Thiol-yne click crosslink hyaluronic acid/chitosan hydrogel for three-dimensional in vitro follicle development

There is a great deal of potential for in vitro follicle growth to provide an alternative approach to fertility preservation. This strategy reduces the possibility of cancer cells re-exposure after transplantation, and it does not require hormone stimulation. Adopting a three-dimensional (3D) culture method helps preserve the architecture of the follicle and promotes the maturity of oocytes. In order to maintain follicle morphology, enhance the quality of mature oocytes, and facilitate meiotic spindle assembly, the current work aimed to develop the 3D in vitro preantral mouse follicle culture method. Thiolated chitosan-co-thiolated hyaluronic (CSHS) hydrogel was designed to evaluate the effects of biomaterials on ovarian follicle development. Isolated follicles from mouse ovaries were randomly divided into alginate (Alg) as a 3D control, thiolated hyaluronic acid (HASH), and CSHS groups. Single follicle was encapsulated in each hydrogel, and performed for 10 days and subsequently ovulated to retrieve mature oocytes on day 11. CSHS hydrogel promoted follicle survival and oocyte viability with maintained spherical morphology of follicle. Matured oocytes with normal appearance of meiotic spindle and chromosome alignment were higher in the CSHS group compared with those in the Alg and HASH groups. Furthermore, CSHS increased expression level of folliculogenesis genes (TGFβ-1, GDF-9) and endocrine-related genes (LHCGR, and FSHR). With various experimental setups and clinical applications, this platform could be applied as an alternative method to in vitro follicle culture with different experimental designs and clinical applications in the long-term period.