Abstract
The first live birth to occur after ovarian-tissue transplantation between two genetically different sisters was reported in 2011. Since this is an acceptable practice with monozygotic twins, there is no apparent reason to refrain from using it with genetically different sisters, especially if one of the sisters previously received bone marrow from the other, leading to complete chimerism (HLA compatibility) between donor and recipient, thus obviating the need for immunosuppressive treatment. This approach allows for natural conception, which could be important on moral, ethical or religious grounds.
Introduction
Studies have shown that more than two-thirds of former pediatric patients who had received allogeneic hematopoietic stem cell transplantation (SCT) showed signs of impaired fertility [1]. Moreover, a large retrospective survey of pregnancy outcomes after SCT (peripheral blood or bone marrow) involving 37,362 patients revealed that only 0.6% of patients conceived after autologous or allogenic stem cell transplantation [1–4]. Total body irradiation (TBI) required before bone marrow transplantation (BMT), associated with chemotherapy, constitutes the treatment combination presenting the greatest risk of premature ovarian insufficiency (POI). For these patients at risk of fertility impairment and POI, several fertility preservation strategies could be offered before starting cancer treatment such as: oocyte and embryo cryopreservation, ovarian tissue cryopreservation or a combination of oocyte and ovarian tissue cryopreservation depending on the context of each patient (sperm availability, sexual maturity and urgency of treatment). Ovarian tissue cryopreservation is the only option for prepubertal patients or those in need of immediate cancer treatment.
For some patients, however, the cryopreservation of ovarian tissue was not possible, as in the majority of cases; the technique was not available at the time of treatment. This is still the case in many countries. When none of these options was implemented or available at the time of treatment, ovarian tissue allotransplantation from the previous bone marrow donor now represents a possibility. This chapter focuses on fertility restoration by means of ovarian tissue allotransplantation.
Allotransplantation of Ovarian Tissue
Between Monozygotic Twins
The first successful ovarian allograft between monozygotic twins resulting in pregnancy was published by Silber et al. in 2005 [5]. This technique was further validated after reports of a series of ovarian allografts between monozygotic twins subsequently published by the same authors. In Silber’s series, a total of 11 healthy babies were born from 9 fresh tissue transplants [6–8].
Ovarian allografting between monozygotic twin sisters affected by Turner syndrome was also successful in our team (Figure 36.1) [9]. In patients with Turner syndrome, oocyte or embryo crypopreservation as fertility preservation techniques are less likely to work, as POI occurs rapidly and most often before adolescence. Although ovarian cortex cryopreservation can be performed with relatively minimal complications at any age, and particularly during childhood [10], we have no guarantee that there are still primordial follicles present at the time of biopsy because it is difficult to evaluate the ovarian reserve during childhood in girls with Turner syndrome. A large study [11] investigated 57 girls with Turner syndrome aged from 8 years of age. The authors concluded that spontaneous puberty, mosaicism, and normal follicle stimulation hormone (FSH) and/or anti-Müllerian hormone (AMH) levels are positive and statistically significant signs but not exclusive prognostic factors for finding follicles in case of Turner syndrome. Like our group [10], they recommend ovarian cortex cryopreservation in girls with mosaic Turner syndrome, girls with spontaneous onset of puberty (45, X or mosaic), and those with normal serum FSH and/or AMH levels, with or without spontaneous onset of puberty. In the case described in 2011, only egg donation could be an alternative. The patient underwent two attempts of egg donation in another country but failed to become pregnant. This case also highlighted the difficulty of predicting ovarian function in girls with Turner syndrome. Although our patients were monozygotic twins and had approximately the same percentage of monosomy X cells, their ovarian function was completely different. Indeed, one presented with POI before the age of 15 and the other still had normal function, delivered twice and had a good follicular reserve at the age of 37. Twins are at higher risk of POI and can be highly discordant for menopausal age [12].
Figure 36.1 The donor sister underwent laparoscopy (A-B), and the recipient sister minilaparotomy (C-D). The procedures were carried out as shown in the figures. Ovaries of the donor had a normal appearance (A). A biopsy was taken from one of the ovaries by removing the cortex and hemostasia was achieved by suturing the edges (B). The ovaries of the recipient consisted of only a white fibrotic area without any recognizable ovarian tissue (streak gonad syndrome) (C), which was removed to allow access to the ovarian hilus vessels. When the area was ready to receive donor ovarian cortex, the edges of the donor cortical fragments were immediately sutured to the ‘‘ovarian’’ area of the recipient (D). Contact between the donor cortex and recipient ovarian hilus was optimal (D)
Discordant ovaries in monozygotic twins may develop from variations in the plane of splitting. Specifically, if early postimplantation embryos split unequally during the critical period when progenitor germ cells (or extraembryonic ectoderm cells that provide morphogens for germ cell multiplication) form in the epiblast, a subnormal follicle store might be formed in one of the twins [13]. According to Lebl et al. [14], it may also result from an unequal distribution of distinct cell lines with different tissues. Furthermore, stress from in utero competition between twins might encourage epigenetic abnormalities during a crucial period of germ cell development when the epigenome is being programmed [15, 16]. In the case reported, being a monozygotic twin might have accelerated her POI leading to discordancy in ovarian function. Reports of monozygotic twins with discordant phenotypes are uncommon.
Ovarian cortex allografting was considered in the patient with Turner syndrome as she had an identical twin sister with persistent ovarian function. The presence of only atrophic ovarian tissue (streak ovaries) backs the assumption that the source of the oocytes that resulted in two pregnancies and two live births was the transplanted tissue from the unaffected twin. In this specific case, follicular density in the donor ovarian cortex was high and recovery of ovarian endocrine function in the recipient was achieved 3 months after transplantation [9] (Figure 36.2).
Figure 36.2 Restoration of endocrine function in a patient allografted with ovarian tissue from her monozygotic twin with Turner syndrome and discordant ovarian function.
In Silber et al. [6, 7] and Donnez et al. [17], was reported that the time to first menses after transplantation of fresh tissue is slightly sooner than after grafting of frozen–thawed tissue (approximately 3 vs. 4–5 months). This interval corresponds to the development of primordial follicles to the antral follicle stage, but it is also possible that one or two growing follicles, having survived the reimplantation procedure and subsequent ischemic period (estimated to be between 3 and 5 days) [18], could reach the pre-ovulatory stage earlier than would be expected of primordial follicles [9].
Between Genetically Different Sisters
Our group reported the first three cases in the world of orthotopic allotransplantation of fresh ovarian tissue between two genetically non-identical sisters, for whom immunosuppression was not required [19]. One of these cases, with this approach, resulted in the first pregnancy and live-birth worldwide [20].
I. Patient 1
This patient was partially reported previously as the first allograft of human ovarian tissue [21]. In 1990, a 20-year-old woman presenting with b-thalassemia major underwent chemotherapy (busulfan 16 mg/kg and cyclophosphamide 200 mg/kg) and TBI (750 cGy) before BMT, with the donor being her 17-year-old HLA-compatible sister. The patient was successfully treated but, at the time, no procedure was available to preserve her fertility. She became amenorrheic shortly after initiation of chemotherapy and radiotherapy. Six weeks later, concentrations of FSH were 108 mIU/ml, luteinizing hormone (LH) 54 mIU/ml and estradiol 10 pg/ml. This ovarian failure profile was confirmed one month later and hormone replacement therapy (HRT) was initiated. The bone marrow donor was 32 years of age at the time of ovarian tissue implantation to her menopausal 35-year-old non-identical sister.
II. Patient 2
In 1988, a 12-year-old girl presenting with acute myelogenous leukemia underwent chemotherapy (busulfan 16 mg/kg and cyclophosphamide 200 mg/kg) and TBI (750 cGy) before BMT, with the donor being her 14-year-old HLA-compatible sister. Thereafter, she quickly (after 2 months) experienced POI, evidenced by FSH 50 mIU/ml and estradiol 20 pg/ml.
In subsequent years, the patient was prescribed HRT and underwent two attempts at oocyte donation, but no pregnancy occurred after transfer.
The bone marrow donor, at 34 years of age with two children, wished to donate an ovary to her now 32-year-old menopausal non-identical sister.
III. Patient 3
In 1992, a 15-year-old girl presenting with homozygous sickle cell anemia underwent chemotherapy (busulfan 16 mg/kg and cyclophosphamide 200 mg/kg) and TBI (750 cGy) before BMT, with the donor being her 19-year-old HLA-compatible sister. Shortly (2 months) after initiation of chemo- and radiotherapy, the patient showed a typical ovarian failure profile, as confirmed by an FSH level 100 mlU/ml and estradiol 20 pg/ml. HRT was therefore initiated. The bone marrow donor was 36 years of age at the time of ovarian allografting and had two children, whereas her menopausal non-identical sister was 32 years of age.
Chimerism and HLA Compatibility
Quantitative chimerism analysis was used to reflect the proportion of recipient and donor genotypes. It is based on the identification of genetic markers characteristic of a given transplant pair. In our study, this genetic fingerprinting was done by PCR amplification of short tandem repeats [22, 23]. HLA group analysis revealed complete chimerism (HLA compatibility) between the sisters in each group, proving that no immunosuppressive treatment would be necessary, even though they were genetically non-identical.
Surgery
Twenty days before surgery, recipient patients were given gonadotropin-releasing hormone (GnRH) agonist and estroprogestative therapy for a period of two months to decrease endogenous FSH levels, as previously suggested in cases of reimplantation of cryopreserved ovarian tissue. The operating rooms of the gynecology department are two communicating surgical suites (Figure 36.3) (ORI, Storz, Tuttlingen, Germany). The surgical procedure is illustrated in Figure 36.4 [19]:
Figure 36.4 Simultaneous laparoscopic surgeries in the donor (A-B) and recipient (patient 1) (C-D). Right donor ovary (A). A large biopsy (8–6 mm) was taken from the right ovary, respecting the medulla and its vascularization. Note the absence of bleeding (B). Left ovary measuring 15–10 mm, decortication was carried out removing approximately two-thirds of the cortex, exposing the grafting site (C).
Both atrophic ovaries of the recipient (measuring 1.5 × 1 cm) were decorticated by laparoscopy (n=1) or minilaparotomy (n=2). Since the ovaries were atrophic, approximately two-third of the cortical tissue was removed in order to accommodate the graft, and sent for histological analysis. When the ovaries of the recipients were ready to receive donor ovarian cortex, for Patient 1, one large biopsy measuring 10 × 6 mm was taken from the right donor ovary and immediately sutured laparoscopically to the recipient ovary. A second biopsy measuring 6 × 4 mm was taken from the left ovary and also immediately sutured as before. This laparoscopic technique has been previously described [21].
From the donor sisters of Patients 2 and 3, a very large biopsy (2 × 2 cm;) was taken from their left ovary and divided into two parts measuring 2 × 1 cm. Each part was immediately sutured to the decorticated recipient ovary.
The fragments were sutured to the recipient ovarian medulla as soon as they were recovered. No medium or ice was used. The time interval between cortex removal and the start of suturing was 1 minute, and both sutures were achieved within 30 minutes of the fragment being excised. The edges of the cortical fragments were sutured to the decorticated edges, so that contact between the donor cortex and receiver medulla was optimal. The minilaparotomy technique used was similar to that recommended by our group [24, 25] and Silber [5, 26]. A small biopsy (4 × 1 mm) was also taken from the donor ovary for histological analysis in order to evaluate the ovarian reserve.
Results
Restoration of Endocrine Function
I. Patient 1
Six months after reimplantation, a first increase in estradiol (65 pg/ml) was observed, concomitant with follicular development (11 mm) (Figure 36.5A). Seven months after reimplantation, a second estradiol peak was observed, reaching almost 100 pg/ml, which was followed by menstrual bleeding. Cycle length was between 30 and 48 days [19].
