Quantification of Ovarian Granulosa Cells and Different Level Follicles in the Mouse Ovary Using Stereology Method, for Potential Application in 3D Bioengineered Ovary.

Background: Stereology is an accurate method to obtain 3D quantitative information of microstructure based on the comprehensive observation of two-dimensional sections. Three-dimensional bioprinting of bioink to rebuild a bioengineered ovary has been proved to be a promising technique in preserving fertility. Fully understand the structure of the ovary, the distribution of the main cells and the numerical density of cells using stereology method is of great significance to the computer 3D reconstruction technology. Results: The Cavalieri method gave the estimations that the ovary was (2.49 ± 0.32)mm 3 , the volume fraction of cortex, medulla, follicles and ovarian granulosa cells ( OGCs ) were 86.80%±2.82%, 13.20%±2.82%, 5.60%±0.25% and 81.19%±2.57%, respectively. From exact counts on serial sections, OGCs, primordial, preantral and antral follicles numerical density were estimated at ( 2.11 ± 0.28 ) × 10 6 /mm 3 , 719.57 ± 18.04/mm 3 , 71.84 ± 3.93/mm 3 and 17.29 ± 3.54/mm 3 , respectively. We estimated that there would be (2.11 ± 0.28) × 10 9 OGCs, (719.57 ± 18.04) × 10 3 primordial follicles, (71.84 ± 3.93) × 10 3 preantral follicles, (17.29 ± 3.54) × 10 3 antral follicles in one milliliter 3D bioink when 3D encapsulated mice ovarian constructs. The total number of the three stages follicles per ovary were 1791.74 ± 5.77, 178.88 ± 1.26, 43.04 ± 1.13, and the respective total number of OGCs and follicles per ovary were (5.26 ± 0.09) × 10 6 , 2013.66 ± 8.16. Conclusions: OGCs and follicles can be accurately measured using either the optical or physical disector. The application of these approaches for the study of 3D bioengineered ovary may contribute to a deeper understanding of the artificial ovary.