O-034 Xenotransplantation of ovarian cortex tissue from young girls with Turner Syndrome in a mice model: is normal follicular development possible?
Authors: Nadesapillai S , Peek R , Nguyen T , Vassart S , Smeets D , Van de Zande G , Braat D , Van der Velden J , Fleischer K , Dolmans M M 
Published: June 2022

Abstract

Study question
Does aneuploidy of granulosa and stromal cells affect folliculogenesis of small follicles in girls with mosaic Turner Syndrome (TS)?

Summary answer
Our results showed that despite high level aneuploidy in granulosa cells of small follicles and stromal cells folliculogenesis was not affected in mosaic TS patients.

What is known already
In 2018 the TurnerFertility study was initiated to explore if ovarian tissue cryopreservation (OTC) is a viable option for TS patients to preserve their fertility at an early age. In this study, karyotyping of ovarian cells in 7 mosaic TS patients showed that the majority of oocytes had a normal X chromosomal content, while granulosa and stromal cells were aneuploid. Until now, it remains unclear if follicular development is affected, and consequently the success rate of autotransplantation after OTC in girls with TS.

Study design, size, duration
A collaboration project was initiated between Radboudumc, The Netherlands and UCLouvain, Belgium to assess folliculogenesis of small follicles in TS patients by using a xenograft mice model. Ovarian cortex tissue was obtained from 18 mosaic TS patients and 13 age-matched controls.

Participants/materials, setting, methods
After unilateral ovariectomy, one fragment of the ovarian tissue was used for research. One part of this fragment was used for fluorescence in situ hybridization (FISH) to determine the X chromosomal content of ovarian cells in non-grafted tissue. The other part was xenografted into severe combined immunodeficient mice. After 5 months, grafts were retrieved and analysed for aneuploidy by FISH. The expression of 6 proteins essential for folliculogenesis was analysed by immunohistochemistry and immunofluorescence.

Main results and the role of chance
The mean follicle density in ovarian tissue of TS patients before grafting was significantly lower compared to controls. FISH analysis showed that 97% of the oocytes in non-grafted tissue had a normal X chromosomal content. Follicles of TS patients contained mainly or exclusively 45,X granulosa cells, but different levels of X chromosome mosaicism between TS patients and between follicles of the same patient were observed.
In total, 12/18 grafts contained follicles after 5 months xenografting. Follicle density of both TS patients and controls decreased significantly after xenotransplantation. Despite the presence of high level aneuploidy in granulosa and stromal cells in the tissue before grafting, secondary and even antral follicles were observed after xenotransplantation. Remarkably, a shift from high to low percentage of 45,X granulosa cells was observed during folliculogenesis. Immunohistochemistry showed that proliferating cell nuclear antigen (PCNA) positive follicles from TS patients increased during grafting to almost 100%. Secretion of anti-Müllerian hormone by granulosa cells was impaired before grafting in peri/postpubertal TS girls, but recovered after grafting. Expression of c-kit receptor and bone morphogenetic protein 15 (BMP15) in peri/postpubertal TS patients remained abnormal after xenotransplantation, while secretion of growth differentiation factor 9 (GDF9) and kit ligand was similar to controls.

Limitations, reasons for caution
In this study, only ovarian tissue of mosaic TS patients was examined, because the chances of finding ovarian follicles in this subgroup is considerably higher than in other TS patients. Subtle effects of reduced expression of c-kit receptor and BMP15 on folliculogenesis might have gone unnoticed.

Wider implications of the findings
Small follicles of mosaic TS patients are able to grow to secondary and antral follicles, despite the presence of aneuploid granulosa and stromal cells before grafting. Therefore, OTC could be a realistic option for young mosaic TS patients to preserve their fertility provided that sufficient numbers of follicles are present.

Trial registration number
NCT03381300