Hum. Reprod. Advance Access originally published online on December 2, 2007
Human Reproduction 2008 23(2):336-339; doi:10.1093/humrep/dem307
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Cryopreservation of ovarian tissue and in vitro matured oocytes in a female with mosaic Turner syndrome: Case Report
1 Department of Obstetrics and Gynecology, McGill University Health Center, McGill University, Montreal, Quebec, Canada H3A 1A1 2 Department of Pediatrics, McGill University Health Center, McGill University, Montreal, Quebec, Canada H3A 1A1
3 Correspondence address. Women’s Pavilion F3.46, Royal Victoria Hospital, 687 Pine Avenue West, Montreal, Quebec, Canada H3A 1A1. Fax: +1-514-843-1662; E-mail: ri-cheng.chian{at}muhc.mcgill.ca
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Abstract |
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We report a novel approach of fertility preservation in a young woman with mosaic Turner syndrome. A 16-year-old female with 20% 45XO and 80% 46XX karyotype underwent laparoscopic ovarian wedge resection. Before performing ovarian tissue cryopreservation, all visible follicles on the ovarian surface were aspirated. We recovered 11 immature germinal vesicle stage oocytes, which were subjected to in vitro maturation (IVM). Eight oocytes that matured (73% maturation rate) were cryopreserved by vitrification. The combination of ovarian tissue cryobanking and immature oocyte collection from the tissue followed by IVM and vitrification of matured oocytes represent a promising approach of fertility preservation for young women with mosaic Turner syndrome.
Key words: Turner syndrome/fertility preservation/in vitro maturation/vitrification/oocyte
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Introduction |
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TopAbstractIntroductionDiscussionFundingAcknowledgementsReferences |
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Accelerated loss of ovarian primordial follicles from the 18th week of fetal life, resulting in gonadal dysgenesis, characterizes classical Turner syndrome. However, in women with mosaic Turner syndrome, follicular development can persist beyond puberty, leading to a spontaneous pubertal development, regular menses and even pregnancy before the onset of premature menopause (Hovatta, 1999
).
The possibility of fertility preservation in young women with gonadal dysgenesis was raised by Abir et al. (2001). Hreinsson et al. (2002) reported ovarian cryopreservation in adolescent girls with Turner syndrome. Examining ovarian tissue histopathologically, the authors noted that follicles exist in most of the ovaries in these young women. However, cryopreservation of ovarian tissue would only preserve primordial and primary follicles. In addition, pre-ovulatory antral follicles, which contain immature oocytes at the germinal vesicle (GV) stage, do not usually survive the cryopreservation procedure of ovarian tissue (Gosden, 2000). This is due to high water content in these antral follicles.
We report a novel approach of fertility preservation in a young woman with mosaic Turner syndrome by ovarian tissue cryobanking combined with immature oocyte collection from the tissue followed by in vitro maturation (IVM) and vitrification of mature oocytes.
Case report
A 16-year-old patient was recently diagnosed with mosaic Turner syndrome and referred to the McGill Reproductive Center for fertility preservation. Her past medical history indicated that menarche occurred spontaneously at the age of 11 years but her menses were irregular. At the age of 13 years, she presented with a 2-week history of severe menorrhagia. Following exclusion of bleeding disorders, thyroid dysfunction and pregnancy, her pediatrician diagnosed anovulatory bleeding and the patient was started on combined oral contraceptives (OCPs). At the age of 16 years, she stopped taking OCP, and she was found to be amenorrheic for 6 months. Subsequently, the patient underwent karyotypic evaluation of peripheral blood lymphocytes and was found to have 20% 45XO and 80% 46XX chromosomes.
On the examination, the patient measured 150 cm in height and weighed 50 kg. The breast development and pubic hair were both Tanner stage 5. Aside from her short stature and cubitus valgus deformity, the patient did not exhibit other features of Turner syndrome.
Basal serum FSH, LH, estradiol and thyroid-stimulating hormone levels were 9.8 IU/l, 1.4 IU/l, 119 pmol/l and 1.30 mIU/l, respectively. Abdominal ultrasonography revealed the presence of bifid renal calyces but otherwise normal kidneys.
The patient, accompanied by her parents, was counseled about the benefits, risks and experimental nature of the available options for fertility preservation and consented to undergo laparoscopic ovarian wedge resection. The surgery was performed on day 21 of her menstrual cycle. Intra-operative findings included a 2 cm functional cyst in the right ovary and a corpus luteum cyst in the left ovary. The uterus, fallopian tubes and peritoneum were normal in appearance and size. Laparoscopic ovarian wedge resection of the left ovary was performed. Approximately 2.5 x 2 x 1 cm of the ovary was excised, suspended in cold (4°C) Leibovitz L-15 medium (Gibco Invitrogen, Carlsbad, CA, USA) and transferred to the clinical embryology laboratory (Fig. 1).
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All visible antral follicles on the surface of the ovarian tissue were punctured and aspirated with a 20-gauge syringe needle that was attached to a 10 ml syringe. The aspirates were flushed in Oocyte Washing Medium (CooperSurgical/SAGE, Turnbull, CT, USA) and searched for immature, GV stage oocytes under the dissecting microscope. Following follicle aspiration, the ovarian cortex was isolated from the underlying stromal tissue in the Oocyte Washing Medium, cut into 4 mm3 (2 x 2 x 1) pieces and searched for the presence of additional oocytes.
A total of 11 immature GV stage oocytes were recovered and subjected to IVM as previously described (Chian et al., 1999, 2004). Briefly, the immature oocytes (Fig. 2a) were cultured in 1.0 ml of IVM-Medium (Cooper Surgical/SAGE, Turnbull, CT, USA) supplemented with a final concentration of 75 mIU/ml of FSH and LH at 37°C in an atmosphere of 5% CO2 in air with high humidity. After 24 h of culture, six oocytes matured to metaphase-II (MII) stage (Fig. 2b). The remaining five GV oocytes were cultured for an additional 24 h. Two additional oocytes matured to MII stage after 48 h of culture, yielding an overall oocyte maturation rate of 73% (8/11) following IVM culture.
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Eight in vitro matured oocytes were cryopreserved using the Vitrification kit (Medicult, Jyllinge, Denmark) as per instruction (Huang et al., 2007
). For the vitrification procedure, the oocytes were suspended in equilibration medium containing 7.5% (v/v) ethylene glycol (EG) + 7.5% (v/v) 1,2-propanediol (PROH) for 5 min at room temperature and then transferred to the vitrification medium containing 15% (v/v) EG + 15% (v/v) PROH + 0.5 M sucrose at room temperature for 45–60 s. Oocytes were loaded on the McGill Cryoleaf and then plunged immediately into liquid nitrogen (LN2) for storage.
Cryopreservation of ovarian tissue was based on a previously published method (Gosden, 2002). Pieces of ovarian cortical tissues were transferred into 2.0 ml cryovials containing 1.0 ml of 1.5 M dimethyl sulfoxide, 0.1 M sucrose and 10% (v/v) human serum albumin in Leibovitz L-15 medium at 4°C. The ovarian tissue was equilibrated for 30 min at 4°C. Subsequently, the cryovials were frozen using a programmable freezer (Planer Kryo 360, Planer PLC, Middlesex, UK) with a cooling rate of 2°C/min from 4 to –9°C. At –9°C, seeding was manually performed with a pair of forceps that was pre-cooled in LN2. Cooling resumed at a rate of 0.3°C/min from –9 to –40°C and then 10°C/min from –40 to –140°C before plunging the cryovials into LN2.
A 1 x 0.5 x 0.5 cm piece of ovarian tissue was submitted to histopathological evaluation using haematoxylin and eosin staining which showed mild fibrosis, primordial and primary follicles, one secondary follicle and one luteinized follicle (Fig. 3).
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Discussion |
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This case demonstrates that in mosaic Turner syndrome, immature oocytes can be retrieved from the excised ovarian tissue, matured by IVM and cryopreserved by vitrification. The combination of ovarian tissue cryobanking with immature oocyte collection from the tissue followed by IVM and vitrification of oocytes represents a novel strategy of fertility preservation for pubertal girls with mosaic Turner syndrome.
Normal menstruation can occur in some girls with mosaic Turner syndrome (Hovatta, 1999). Many of these young women can have normal sonographic appearance of the ovaries and normal ovarian functions based on plasma FSH and LH and GnRH stimulation test (Abir et al., 2001). In this group of Turner patients, gonadotropin stimulation followed by transvaginal oocyte retrieval and oocyte cryopreservation is another possible and less invasive option of fertility preservation. Oocyte cryopreservation by vitrification is a promising new technique and appears to be more effective than the conventional slow-freezing method (Oktay et al., 2006). Vitrification results in high survival rates of 89–100% and more than 100 successful live births worldwide including 20 from our institution (Huang et al., 2006). However, our patient elected to undergo laparoscopic ovarian wedge resection and cryopreservation of ovarian tissues. The patient refused to undergo any vaginal examination or procedure and was concerned about the possibility of developing side effects, such as ovarian hyperstimulation syndrome, and the cost of medications.
The goal of ovarian cryobanking is to cryopreserve the primordial follicles in the ovarian cortex before follicular atresia is completed (Gosden, 2002). In order to salvage an optimal number of primordial follicles, it has been suggested that the ideal age to perform ovarian tissue cryopreservation is 12–13 years old in cases of non-mosaic Turner syndrome (Hreinsson et al., 2002). Although a large population of primordial follicles can be preserved by cryobanking ovarian cortical tissue, the efficacy of the current ovarian cryobanking techniques remains to be improved. Transplantation of thawed ovarian tissues was shown to hasten the follicular atresia due to ischemic damage to the ovarian vasculature (Revel and Schenker, 2004). In fact, only two live births have been reported following transplantation of thawed ovarian tissue to date (Donnez et al., 2004; Meirow et al., 2005). Moreover, transplantation of cryopreserved ovarian tissue has not been reported in patients with Turner syndrome. Therefore, further studies are required to investigate the long-term survival and functions of the transplanted ovarian tissues following freezing and thawing before this procedure can be offered as a standard fertility preservation strategy to young patients with Turner syndrome. Another possible strategy of fertility preservation is in vitro culture of the primordial follicles isolated from the ovarian cortex (Abir et al., 2001). However, whether accelerated follicular atresia normally found in the ovaries of females with Turner syndrome occurs in vitro remains unknown.
IVM has become an effective treatment option for many infertile women, including those with polycystic ovaries or polycystic ovary syndrome, high antral follicle count, and for those at risk of developing ovarian hyperstimulation syndrome (Chian et al., 1999) To date, over 500 healthy infants have been born following IVM (Chian et al., 2004). In the patient reported here, a maturation rate of 73% was achieved following IVM, suggesting that a high percentage of GV stage oocytes can mature in patients with mosaic Turner syndrome. However, the relatively high number of pre-antral follicles and GV stage oocytes identified may be related to the low percentage of 45XO karyotype (20%). In patients with a higher percentage of 45XO karyotype, it is possible that the ovaries would contain fewer follicles.
One important concern relates to the chromosome status of oocytes retrieved from young women with mosaic Turner syndrome, namely an increased incidence of meiotic non-disjunctions (Hreinsson et al., 2002). Although spontaneous pregnancy has been reported in 5% of women with Turner syndrome, a high risk of miscarriage, stillbirth and congenital malformations has been reported (Hovatta, 1999). In addition, their offspring have a higher incidence of chromosomal abnormalities (Abir et al., 2001). Therefore, preimplantation genetic diagnosis of the resulting embryos should be offered to patients with mosaic Turner syndrome (Ao et al., 2006).
In conclusion, retrieval of immature oocytes from excised ovarian tissue followed by IVM and oocyte vitrification can be offered as an adjunct to ovarian tissue cryobanking, providing an additional possibility of fertility preservation in pubertal girls with mosaic Turner syndrome.
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This study was supported by a grant from Ton Yen General Hospital, Taiwan.
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We would like to thank Dr Moy Fong Chen from the Department of Pathology at the McGill University Health Center for providing us with histopathology images of the ovarian tissue.
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References |
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Submitted on April 10, 2007; resubmitted on July 29, 2007; accepted on August 29, 2007.
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