New hyaluronan-based biomatrix for 3-D follicle culture yields functionally competent oocytes
Authors: Desai Nina , Spangler Maribeth , Nanavaty Vaani , Gishto Arsela , Brown Alyssa 
Published: July 2022

Background
Within the ovary, bidirectional communication between oocytes and surrounding granulosa cells is required for appropriate nuclear and cytoplasmic maturation (1, 10–14). The 3-D architecture of the growing follicle is however difficult to maintain during in vitro maturation using conventional 2-D tissue culture systems. Encapsulation of follicles within a biomatrix is one approach to maintaining 3-D follicle architecture during culture. Hyaluronan is one component of natural extracellular matrices (ECM) that provide support to cells in vivo. This report describes the application of hyaluronan for 3-D in vitro follicle culture and the production of developmentally competent metaphase II oocytes.
Materials and Methods
A novel tyramine-linked hyaluronan (HA) hydrogel was used for follicle encapsulation. Enzymatically isolated mouse preantral follicles or follicle clusters (FL-C) from fresh or vitrified ovaries were encapsulated in 3 mg/ml HA gel for in vitro follicle maturation. Follicle growth, antrum formation and meiotic maturation to metaphase II oocytes was monitored. Chromatin staining was used to assess GV oocyte progression towards meiotic competence. Spindle morphology was evaluated in metaphase II oocytes using immunostaining and polarized light. Functional competence of in vitro matured oocytes was evaluated by in vitro fertilization and ability to develop to blastocyst. Modifying the HA gel by inclusion of laminin (HA-LM), mouse sarcoma extracellular matrix (Matrigel;HA-MG) or placental extracellular matrix (HA-PM) was also tested to see if this might further enhance IVM outcomes.
Results
A total of 402 pre-antral follicles were embedded in HA gel. Antrums were observed in 55% of follicles by day 8. Post-hCG trigger, 314 oocyte-cumulus complexes ovulated from the cultured follicles and 84% (264/314) underwent GVBD. The maturation rate to MII was 72.6%. (228/314). The fertilization rate was 82.5% (188/228). The subsequent blastulation rate with IVM oocytes was 46.3% (87/188). Blastocysts were vitrified for later use. Thawed blastocysts were transferred into three recipient and resulted in the birth of a single pup. HA gel supported individually isolated follicles as well ovarian tissue fragments containing clusters of 6–8 preantral follicles. Meiotic maturation to the metaphase II stage was significantly lower with FL-clusters from vitrified versus fresh ovaries (34% and 55%, respectively; p < 0.007). Modification of the HA gel with ECMs or laminin affected antrum formation and follicle retention within the gel during in vitro culture. Significantly lower antrum formation was noted amongst follicles in HA-LM (29%), HA-MG (18%) and HA-PM (26%) as compared to HA alone, 48%; p = 0.006). The HA-Matrigel encapsulated follicle treatment group had a lower rate of ovulation (37%) as compared to all three of the other treatment groups ( 65–67%; P = 0.001). The rate of maturation to metaphase II oocytes was 74% for HA gel alone as compared to HA-LM (67%), HA-MG (56%) and HA-PM (58%) but these differences were not statistically significant. HA alone proved to be entirely sufficient for supporting the development of mature competent metaphase II oocytes.
Conclusion
Tyramine-linked hyaluronan gel is an effective ECM based biomaterial for 3-D culture of ovarian follicles in clusters or individually. In vitro matured metaphase II oocytes are functionally competent, capable of being fertilized and developing to the blastocyst stage. The versatility, simplicity of use and ability to be used at various rigidities give this new biomatrix numerous advantages, making it particularly attractive for ovarian follicle culture.