The development of fertility preservation protocols for oncology patients requires the isolation of follicles from ovarian tissue for in vitro maturation. Ovarian mechanical disaggregation using needles is time-consuming compared to disaggregation using enzymes such as collagenase IV (Col-IV), or FDA-approved purified collagenase ‘Liberase’[1]. Ovarian disaggregation requires optimisation to maximise follicle yield whilst minimising damage. Follicle damage can be evaluated in a DAPI-stained follicle grading system[1] that defines M1 follicles as having viable normal morphology, and M4 as non-viable abnormal morphology. We aimed to optimise follicle harvest and test a newly available animal origin free (AOF) collagenase IV, which has the potential for TGA approval.
The ovaries from 3 month mice (n=7) were halved, weighed, and disaggregated using Col-IV (590U/mL), or AOF590U/mL or AOF1180U/mL. Control half-ovaries were disaggregated mechanically without enzyme. Isolated follicles were stained with DAPI and CMXRos, and images were captured with an Olympus Brightfield BX50 microscope & Micro-Manager software. Follicular diameters and staining were measured using Image J. Follicle yields analysed by 1-way ANOVA, and follicle quality grades by 2-way ANOVA with Bonferroni post-test.
The ovarian weights were 6.6±2.3mg. Most of the follicles were secondary (65%)>antral (29%) >primary (6%). Follicle yields were similar for all disaggregation methods; control 13±7 follicles/ mg ovarian tissue, Col-IV 17±10, AOF590u/mL 15±11, AOF1180u/mL 13±3. For each mouse, the highest number of M1(4.6±2.6) and M2(4.7±1.6) follicles were obtained after Col-IV disaggregation, and the lowest M1(2.4±2.5) and M2(2.7±1.9) after mechanical disaggregation. AOF590u/mL yields were M1(3.7±2.2) , M2(3.3±2.5), M3(3.7±2.5) and M4(3±1.7) follicles/mg.
Previously yields were 30-40 follicles per immature mouse ovary[2] whereas our yields from more fibrous adult ovaries were higher, ~90 follicles/ovary. Our method ‘selected’ for secondary follicles, and did not yield a population representative of the original tissue. Col-IV disaggregation yielded higher proportions of high quality M1&M2 follicles than the AOF preparation.