Ovarian follicles of young patients with Turner's syndrome contain normal oocytes but monosomic 45,X granulosa cells

Ronald Peek, Myra Schleedoorn, Dominique Smeets, Guillaume van de Zande, Freek Groenman, Didi Braat, Janielle van der Velden, Kathrin Fleischer, Ronald Peek, Myra Schleedoorn, Dominique Smeets, Guillaume van de Zande, Freek Groenman, Didi Braat, Janielle van der Velden, Kathrin Fleischer

Abstract

Study question: What is the X chromosomal content of oocytes and granulosa cells of primordial/primary (small) follicles and stromal cells in ovaries of young patients with Turner's syndrome (TS)?

Summary answer: Small ovarian follicles were detected in one-half of the patients studied, and X chromosome analysis revealed that most oocytes were normal, granulosa cells were largely monosomic, while stromal cells showed a high level of mosaicism.

What is known already: Most women with TS experience a premature reduction or complete loss of fertility due to an accelerated loss of gametes. To determine whether fertility preservation in this group of patients is feasible, there is a strong need for information on the X chromosomal content of ovarian follicular and stromal cells.

Study design, size, duration: Small follicles (<50 μm) and stromal cells were isolated from ovarian tissue of young TS patients and analysed for their X chromosomal content. In addition to ovarian cells, several other cell types from the same patients were analysed.

Participants/materials, setting, methods: After unilateral ovariectomy, ovarian cortex tissue was obtained from 10 TS patients (aged 2-18 years) with numerical abnormalities of the X chromosome. Ovarian cortex fragments were prepared and cryopreserved. One fragment from each patient was thawed and enzymatically digested to obtain stromal cells and primordial/primary follicles. Stromal cells, granulosa cells and oocytes were analysed by FISH using an X chromosome-specific probe. Extra-ovarian cells (lymphocytes, buccal cells and urine cells) of the same patients were also analysed by FISH. Ovarian tissue used as control was obtained from individuals undergoing oophorectomy as part of their gender affirming surgery.

Main results and the role of chance: Ovarian follicles were detected in 5 of the 10 patients studied. A method was developed to determine the X chromosomal content of meiosis I arrested oocytes from small follicles. This revealed that 42 of the 46 oocytes (91%) that were analysed had a normal X chromosomal content. Granulosa cells were largely 45,X but showed different levels of X chromosome mosaicism between patients and between follicles of the same patient. Despite the presence of a low percentage (10-45%) of 46,XX ovarian cortex stromal cells, normal macroscopic ovarian morphology was observed. The level of mosaicism in lymphocytes, buccal cells or urine-derived cells was not predictive for mosaicism in ovarian cells.

Limitations, reasons for caution: The results are based on a small number (n = 5) of TS patient samples but provide evidence that the majority of oocytes have a normal X chromosomal content and that follicles from the same patient can differ with respect to the level of mosaicism of their granulosa cells. The functional consequences of these observations require further investigation.

Wider implications of the findings: The results indicate that despite normal ovarian and follicular morphology, stromal cells and granulosa cells of small follicles in patients with TS may display a high level of mosaicism. Furthermore, the level of mosaicism in ovarian cells cannot be predicted from the analysis of extra-ovarian tissue. These findings should be considered by physicians when offering cryopreservation of ovarian tissue as an option for fertility preservation in young TS patients.

Study funding/competing interest(s): Unconditional funding was received from Merck B.V. The Netherlands (Number A16-1395) and the foundation 'Radboud Oncologie Fonds' (Number KUN 00007682). The authors have no conflicts of interest.

Trial registration number: NCT03381300.

Keywords: Turner’s syndrome; fertility preservation; karyotyping; mosaicism; primordial/primary follicles.

© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.

Figures

Figure 1
Figure 1
Mosaicism in extra-ovarian tissues and ovarian stromal cells, granulosa cells and oocytes from patients with Turner syndrome. In patients with Turner’s syndrome (TS), lymphocytes (n = 30), cells from buccal smears (n = 100) and urine (n = 100) were analysed by FISH with an X chromosomal peri-centromeric probe. The percentage of cells with a particular karyotype is indicated. In Patient A three different cell lines (45X/46,XX/47,XXX) were detected, Patients B–E were mosaic for 45,X and 46,XX cell lines, while in Patients F–J only 45,X cells were found. In addition, ovarian cortex components including stromal cells (n = 100), granulosa cells (n ≥ 100) and oocytes were karyotyped. The number of oocytes was 19, 12, 2, 9 and 4 for Patients A, B, C, D and E, respectively. In Patients F–J no follicles were detected. ND = not done; NA = not applicable.
Figure 2
Figure 2
Macroscopic images of ovaries from TS patients. After unilateral ovariectomy intact ovaries were transported to the laboratory and photographed. A representative example of an ovary from a mosaic 45,X/46,XX girl (Patient D) with normal morphology and volume is shown in panel A. The small fibrous streak ovary shown in photo B is from a girl with 45,X monosomy (Patient I). The brown discoloration of the tissue is due to electrocauterization during surgery. Bars represent 1 cm.
Figure 3
Figure 3
Flow scheme for separating ovarian cortex cellular constituents prior to FISH analysis. After the surgical removal of the intact ovary (A), cortical fragments were prepared. Part of one representative cortex fragment (B) was analysed by standard haematoxylin–eosin staining for the presence of follicles. When no follicles were present (C), the remaining part of the fragment was used to make a suspension of stromal cells (D), for interphase FISH with chromosome X (green) and chromosome 18 (red)-specific probes (E). When follicles were present (F), the remaining part of the cortex fragment was used to make a cell suspension (G) from which small follicles were manually picked up. These isolated follicles were subjected to further digestion (J) and subsequently analysed by FISH. The white arrowhead points to the signals from the oocyte (K). Part of the remaining cell suspension (H) was used for FISH analysis of stromal cells (I). Bars represent 1 cm (A and B) or 100 μm (C, D, F–H and J). Original magnification of FISH signals was ×630 (E, I and K).
Figure 4
Figure 4
After treatment of isolated follicles with trypsin the follicular cells become more available for FISH. Cells of individual small follicles isolated from a suspension of ovarian cortex remain clumped during preparation for FISH, obscuring signals of individual granulosa cells and the oocyte (panels A and B). Treatment with trypsin of isolated follicles prior to FISH resulted in less cell clumping and allowed karyotyping of granulosa cells and oocyte of the same follicle (panels C and D). Note that the DAPI counterstain in the trypsin treated follicles reveals that the DNA of the oocyte is much more diffuse and can be easily distinguished from DNA of the granulosa cells. FISH signals for the X chromosome (green) and chromosome 18 (red) of the oocytes are indicated by arrowheads. Original magnification was ×630.
Figure 5
Figure 5
Histological sections of ovarian cortex from patients with TS. Haematoxylin–eosin stained 4-μm sections were prepared from cortical tissue from ovaries of mosaic (panels A and B) and 45,X monosomy TS patients (panel C). In addition to the variable number of small follicles in the tissue of the mosaic patients (panel A), a low number (<2%) of secondary follicles were observed (panel B). The ovarian tissue of the monosomic 45,X patients contained no follicles and showed a fibrous texture with relatively low number of cells (panel C). Bars represent 100 μm.
Figure 6
Figure 6
FISH analysis of granulosa cells from individual follicles. The ratio between 46,XX and 45,X granulosa cells from the same small follicle varied considerably. FISH analysis of six individual small follicles from Patient B revealed that the percentage of 45,X granulosa cells varied from 0% to 59% (top row; four follicles are shown). In Patients D (337 granulosa cells from 10 follicles) and E (152 granulosa cells from 5 follicles) all granulosa cells were 45,X. For Patients B and D at least 25 granulosa cells per follicle were analysed and for Patient E at least 22 granulosa cells per follicle.
Figure 7
Figure 7
The surface of FISH signals can be used to karyotype oocytes from small follicles. To assess whether the surface of FISH signals can be used to determine the X chromosomal content of oocytes in the prophase of meiosis I, we measured the surface of split signals for the X chromosome (panel A), for chromosome 18 (panel B) or for both chromosomes (panel C) and compared these to the signal surface of the other chromosome. The surface ratio is shown in panel D and indicates that the surface of a split FISH signal is approximately 0.5 compared to the non-split signal of the other chromosome in the same oocyte. A 50% reduction in the number of X chromosomal sister chromatids will therefore lead to a reduction in the surface ratio between FISH signals for the X chromosome and chromosome 18.
Figure 8
Figure 8
Most oocytes of small follicles from mosaic TS patients are 46,XX. The ratio between FISH signals from the X chromosome and chromosome 18 was measured for 46 oocytes from 5 mosaic TS patients (pt) and 13 oocytes from 2 controls (ctrl). The ratio in the controls varied between 0.9 and 1.5. In TS Patients B, C and E, a similar distribution was seen, indicating that these oocytes had a normal X chromosomal content. In Patients A and D the majority of the oocytes displayed a normal ratio, while in three oocytes from Patient A and one oocyte from Patient D, the ratio varied between 0.4 and 0.6, which is indicative of a reduction in the number of the X chromatids by half.

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