placed in each pocket which were closed with 4–0 prolene sutures; (ii) on the right pelvic wall, adjacent to the site of the removed ovary, a peritoneal pocket was made and four pieces of cortex were enclosed with staples; (iii) an additional pocket was made in the subperitoneal tissue beneath the abdominal fascia between the umbilicus and the pubic bone. Four pieces were enclosed and the pocket was closed with staples and one vicryl-rapid 3–0 suture.
As previously published, the autotransplanted tissue resumed hormone production, hot flashes disappeared and follicle development was detected in the subperitoneal transplant under the abdominal wall. Twice transabdominal aspirations were unsuccessfully performed (Schmidt et al., 2005). Figure 1 summarizes the time course. Fifteen and a half months after autotransplantation (MAT), a single 17 mm follicle was seen on cycle day 18 in the same location. For the next 2 days, recombinant FSH (rFSH) 150 IE/day (Puregon, Organon AS, Skovlunde, Denmark) and the GnRH antagonist ganirelix (Orgalutran, Organon AS) 0.25 mg/day were given s.c. On cycle day 22, 36 h after 10 000 IE hCG s.c. (Pregnyl, Organon AS), a metaphase-II oocyte was aspirated transabdominally. Due to a persistent low sperm count, intracytoplasmatic sperm injection (ICSI) was performed. Forty-eight hours after ICSI, a 4-cell embryo (<20% fragmentation, unequal blastomere size) was transferred to the patient’s uterus (Figure 2).
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On cycle day 18, serum estradiol was 0.33 nmol/l and progesterone was 1.0 nmol/l. On cycle day 24 – the day of embryo transfer – estradiol had decreased to 0.08 nmol/l, but progesterone had increased to 2.3 nmol/l. From this day, 200 mg of vaginal progesterone (Progestan, Organon AS) and 2 mg of oral estradiol (Oestradiol, Nycomed AS, Roskilde, Denmark) was administered three times daily. Regrettably, no blood sample was obtained in the mid-luteal phase. Two weeks after transfer, serum-hCG was 22 IU/l confirming a biochemical pregnancy. Two days later, however, serum-hCG decreased to 13 IU/l. Hormonal substitution was cancelled, vaginal bleeding occurred and serum-hCG subsequently decreased to <2 IU/l.
The following month (17.5 MAT), sex hormones and a growing hypoechogenic endometrium suggested follicular phase. Follicles were visible neither in the ovary nor in the abdominal wall transplant. However, on the right pelvic wall, a 17 mm follicle-like structure developed, suggesting that the transplant here was now producing hormones and maturing follicles. Aspiration was considered to be perilous due to adjacent and partly overlying intestines, as well as the proximity to the iliac vessels.
One month later (18.5 MAT), a new follicle developed under the abdominal wall. From cycle day 11–15, rFSH and ganirelix were administered in the doses described above; however, on cycle day 15, rFSH 300 IE/day was given. Thirty-six hours after hCG injection, aspiration of a 22 mm follicle yielded a metaphase-II oocyte. ICSI resulted in a 5-cell embryo (<20% fragmentation, unequal blastomere size) (Figure 3) that was transferred on cycle day 20. On the aspiration day, serum progesterone was 2.7 nmol/l and serum estradiol had dropped to 0.30 nmol/l from 0.93 nmol/l 2 days earlier. From the aspiration day, estradiol and progesterone were administered as described above, but estradiol was increased to 4 mg twice daily to support possible poor luteal phase hormone production. At the day of transfer, estradiol and progesterone levels were 0.95 and 69.0 nmol/l, respectively. Seven days after transfer, the endometrium was 6.5 mm and hyperechogenic. Serum estradiol and progesterone levels were 1.43 and 123.0 nmol/l, respectively, indicating adequate hormone availability. Inhibin-B was 52 pg/ml on the aspiration day and declined to <20 pg/ml 7 days after aspiration. Fourteen days after transfer, hCG was <2 IU/l.
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Discussion |
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This study demonstrates three notable points: (i) cryopreserved ovarian tissue possesses the ability to re-establish endogenous hormone production and sustain follicular development at least up to 2 years after being autotransplanted to a heterotopic location; (ii) development of mature follicles containing oocytes, capable of undergoing embryo development and hCG production, is feasible; (iii) a corpus luteum, which develops in a heterotopic location, may not have the optimal conditions to produce adequate levels of luteal phase hormones. So far, two live births have been described in humans after autotransplantation of cryopreserved ovarian tissue. In both cases, fertile women treated for Hodgkin’s lymphoma and non-Hodgkin’s lymphoma, respectively, had ovarian tissue cryopreserved prior to receiving gonadotoxic treatment. Both became amenorrhoeic during treatment and had elevated levels of serum gonadotropins. In the first case, the cryopreserved tissue was orthotopically transplanted to a peritoneal window beneath the ovarian hilus. The tissue resumed ovarian function, and spontaneous ovulation and intercourse resulted in a pregnancy, leading to the birth of a healthy child in the summer of 2004 (Donnez et al., 2004
). In the second case, ovarian tissue was transplanted beneath the ovarian cortex. Aspiration and transfer of a fertilized oocyte resulted in a pregnancy and the birth of a healthy child in the summer of 2005 (Meirow et al., 2005). However, in both cases, the origin of the fertilized oocytes can be questioned. Although unlikely, it cannot be ruled out that the fertilized oocytes originated from the remaining ovarian tissue rather than in the thawed ovarian transplants.
Definite proof that the oocyte does in fact originate from the autotransplanted tissue can only be obtained if the follicle develops in a heterotopic transplant. Oktay et al. (2004) achieved embryo development in an oocyte aspirated from cryopreserved ovarian tissue autotransplanted s.c. to the patient’s lower abdominal wall. Transfer of the embryo, however, failed to result in a pregnancy.
Studies on the pharmacokinetics of exogenous hCG allow us to conclude that the hCG measured in the patients’ serum, 18 days after the administration of hCG for ovulation induction, was in fact endogenous and thus an indication of an early pregnancy (Weissman et al., 1996; Mannaerts et al., 1998). The present pregnancy did not develop clinically but does provide definite proof that even after heterotopic autotransplantation of cryopreserved ovarian tissue, pregnancy is within reach in humans.
The main reason for the early termination of pregnancy could be related to an insufficient corpus luteum function. A heterotopically situated corpus luteum may lack sufficient support to thrive and produce adequate amounts of corpus luteum-specific substances including estradiol and progesterone. Despite administration of 10 000 IU of hCG, and pre-ovulatory serum estradiol levels of 0.33 nmol/l, there was a very low estradiol level on the day of the first transfer (0.08 nmol/l). Therefore, in the case of the second transfer, where pre-ovulatory estradiol levels reached 0.93 nmol/l, exogenous estradiol and progesterone were administered from the day of aspiration. This time, mid-luteal phase hormone measurements combined with sonography of the endometrium indicated appropriate conditions for implantation, which, however, did not occur. Luteal phase support should be carefully considered in these cases and should include estradiol, progesterone and/or hCG injections.
Further challenges include defining the optimal site of autotransplantation. Only once did we observe follicular development on the right pelvic wall. Even with adequate follicular development, oocyte aspiration here is perilous due to adjacent intestines and large iliac vessels. Therefore, in addition to the remaining ovary, we propose the use of the subperitoneal midline location below the abdominal wall. It is easily accessed by abdominal ultrasonography and probably has advantages over a subcutaneous location as proposed by Oktay (Oktay et al., 2004), as it benefits from the rich vascularization of the peritoneum and from a higher and more constant temperature.
The longevity of autotransplanted, cryopreserved ovarian tissue is unknown. In this case, the tissue was autotransplanted almost 2 years ago (as of April 2006) and is still functioning. Others have described ceased ovarian function 9 MAT (Radford et al., 2001). Biological age at the time of cryopreservation, previous chemotherapy and the quantity of autotransplanted primordial follicles probably play a role, but other factors, such as vascularization and innervation of the transplant, may also affect the result.
In conclusion, this study demonstrates that cryopreserved ovarian tissue, autotransplanted to a heterotopic location, can result in the production of mature, fertilizable oocytes capable of initiating pregnancy.
Although a number of questions remain to be answered, the present data support the clinical potential of cryopreservation of ovarian tissue from young women prior to gonadotoxic treatment.
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Donnez J, Dolmans MM, Demylle D, Jadoul P, Pirard C, Squifflet J, Martinez-Madrid B, van Langendonckt A. (2004) Livebirth after orthotopic transplantation of cryopreserved ovarian tissue. Lancet 364:1405–1410.[CrossRef][Web of Science][Medline]
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Submitted on January 18, 2006; resubmitted on March 28, 2006; accepted on March 30, 2006.
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