The study of sperm head vacuoles using deep learning algorithm and its correlation with protamine mRNA ratio
Abstract BackgroundAs regards the routine semen analysis is not sufficient to assess male fertility status, is it necessary to use other morphological sperm examination that may be more relevant in regard to the promotion of assisted reproduction outcomes? This study was designed for examination of sperm vacuole characteristics, its association with other sperm parameters and protamine 1 to protamine 2 ratio, and predict assisted pregnancy outcomes. Methods 98 Semen samples from subfertile men were classified based on Vanderzwalmen's criteria as follows: grade I, no vacuoles; grade II, ≤ 2small vacuoles; grade III, ≥ 1 large vacuole; grade IV, large vacuole with other abnormalities. The location, frequency and size of vacuoles were assessed using high magnification, a deep learning algorithm, and scanning electron microscope methods. The chromatin integrity (toluidine blue staining), condensation status (aniline blue), viability and acrosome integrity (triple staining), and protamination status (CMA3 staining) was evaluated for vacuolated samples. Protamine 1 and protamine 2 gene expression was analyzed by quantitative real-time PCR. The assisted reproduction outcomes were also followed for each cycle. Results The results show a significant correlation between the vacuole size (III and IV) and abnormal sperm chromatin condensation (p<0.05), and protamine-deficient (p<0.05). The percentage of reacted acrosomes was significantly higher in spermatozoa with grades III and IV compared with normal group (p<0.05). A high protamine mRNA ratio ( prm-2 was underexpressed) was observed in the vacuolated spermatozoa with grade IV (p<0.01). The sperm head vacuole was negatively associated with the fertilization rate (p<0.01) under IVF cycles. This association was also significantly observed in pregnancy and live birth rate in the groups with grade III and IV (P<0.05). Conclusions The results of our study highlight the importance of follow up of more sperm parameters such as sperm head vacuole characteristics, because may reflect protamine-deficient and poor IVF/ICSI outcomes.