Culture and cumulus expansion was calculated as ratio between final cumulus
Culture and cumulus expansion was calculated as ratio between final cumulus area and initial cumulus area. Cumulus area was measured by ImageJ I-BRD9 biological activity pubmed ID:https://www.ncbi.nlm.nih.gov/pubmed/25112874 1.48v (National Institute of Health, USA; [52]) tools. Oocytes were then mechanically freed from cumulus cells and fixed in 500 l of 60 Methanol in Dulbecco’s Phosphate Buffered Saline for 30 min at 4 . The oocytes were then stained with 0.5 mg/ml of Propidium Iodide to evaluate meiotic stage by observation at 200?00?under fluorescence microscopy [50]. About 30 COCs were analyzed for each group during three different runs.Statistical analysisanimals was not statistically different between groups (Fig. 2). Mice increased in weight during the treatment phase of this experiment but those treated with DHEA gained approximately 10 more than controls (Fig. 2).Hormonal analysis of serumThe effect of low dose administration of rhFSH on the serum hormonal profiles was evaluated using immunoassay techniques. Results are illustrated in Fig. 3. Testosterone and P4 concentration were statistically higher in all the PCOS-induced animals (DHEA treated) compared to CTRL, irrespectively to rhFSH administration (Fig. 3a and b respectively). E2 analysis revealed that while DHEA significantly increased serum E2 concentration compared to CTRL, DHEA+rhFSH treatment was able to restore E2 concentration statistically similar to the CTRL (Fig. 3c). Finally DHEA alone or with rhFSH resulted in a statistical decrease of LH concentration respect to CTRL (Fig. 3d).Morphological evaluation of ovariesStatistical analyses were performed using Prism GraphPad (GraphPad Software, version 6.0f, San Diego, CA, USA). In vivo data were analyzed by one-way ANOVA, followed by Fisher’s Least Significant Difference (LSD) multiple comparison test. Fisher’s exact test was PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26100631 used to compare the percentages of atretic/cystic follicles on the total follicle population. Data of cumulus expansion assay were tested for Gaussian distribution using KolmogorovSmirnov test. Since these data were not normally distributed, Kruskal-Wallis test, followed by Dunn’s multiple comparison test was used to analyze the cumulus expansion data. Regardless of the test P values <0.05 were considered significant.ResultsBody weight increaseTo investigate the effect of different hormonal treatments on the body mass, mice were weighted during experimentation. Before treatment the body mass ofFig. 2 Effect of different hormonal treatments on body weight of mice at day 0 and 21. Data were analyzed by one-way ANOVA, followed by Fisher's LSD multiple comparison test; different letters indicate significant differences between groups (P <0.05)To study the potential role of the oral administration of low doses of rhFSH on follicle development in DHEAtreated animals, the number of follicles 150?00 m in diameter and of follicles >300 m in diameter was monitored in each ovary. DHEA statistically reduced the number of small follicles per ovary independently from rhFSH (Fig. 4a). On the other hand, DHEA in the presence or absence of rhFSH importantly increases the number of larger follicles per ovary, but rhFSH attenuated DHEA’s actions, significantly reducing large follicle population (Fig. 4b). The number of granulosa cells as assessed by the thickness of the theca and granulosa cell layers was decreased by DHEA (P <0.05), irrespective of rhFSH administration, in the large (>300 m) follicles (Fig. 5) but not in 150?00 m follicles (data not shown). Finally, morphological signs.
