Two successful pregnancies following autotransplantation of frozen/thawed ovarian tissue

Claus Yding Andersen 2 Mikkel Rosendahl 1 2 Anne Grete Byskov 2 Anne Loft 1 Christian Ottosen 0 Margit Dueholm 4 Kirsten L.T. Schmidt 1 2 Anders Nyboe Andersen 1 Erik Ernst 3 4 0 Gynaecological Department at The Juliane Marie Centre for Women , Children and Reproduction , University Hospital of Copenhagen , Copenhagen , Denmark 1 The Fertility Clinic, University Hospital of Copenhagen , Copenhagen , Denmark 2 Laboratory of Reproductive Biology, Section 5712, University Hospital of Copenhagen , Blegdamsvej 9, Rigshospitalet, DK-2100 Copenhagen , Denmark 3 Reproductive Laboratory, Institute of Anatomy, University of Aarhus , Aarhus , Denmark 4 Department of Gynaecology and Obstetrics, Aarhus University Hospital , Aarhus , Denmark BACKGROUND: Cryopreservation of the ovarian cortex with subsequent autotransplantation has, on an experimental basis, been performed to preserve fertility in women being treated for a malignant disease. The present study reports ovarian activity and pregnancies following autotransplantation of frozen/thawed ovarian tissue. METHODS: One complete ovary was cryopreserved from each of six patients who were 26 - 35 years old prior to treatment. Tissue from three of the patients was transported 4 - 5 h on ice prior to cryopreservation. After a period of 17 - 32 months, orthotopic autotransplantation was performed replacing 20 - 60% of the tissue. Two patients received additional heterotopic transplants. RESULTS: In all cases, the tissue restored menstrual cyclicity 14- 20 weeks following transplantation. Four of the six women conceived following assisted reproduction: two women (who had the tissue transported 4 - 5 h prior to cryopreservation) each, based on the orthotopic transplanted tissue, delivered one healthy child (February 2007 and January 2008); one woman miscarriaged in gestational Week 7; and the other had a positive hCG test but no clinical pregnancy. The remaining two women did not become pregnant. CONCLUSIONS: Two additional healthy children have been born as a result of the ovarian cryopreservation procedure. In both cases, the ovarian tissue was transported 4 - 5 h prior to freezing demonstrating that hospitals may offer cryopreservation without having the necessary expertise locally. Introduction Girls and women suffering from malignant diseases, such as cancer, that require treatment with irradiation and/or gonadotoxic drugs, may as a consequence lose ovarian function and become infertile. The efficacy of treatment and survival rates for many types of such malignancies have increased over the past decades (McVie, 1999) and as a consequence, more women will face the risk of premature ovarian failure. With prospects of living beyond cancer, salvage of fertility becomes of priority to both patients and physicians (Lee et al., 2006). In addition to cryopreservation of oocytes and embryos, cold storage of the ovarian cortical tissue, which harbours the majority of the ovarian pool of follicles, has recently been developed in an attempt to circumvent the long-term ablative effect on reproductive performance by gonadotoxic treatment. When the patients are cured from their malignancy, the thawed tissue can be transplanted to those with treatment imposed ovarian failure, to restore ovarian function and normalize levels of gonadotrophins (Donnez et al., 2004, 2006; Oktay et al., 2004; Schmidt et al., 2005; Demeestere et al., 2006; Rosendahl et al., 2006). Worldwide, three children have been reported to have been born thus far as a result of autotransplanting frozen/thawed ovarian tissue (Donnez et al., 2004, Meirow et al., 2005; Demeestere et al., 2007). One additional pregnancy has been reported following transplantation of fresh tissue first and subsequently frozen/thawed tissue (Silber et al., 2008). Although the procedure was introduced .10 years ago, experience with autotransplantation of frozen/thawed tissue is still very scarce and only a few cases have been reported, reflecting its experimental nature (Donnez et al., 2006, 2008). Further, a considerable amount of time often elapses after cancer treatment before patients feel prepared for autotransplantation and are considered cured and fit by their physicians. In addition, many hospitals lack the expertise to perform ovarian cryopreservation. The present paper documents that autotransplantation of frozen/thawed ovarian tissue in combination with assisted reproduction may prove a valid method of fertility preservation. Materials and Methods Cryopreservation of ovarian tissue In Denmark, recovery of ovarian tissue for cryopreservation and autotransplantation of frozen/thawed tissue takes place in three centres, whereas the actual cryopreservation procedure itself is centralized to just one laboratory. The procedure has been approved by the Minister of Health and by the ethical committee of Copenhagen and Frederiksberg (J/KF/01/170/99). All patients had one entire ovary removed laparoscopically, except patient E who had her left ovary removed in 1992 due to a benign cyst and therefore only had one third of the remaining ovary removed for cryopreservation. The cortex was isolated, cut into fragments of 5 5 (1 2) mm and cryopreserved as previously described (Schmidt et al., 2003b). Out of 252 women having ovarian tissue cryopreserved, a total of six women have so far requested autotransplantation. The number of fragments cryopreserved from each of these patients A, B, C, D, E and F was 34, 29, 20, 20, 13 and 19, respectively. In addition, one small biopsy of the cortex was taken for histological evaluation. Transportation of ovarian prior to cryopreservation Patients D, E and F had the ovarian tissue transported on ice 4 5 h prior to cryopreservation (Schmidt et al., 2003a). Prior to transportation, the ovary was cut into two halves, rinsed for blood cells in PBS and, using sterile forceps, transferred into a 50 ml sterile tube containing 20 ml ice-cold IVF medium. The tube was placed in an aluminium container with an aluminium-covered lid in order to protect the tissue from damaging effects of the irradiation used to screen packages before airfreight. The package contained enough ice to maintain low temperatures during the transport. Our usual procedure for cryopreservation of ovarian tissue was followed when the tissue arrived at the cryopreservation unit. Autotransplantation The gonadotoxic treatment given to each woman is given in Table I. Following chemotherapy, the remaining ovary showed no activity in any of the patients. All were amenorrhoeic and had experienced typical menopausal symptoms including hot flushes and consistent menopausal levels of FSH (37 135 IU/l at transplantation). All patients requested autotransplantation in order to become pregnant and after individual counselling and approval from the patients oncologist or haematologist, transplantation was performed 17 32 months after cryopreservation (Table II). Patient A had given birth once prior to chemoth (...truncated)