We report a case of oocyte cryopreservation and ovarian tissue cryopreservation in a woman with mosaic Turner syndrome in whom, for the first time in the literature, a detailed assessment was made of the genetic composition of the oocytes. The aim was to preserve fertility in a woman considered at high risk of premature ovarian failure.
The patient presented at the age of 12 years with short stature. Her parents had been concerned about her rate of growth compared with her contemporaries. She had no other stigmata of Turner syndrome, but investigations confirmed mosaicism with 45,X/46,XX, with only two of the 30 cells examined being 46,XX and the remainder being 45,X. All other investigations were normal and, in particular, an ultrasound scan revealed a prepubertal uterus with ovaries clearly visualised and of normal volume.
Treatment was commenced with growth hormone under the care of a paediatric endocrinologist. This produced a good response with a final height of 1.55 m. Puberty occurred naturally and menarche was achieved at the age of 15 years and 0 months, followed by a regular menstrual cycle. There was fluctuating thyroid function and, eventually, the girl became hypothyroid and is now on thyroxine replacement.
The patient consulted at the age of 17 years to talk about the possibility of ovarian cryopreservation. At that stage, we were undertaking research into ovarian tissue cryopreservation for women about to undergo chemotherapy.1,2 Therefore, counselling was provided with regard to the risks and limitations of this process.3
The patient continued to have a regular menstrual cycle with normal gonadotrophin concentrations and, therefore, in February 1998, laparoscopy was performed and bilateral ovarian biopsies were taken. Both ovaries appeared to be of normal size and appearance. Multiple samples of ovarian cortical tissue were collected laparoscopically using punch biopsy and the tissue was cryopreserved by slow freezing following equilibration in 1.5 m dimethylsulphoxide and 0.5 m sucrose, according to the protocol described by Newton et al.,1 and stored under liquid nitrogen. A specimen from each ovary was karyotyped. The analysis of cultures from the left ovarian biopsy showed 16 cells with a normal female pattern and 14 cells with only a single X chromosome. From the right ovary, eight cells had a normal female pattern and 22 cells had a single X chromosome. Therefore, these ovarian tissue biopsies showed a significantly higher proportion of cells with a normal female pattern. The patient was reviewed annually and continued to have a fairly regular 28–30-day menstrual cycle with normal serum gonadotrophin concentrations.
At the age of 25 years, the serum follicle-stimulating hormone concentration was 3.3 iu/l with a luteinising hormone level of 1.3 iu/l. A measurement of inhibin B yielded 176 pg/ml. These were all within the normal range. Further discussions were held regarding the possibility of ovarian stimulation and the creation of embryos with donor sperm. Again, extensive counselling was provided and this situation was discussed with the hospital Clinical Ethics Committee. In the end, the patient elected not to go down this route.
At the same time, the oocyte cryopreservation programme was becoming established. At the age of 28 years, the patient’s gonadotrophins were still normal, the anti-mullerian hormone level was 43.8 pmol/l (normal range, 12–75 pmol/l) and the inhibin B level was 106.2 pg/ml (normal range, 5–200 pg/ml).
It was decided that she would first undergo a cycle of ovarian stimulation to assess oocyte quality and genetics. A cycle of stimulation was commenced using recombinant follicle-stimulating hormone 150 units, combined with a gonadotrophin-releasing hormone antagonist started on day 6 of stimulation. After 9 days of stimulation, oocyte retrieval was planned 36 hours after the administration of 10 000 units of human chorionic gonadotrophin. Of the 12 cumulus-enclosed oocytes that were analysed, the morphology of both the cumulus and oocytes appeared normal. The greater majority (92%) of the cumulus complexes were fully expanded and mucified. All of the oocytes and their companion cumulus cells were morphologically normal. Denudation revealed that nine oocytes (75%) had progressed to metaphase II (MII: mature). The remaining oocytes in the cohort were classified as germinal vesicle (immature, n = 1) and metaphase I (MI: immature, n = 2). There was no evidence of any irregularities in egg morphology and there were no excessive cytoplasmic inclusions. The polar bodies of the MII oocytes all appeared normal and were not fragmented.
Cytogenetic analysis was conducted on the 11 MI and MII oocytes. Metaphase chromosome preparations were obtained using the methodology previously detailed by Clyde et al.4,5 Briefly, oocytes were incubated in a hypotonic solution of 1% w/v sodium citrate for 18–20 minutes at 30 °C before transfer to fresh, ice-cold Carnoys fixative. Using a finely pulled glass pipette, oocytes were then immediately dropped onto ice-cold, positively charged slides (VWR UK Ltd, Lutterworth, Leicestershire, UK). Oocyte spreads were air dried at 30 °C and 50% humidity, and the location of the oocyte chromosomes was visualised under phase contrast microscopy and marked with a diamond-tipped slide marker (Zeiss, Welwyn Garden City, Hertfordshire, UK). The chromosome spread was allowed to age at room temperature overnight before storage at 4 °C until karyotype analysis. Accurate chromosome counts were obtained before further analysis by staining in 60 ng/ml 4′,6-diamidino-2-phenylindole in Vectorshield mounting medium (Vectorlabs, Burlingame, CA, USA). Chromosome spreads were then soaked for 30 minutes in 2× sodium chloride/sodium citrate (SSC) solution and washed in a further two changes of 2× SSC. Oocytes were karyotyped using multifluor in situ hybridisation (mFISH) whole chromosome paints (SpectraVysion, Abbott Molecular Inc, Maidenhead, Berkshire, UK) in which chromosomes are labelled with a unique combination of six different fluorophores. The manufacturer’s protocol for mFISH was followed with the following minor modification: prior to pepsin treatment, oocyte spreads were exposed to 1 m sodium thiocyanate at 80 °C for 0, 4 or 8 minutes, depending on chromosome morphology. Fluorescent images were captured and oocyte karyotypes were analysed using Genus software (Applied Imaging International, Genetix Ltd, New Milton, Hampshire, UK). Gamete chromosomal health was further investigated by screening all MI and MII oocytes for sex chromosome aneusomies by interphase FISH for chromosomes 13, 18, 21, X and Y (AneuVysion, Abbott Molecular Inc), according to the manufacturer’s recommendations.
The analysis of 4′,6-diamidino-2-phenylindole-stained chromosomes from MI and MII oocytes indicated that all 11 oocytes contained 23 chromosomes. Informative FISH results were obtained from ten oocytes. No alteration in chromosome X number was detected in any of the oocytes analysed. Full karyotypes were obtained from five MII oocytes, all of which were normal. Overall, these data indicate that the cohort of eggs analysed were genetically normal. Representative images of a karyotypically normal (23,X) MII oocyte and its first polar body are shown in Figure 1.
With the reassurance of normal oocytes, a second cycle of controlled ovarian stimulation was undertaken with a view to collecting oocytes for freezing. A similar protocol with the same dose of ovarian stimulation was performed, as detailed above. Following stimulation, ten oocytes were collected by transvaginal, ultrasound-guided, oocyte pick-up, seven of which were at MII. Mature MII oocytes were cryopreserved by slow freezing in 1.5 m 1,2-propanediol and 0.3 m sucrose (OocyteFreeze, Medicult, Redhill, Surrey, UK), according to the manufacturer’s recommendations, which are based on the methods published by Fabbri et al.6 At the time of writing, the cryopreserved oocytes remain stored under liquid nitrogen.
The patient is now in a stable relationship and has elected to have embryo cryopreservation. Using a similar protocol, a total of 20 oocytes was collected. Of these, 13 fertilised and 11, day 2, four-cell embryos with high-grade morphology have been cryopreserved. She is now using no contraception and awaiting natural conception.