(328) Ovarian Tissue from Girls and Adolescents with Turner Syndrome Contain Oocytes with DDX4 Expression

Margaret A. Brunette¹; Jacqueline C. Yano Maher¹; Natalie Hanby¹; Ramya Balasubramanian¹; Mary Soliman¹; Taylor Badger¹; Hong Lou¹; Maria De La Luz Sierra¹; Raghu Kavarthapu¹; Tazim Dowlut-McElroy²; Veronica Gomez-Lobo¹

1.Division of Pediatric and Adolescent Gynecology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, Maryland, USA
2. Pediatric and Adolescent Gynecology, Department of Surgery, Children's Mercy Hospital, Kansas City, MO, USA.

Abstract Text:

Turner syndrome (TS) is caused by chromosome abnormality that affects 1 in 2500 live female births. The impact of TS on pubertal health and fertility is significant, with most girls losing their ovarian reserve by adolescence. Clinically, it is uncertain whether Ovarian Tissue Cryopreservation (OTC) is a viable procedure to preserve fertility for these patients. Not only is the quantity of follicles lower than average, but the quality of the follicles before cryopreservation and after implantation is generally unknown. Recent work has started investigating TS follicles on a molecular level, but unanswered questions remain; notably, are oocytes from TS patients of similar quality to those from non-TS patients? Ovarian tissue from 21 females with TS was collected via laparoscopic unilateral oophorectomy under The National Institutes of Health IRB approved protocol from October 2021-October 2024. Patients with Y material (n=6) were not included in this study. The remaining patients had a 45,X karyotype (n=6) or mosaic/X deletion karyotype (n=9).  Initial histological analysis of tissue was conducted for all included patients (n=16), and follicles were found in the tissue of 43.8% of patients (n=7). Of the 7 patients with follicles, 57.1% (n=4) had a 45,X karyotype. The mean follicle density (MFD) was calculated for these patients. One outlier patient, with a mosaic karyotype, had a MFD of 16.2 follicles/mm², and 4.5% of follicles were of abnormal morphology. The remaining 6 patients had an average MFD of 0.45 follicles/mm² (ranging from 0.12 – 1.09 follicles/mm²), and an average of 55% of follicles were of abnormal morphology (ranging from 0 - 100%). After the initial histological screening described above, we conducted immune-histochemistry (IHC) on ovarian tissue from 5 TS patients with observed follicles. The first set of tissues were from patients A-C, with 45,X karyotype. The second set was from patients D and E, with mosaic karyotypes. Deceased donor tissues were used as controls. We assessed two markers (1) DDX4, a germ cell marker, and (2) phosphorylated-p63 (p-p63), a marker of DNA damage and oocyte integrity that is implicated in the apoptosis pathway. DDX4 positive staining was observed in 81%, 100%, 87%, and 100% of follicles from patients C, D, E, and controls, respectively. The samples used for patients A and B did not contain follicles for analysis. This staining was found predominantly in the cytoplasm of the oocytes, however there was some nuclear staining as well. Positive p-p63 staining was absent in 100% of oocytes for patients B-E, and controls. The sample used for patient A did not contain follicles. These preliminary results indicate that although there are follicles with abnormal morphology, many oocytes in TS patients express DDX4, as anticipated. DDX4 staining is typically only seen in the cytoplasm of murine oocytes, however DDX4 positive staining in the nucleus has been observed before in human tissues. Furthermore, these results also suggest that the decrease in TS patient follicle number may not be due to ongoing DNA damage. Continuing work will focus on further analysis of various oocyte/follicle parameters to obtain a more comprehensive picture. This information can then be leveraged to help counsel patients whether it is in their best interest to undergo a surgical procedure and perform OTC for fertility preservation. This work was supported by Z1A HD009005 from the Eunice Kennedy Shriver National Institute of Child Health and Human Development.