The in vitro Analysis of Quality of Ovarian Follicle Culture Systems Using Time-Lapse Microscopy and Quantitative Real-Time PCR.

BACKGROUND: The aim of ovarian follicle in vitro culture is to obtain mature oocytes. To evaluate the efficiency of in vitro culture system, the status of the cultured oocyte can be analyzed. METHODS: The preantral ovarian follicles retrieved from 14-day-old C57Bl/6J mice were cultured in 3D alginate hydrogel. The status of oocytes obtained from mature (3 months old, group A) and immature (3 weeks old, group B) mice was compared to the status of oocytes retrieved from ovarian follicles cultured in vitro (Group C) using qRT-PCR analysis and time-lapse microscopy. In the qRT-PCR analysis, 8 samples for group A (80 oocytes), 8 samples for group B (80 oocytes), and 6 samples for group C (60 oocytes) were included. Time-lapse analysis was performed in group A (oocytes n=31), group B (n=45), and group C (n=21). Statistical analysis was done by Kruskal-Wallis and chi-square tests and differences were considered statistically significant if p<0,05. RESULTS: The diameter of group C oocytes is lower in comparison to group A oocytes (67 µm vs. 75 µm, correspondingly). Groups B and C oocytes exhibited delayed meiosis in comparison to group A oocytes. Expression levels of six oocyte maturation genes (Ccnb, CDK1, Ccnh, Wee2, Mos and Epab) were evaluated using qRT-PCR analysis. Expression levels of Ccnh and Epab are lowered in group C oocytes compared to the expression levels of these genes in groups A and B oocytes (p< 0.05). CONCLUSION: Oocytes obtained after ovarian follicles in vitro culture have reduced development competence, future fundamental changes of in vitro culture systems can be expected.