Assessment of the effects of cryostorage on pre-antral follicle survival and preservation of intercellular connections

Anniek Bus, DVM, PhD

Date: 20 December 2021 at 5pm

Location: ‘Promotiezaal’ (Building Q, Campus Drie Eiken)


Nowadays, an emerging need for (human) fertility preservation (FP) strategies is present, in particular when young girls and woman are confronted with cancer treatment. In addition, the preservation of genetic material from endangered animal species or animals with important genetic traits will also benefit from the development of alternative FP strategies. The strategies in this thesis focus on the use of pre-antral follicles (PAF) as, for prepubertal girls and women whose cancer treatment cannot be postponed, there are no other options. As PAFs account for the vast majority of follicles in the ovarian cortex, they represent an untapped potential, which could be cultivated for reproduction, preservation or research purposes. Vitrification is the most successful preservation method for gametes and embryos. However, a negative consequence of this strategy is the increased probability of injuries caused by exposure of cells to other non-physiological conditions that may impair their further development. Research performed on effects of vitrification on the viability of isolated PAFs remains scarce and protocols still need to be optimized for each specific cell type and species. Due to their small size (30 µm), the isolation and processing of isolated PAFs remains a huge challenge in terms of follicular retrieval and manipulation. Research presented in this thesis shows that the vitrification of isolated PAFs is a promising FP strategy. This is particularly relevant for cancer patients at moderate-to-high risk of ovarian metastasis. Different vitrification techniques including straws, follicle embedding, cell strainers and Cryotop were evaluated for follicle survival and applicability in clinical practice. We however, found that all evaluated techniques, next to their advantages also had their downsides. Several methods were used to assess follicle quality, ranging from gross morphology, simple viability stains as Neutral red and Calcein, immunochemical methods to more complex xenografting procedures. Our main finding is that isolated PAFs can survive the cryopreservation process and maintain their intercellular connections which are crucial for further development. In vitro development of PAFs to the pre-ovulatory stage has not yet been achieved in humans and larger animals. However, in vitro culture systems for PAFs are under development and are expected to become available in the near future. Animal in vitro models such as the bovine that rely on unlimited sources of research material and which are largely free of ethical or moral restrictions, have an important role in contributing to make faster progress in the development of these FP strategies.