Conservation of fertility and oocyte genetics in a young woman with mosaic Turner syndrome

Authors

  • AH Balen,

    1. Reproductive Medicine Unit, Leeds Teaching Hospitals Trust, Leeds General Infirmary, Leeds, UK
    Search for more papers by this author
  • SE Harris,

    1. Division of Reproduction and Early Development, Leeds Institute of Genetics, Health and Therapeutics, University of Leeds, The Light Laboratories, Leeds, UK
    Search for more papers by this author
  • EL Chambers,

    1. Division of Reproduction and Early Development, Leeds Institute of Genetics, Health and Therapeutics, University of Leeds, The Light Laboratories, Leeds, UK
    Search for more papers by this author
  • HM Picton

    1. Division of Reproduction and Early Development, Leeds Institute of Genetics, Health and Therapeutics, University of Leeds, The Light Laboratories, Leeds, UK
    Search for more papers by this author

Dr A Balen, Reproductive Medicine Unit, Leeds Teaching Hospitals Trust, Clarendon Wing, Leeds General Infirmary, Belmont Grove, Leeds LS2 9NS, UK. Email adam.balen@leedsth.nhs.uk

Case report

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.

Figure 1.

 Image from the cytogenetic analysis of a single metaphase II oocyte which was determined to be karyotypically normal (23,X). The images shown include: (A) bright field image of the morphology of the metaphase II oocyte and first polar body; (B) phase contrast image at 40× magnification of oocyte chromosomes (black oval) and first polar body chromosomes (white oval); (C) 4′,6-diamidino-2-phenylindole-stained image of oocyte chromosomes; (D) composite fluorescent image of oocyte chromosomes; (E) pseudo-coloured image of oocyte chromosomes with the position of the X chromosome marked; (F) karyotype analysis of the oocyte chromosomes; (G) 4′,6-diamidino-2-phenylindole-stained image of the polar body chromosomes; (H) composite fluorescent image of the polar body chromosomes; (I) pseudo-coloured image of the polar body with the position of the X chromosome marked; and (J) karyotype analysis of the polar body chromosomes.

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.

Discussion

Turner syndrome is the most common sex chromosome disorder among women, affecting one in 2000 live-born girls.7 The main characteristics of Turner syndrome include short stature and a failure to enter puberty because of an accelerated rate of atresia of ovarian follicles causing gonadal insufficiency and infertility.8 Approximately 30% of individuals with Turner syndrome have mosaicism, most often a mixture of normal cells and cells with only one X chromosome (45,X/46,XX), and the number of girls and women with low-frequency mosaicism may be greater than that previously believed.9 Usually, the different Turner karyotypes (monosomy, mosaicism and structural deletion) are determined by analysing the frequencies of different cell lines using conventional karyotyping on lymphocytes. Spontaneous puberty may occur and ovulatory cycles may provide the potential for fertility, although premature ovarian failure is likely to occur in early adult life.10 Healthy children have been reported from both cases with monosomy X and mosaics; however, spontaneous abortions, stillbirths and chromosomal defects have been reported to occur frequently.8,11

There is now an extensive literature on ovarian tissue and oocyte cryopreservation for women about to undergo sterilising chemotherapy or radiotherapy.12 Oocyte cryopreservation has also become standard practice for surplus oocytes collected during in vitro fertilisation cycles in some Roman Catholic countries, where the law prohibits the exposure of more than three oocytes to sperm, in order to avoid the development of surplus embryos. There is also debate about the potential for ‘social oocyte cryopreservation’ for women who wish to attempt to preserve fertility whilst they pursue a career.

There have been a few case reports of oocyte cryopreservation for women who have a high likelihood of premature menopause because of an underlying genetic predisposition, such as Turner syndrome with mosaicism. We believe this to be the first case in which a detailed assessment of ovarian and oocyte genetics has been made. In this case, the results have been extremely reassuring, although the trial cycle cannot guarantee a similar outcome in a therapeutic cycle. Furthermore, it is unlikely that all cases of Turner mosaicism will have similar genetic profiles in either their peripheral or ovarian tissue. The survival and fertility rates for cryopreserved oocytes are still low, and certainly not as good as for cryopreserved embryos, and therefore it is still uncertain how many oocytes need to be frozen in order to provide a realistic prospect for future fertility.12 Nonetheless, this case provides some reassurance for the validity of this approach.

Disclosure of interests

None.

Contribution to authorship

AHB wrote the case history. HP wrote the majority of the laboratory methods with the help of SH and EC, both of whom also performed the laboratory work.

Details of ethics approval

Not specifically required as not a clinical study. We have both a clinical license and a research license granted by the Human Fertilisation and Embryology Authority for oocyte freezing. We also received approval from both the Research Ethics Committee and Clinical Ethics Committee of the Leeds Hospitals Trust for oocyte freezing, ovarian tissue cryopreservation and the specific management of this case.

Funding

None.

Acknowledgements

We would like to acknowledge the laboratory staff of the IVF Unit, Leeds General Infirmary for assisting with oocyte retrieval and the initial preparation of oocytes and embryos.

Permission

The patient has given written permission for the publication of her case history.

Editor's Commentary

Turner syndrome (TS) is the most common sex chromosomal abnormality and affects approximately one in 2000 live-born phenotypic females. Only 0.5% of monosomy X conceptions survive to term. The severity of the phenotypic manifestations of TS depends on, among other factors, the proportion of chromosomally compromised cells in each tissue. Chromosomal mosaicism occurs in up to 30% of cases of TS, where follicular development and the potential for fertility can be maintained, although the risk of premature ovarian failure as a result of accelerated follicular atresia remains considerable.

Provoked by the almost universal need for donated oocytes to achieve pregnancy in cases of non-mosaic TS, interest in fertility preservation in cases of mosaic TS has grown, underscoring the importance of the two case reports published in this issue of BJOG (El-Shawarby SA, et al. BJOG 2010;117:234–7; Balen AH, et al. BJOG 2010;117:238–42). Extensive patient counselling is paramount as this fertility option is fraught with difficulties. In addition to those mentioned in the two case reports and the significant cost implications, the biggest concern is the low likelihood of achieving a live birth following the thawing of vitrified oocytes (approximately 1% per collected oocyte). Furthermore, the outcome of pregnancies achieved using cryopreserved oocytes from cases of mosaic TS is currently unknown.

In common with other sex chromosome aneuploidies, the suggestion by El-Shawarby et al. and other researchers that performing preimplantation genetic diagnosis (PGD) for the resulting embryos ‘should be offered to patients with mosaic TS’ remains contentious. The application of PGD to these embryos is expected to lead to a reduction in the number of available embryos for transfer, and hence could make no difference or may even compromise the overall chance of a successful pregnancy. Therefore, it is imperative that consideration is given to the extent to which the germ cells are affected by the chromosomal abnormality, which is generally lower than that of peripheral cells, and to recent evidence suggesting that only chromosomally competent gametes are capable of progressing through the various stages of meiosis (as has been demonstrated in the report of Balen et al.). Furthermore, the false positive result associated with PGD testing, potentially leading to the exclusion of viable embryos and the 99% chance of a monosomy X embryo miscarrying before term, argue against the use of PGD in such cases.

The two case reports emphasise that the management of controlled ovarian stimulation and oocyte cryopreservation in cases of mosaic TS is feasible, but the management and fulfilment of patient expectations afterwards may prove to be more difficult.

Conflict of interest

I have none to declare.

Ancillary