Autotransplantation of cryopreserved human ovarian tissue

Reported cases of autotransplantation of cryopreserved ovarian tissue, either to an orthotopic or heterotopic site, are summarised on the ISFP (International Society for Fertility Preservation) website ( http://www.isfp-fertility.org ), detailing, in each case, the age of the patient before freezing, whether she received chemotherapy before freezing, the indications for cryopreservation, the graft site and size, the interval before recovery of ovarian function after grafting and the outcome of transplantation.

Orthotopic autotransplantation of cryopreserved human ovarian tissue

In theory, natural pregnancy may be achieved via orthotopic tissue transplantation if the fallopian tubes remain intact.

In 2000, Oktay and Karlikaya reported laparoscopic transplantation of frozen–thawed ovarian tissue in a 29-year-old patient, who had undergone bilateral oophorectomy for intractable menorrhagia. The reason for this bilateral oophorectomy was not made clear and data on restoration of ovarian function were never described for some unknown reason.

Radford et al. reported a patient with a history of Hodgkin’s disease treated by chemotherapy. Ovarian tissue was biopsied and cryopreserved 4 years after chemotherapy. Histological section of the ovarian cortical tissue revealed only a few primordial follicles due to the previous chemotherapy. Eight months after reimplantation, oestradiol was detected and follicle-stimulating hormone (FSH) and luteinising hormone (LH) levels decreased but, 1 month later, FSH and LH concentrations returned definitively to menopausal levels.

We reported the first successful transplantation of cryopreserved ovarian tissue resulting in a pregnancy and live birth. In 1997, a 25-year-old woman presented with clinical stage IV Hodgkin’s lymphoma. Ovarian tissue cryopreservation was undertaken before chemotherapy. After laparoscopy, the patient received MOPP/ABV hybrid chemotherapy (chlorethamine, vincristine, procarbazine, prednisone, doxorubicin, bleomycin and vinblastine) from August 1997 to February 1998, followed by supradiaphragmatic radiotherapy (38 Gy). According to Schilsky et al , the risk of POF after such a regimen in a woman aged 26 years is more than 90%, while according to Wallace et al. and Lobo , the risk of subfertility after Hodgkin’s treatment with alkylating agents is more than 80%. Indeed, not only the type of drug and dose, but also age, are important factors when evaluating the risk of POF after chemotherapy. In 2003, once the patient had been declared completely free of disease, transplantation was carried out (see Donnez et al. for techniques). A large strip and 35 small cubes of frozen–thawed ovarian tissue were implanted into a furrow created by the peritoneal window very close to the ovarian vessels and fimbria on the right side ( Fig. 1 ). Four months after transplantation, a laparoscopy was carried out to check the viability of the orthotopic graft and re-implant the remaining 32 ovarian cortical cubes. From 5 months to 9 months after re-implantation, concentrations of FSH, 17 β-oestradiol and progesterone showed the occurrence of ovulatory cycles. At 11 months, the patient became pregnant and subsequently delivered a healthy baby. This birth was announced in the Lancet in September 2004.

A large strip and 35 small cubes of frozen-thawed ovarian tissue were implanted in a furrow created by the peritoneal window very close to the ovarian vessels and fimbria.

In 2006, restoration of ovarian function after orthotopic (intra-ovarian and para-ovarian) transplantation of cryopreserved ovarian tissue was reported in a woman treated by BMT for a non-cancerous disease (sickle cell anaemia). Vaginal echography and sequential measurement of FSH, LH, 17 β-oestradiol and progesterone concentrations revealed the onset of an ovulatory cycle 4½ months after re-implantation of ovarian tissue.

We applied a technique similar to that used by Silber et al. for the transplantation of fresh ovarian cortex between monozygotic twins in a woman who had also undergone BMT and two regimens of alkylating agents in 2000 for non-Hodgkin’s lymphoma. Cryopreservation of ovarian tissue was carried out 1 year after first-line chemotherapy. One ovary was removed and biopsies of cortical ovarian tissue revealed the presence of histologically normal primordial follicles. Six ovarian cortical pieces measuring 10 × 4–5 mm were then grafted onto the remaining ovary after the cortex of this ovary had been removed ( Fig. 2 ). The goal was to ‘rebuild’ an ovary. It was 5 months before a mature follicle (21 mm) developed and an increase in oestradiol levels (194 pg ml ⁻¹ ) was noted. The patient experienced an ovulatory cycle every 5 weeks, the preovulatory oestradiol level reaching values between 210 and 356 pg ml ⁻¹ .

Ovarian cortical pieces measuring 4–5 mm to 1 cm in size were grafted onto the remaining ovary after the cortex of this ovary had been removed. a: Cortex of the remaining ovary was removed. b: Cortical pieces were sutured with 7-0 stitches.

In total, nine transplantations have been carried out in our department, according to the previously described technique.

Analysis of these cases raises some important points for discussion. First of all, in all cases, it took between 4½ months and 5 months after reimplantation before a follicle could be seen. The process of folliculogenesis takes ∼4–6 months, during which time the oocyte and surrounding somatic cells undergo a series of changes that eventually result in the development of a large antral follicle, capable of producing a mature oocyte. Thus, the appearance of the first follicle originating from the grafted tissue 5 months after re-implantation, proved by laparoscopy in one case, is totally consistent with the expected time course. This time interval between implantation of cortical tissue and the first oestradiol peak is also consistent with data obtained from sheep and humans, although some variations may be observed. Indeed, as discussed in a recent review , the delay between transplantation and follicular development was found to vary from 6 weeks to 8 months. Such a variation could be explained by a difference in follicular reserve at the time of cryopreservation.

Another very interesting finding is the persistence of relatively high FSH levels during the follicular phase. FSH levels remained as high as 25 mIU ml ⁻¹ during the follicular phase until ovulation, and then decreased to less than 15 mIU ml ⁻¹ during the luteal phase ( Fig. 3 ). This may constitute an argument against the use of gonadotropin injections. The relatively high FSH levels may be explained by the relatively low number of surviving primordial follicles in the graft. The patient should be considered a poor responder, with reduced inhibin B secretion. These results are in agreement with those obtained in sheep by Campbell et al.

Mean FSH and 17β-oestradiol levels (±standard deviation) in the 7 cases of frozen-thawed ovarian tissue transplantation. It took between 4 and 6 ½ months after transplantation before a rise in oestradiol and a drop in FSH were observed. (Note that, in the last 4 cases, FSH and oestradiol values at 1 month post-transplantation were not taken into account, as the patients were under GnRH agonist down-regulation).

A further significant observation is the return to an FSH level of >35 mIU ml ⁻¹ immediately after each menstrual bleed, which supports the theory suggested by Baird et al. that some inhibitory mechanisms, such as inhibin B or anti-Müllerian hormone (AMH) normally produced by developing follicles in intact human ovaries, are probably almost non-existent in transplanted tissue. After transplantation, the patient would have been regarded a poor responder because, of the 500–1000 primordial follicles that would have been transplanted, more than 50% would have been lost owing to hypoxia. This raises the question of the evaluation of the ovarian reserve. There is a lack of data on the ovarian reserve in cancer. Qu et al , Gook et al. and Schmidt et al. have all demonstrated an unequal distribution of primordial follicles in ovarian cortex.

In 2005, Meirow et al. also published a live birth after orthotopic autotransplantation of cryopreserved ovarian tissue in a patient with POF after chemotherapy. Eight months after orthotopic transplantation, the patient spontaneously menstruated. The rise in AMH and increased inhibin B levels were consistent with the presence of early growing follicles and ovulation, respectively. Nine months after transplantation, the patient experienced a second spontaneous menstrual period. After a modified natural cycle, a single mature oocyte was retrieved and fertilised. Two days later, a four-cell embryo was transferred. The patient became pregnant from this embryo transfer and delivered a healthy infant weighing 3000 g. The possibility that the oocyte was derived from the native ovary is highly unlikely, given the consistent evidence of POF after high-dose chemotherapy in this patient, from whom ovarian tissue was harvested after administration of a first-line conventional chemotherapy regimen, prior to second-line high-dose chemotherapy.

Demeestere et al. recently reported a pregnancy after natural conception in a woman who had undergone orthotopic and heterotopic transplantation of cryopreserved ovarian tissue. They observed follicular development in all three transplantation sites: large follicles in the ovarian site, only one dominant follicle in the peritoneal site and follicles <13 mm in size in the heterotopic site. Detectable hCG levels and ultrasonography confirmed the presence of a viable intrauterine pregnancy. Unfortunately, this pregnancy, obtained by natural conception, ended in miscarriage at 7 weeks due to aneuploidy. Thereafter, this patient underwent a second transplantation, became pregnant and delivered a healthy baby.

Andersen et al. reported a series of six women (some already described in the paper by Schmidt et al. ). Orthotopic autotransplantation was performed in all patients, two of whom received additional heterotopic transplants. These authors also observed restoration of ovarian function in all women within 20 weeks. Four of the six women conceived following assisted reproduction. One woman miscarried in gestational week 7 and another had a positive hCG test but no clinical pregnancy. Two became pregnant after follicle puncture and ovum pick-up during natural cycles. Each delivered a healthy baby. Both these women had undergone orthotopic transplantation of cryopreserved ovarian tissue to the remaining ovary.