Effects of needle puncturing on re-vascularization and follicle survival in xenotransplanted human ovarian tissue

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  • Hanna Ørnes Olesen
  • Susanne Elisabeth Pors
  • Cristina Subiran Adrados
  • Mette Christa Zeuthen
  • Linn Salto Mamsen
  • Pedersen, Anette Tønnes
  • Stine Gry Kristensen

Background: Ovarian tissue transplantation can restore fertility in young cancer survivors, however the detrimental loss of follicles following transplantation of cryopreserved ovarian tissue is hampering the efficiency of the procedure. This study investigates whether needle puncturing prior to transplantation can enhance revascularization and improve follicle survival in xenotransplanted human ovarian cortex. Methods: Cryopreserved human ovarian cortex pieces (N = 36) from 20 women aged 24–36 years were included. During the thawing process, each piece of tissue was cut in halves; one half serving as the untreated control and the other half was punctured approximately 150–200 times with a 29-gauge needle. The cortex pieces were transplanted subcutaneously to immunodeficient mice for 3, 6 and 10 days (N = 8 patients) and for 4 weeks (N = 12 patients). After 3, 6 and 10 days, revascularization of the ovarian xenografts were assessed using immunohistochemical detection of CD31 and gene expression of angiogenic factors (Vegfα, Angptl4, Ang1, and Ang2), and apoptotic factors (BCL2 and BAX) were performed by qPCR. Follicle density and morphology were evaluated in ovarian xenografts after 4 weeks. Results: A significant increase in the CD31 positive area in human ovarian xenografts was evident from day 3 to 10, but no significant differences were observed between the needle and control group. The gene expression of Vegfα was consistently higher in the needle group compared to control at all three time points, but not statistically significant. The expression of Ang1 and Ang2 increased significantly from day 3 to day 10 in the control group (p < 0.001, p = 0.0023), however, in the needle group this increase was not observed from day 6 to 10 (Ang2 p = 0.027). The BAX/BCL2 ratio was similar in the needle and control groups. After 4-weeks xenografting, follicle density (follicles/mm3, mean ± SEM) was higher in the needle group (5.18 ± 2.24) compared to control (2.36 ± 0.67) (p = 0.208), and a significant lower percentage of necrotic follicles was found in the needle group (19%) compared to control (36%) (p = 0.045). Conclusions: Needle puncturing of human ovarian cortex prior to transplantation had no effect on revascularization of ovarian grafts after 3, 6 and 10 days xenotransplantation. However, needle puncturing did affect angiogenic genes and improved follicle morphology.

TidsskriftReproductive Biology and Endocrinology
StatusUdgivet - 2023

Bibliografisk note

Funding Information:
For the histological processing, especially sectioning of samples, we wish to thank Marianne Sguazzino. Marjo Westerdahl is acknowledged for excellent technical assistance. We thank Oliver Carlsson form Visiopharm for assisting with optimization of the APP for quantification of CD31, and Clara Borelli-Møller and Julie Lee Aastrand from the Department of Technology at the University College Copenhagen for their work with histological staining and analysis, IHC staining and APP analysis. Finally, we are utmost grateful to all the women who donated their cryopreserved ovarian tissue for research and to all involved with the clinical activities in the ovarian tissue cryopreservation program in Denmark.

Funding Information:
We are thankful for the funding from The University Hospital of Copenhagen, Rigshospitalet, The Danish Childhood Cancer Foundation, Doctor Sofus Carl Emil Friis and wife Olga Doris Friis scholarship, Carpenter Jørgen Holm and wife Elisa F. Hansen scholarship, and the Hørslev Foundation.

Publisher Copyright:
© 2023, The Author(s).

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