Exploration of Optimizing Density Gradient Centrifugation in Semen Processing to Improve Fertilization Outcomes / 中山大学学报(医学科学版)

ABSTRACT

ObjectiveThis study aimed to improve the existing semen processing methods in the field of reproductive male medicine， particularly focusing on the 300 ×g 20 min treatment condition in the double-layer density gradient method， to enhance fertilization outcome. MethodsSemen specimens from 1 623 patients undergoing assisted reproductive techniques at the Reproductive Medicine Center of the Sixth Affiliated Hospital of Sun Yat-sen University from July and September 2020 and March and May 2022 were collected for preliminary experiments. Four different double-layer density gradient methods （200 ×g 10 min， 200 ×g 20 min， 300 ×g 10 min， and 300 ×g 20 min） were compared for sperm DNA fragmentation rates and recovery rates after processing. Subsequently， the optimal method was selected as the new approach and compared with the current method in use （300 ×g 20 min double-layer gradient method） to assess any statistical differences in fertilization rates. Further optimization to a single-layer density gradient method was performed based on the new method and compared with the double-layer density gradient method to determine any statistical differences. Experimental conditions were strictly controlled for temperature， centrifugation speed， and duration， with the quantity and processing conditions of each sample recorded. ResultsAmong the four double-layer density gradient methods， the sperm DNA fragmentation rate was lower with the 300 ×g 10 min treatment compared to 300 ×g 20 min while ensuring sufficient sperm recovery rates. Consequently， the 300 ×g 10 min method was selected as the new approach for experimentation. Results indicated that the total fertilization rate and 2 pronuclei （2PN） fertilization rate with the new 300 ×g 10 min method were higher than with the 300 ×g 20 min method， the difference was statistically significant （P < 0.05）. Although the cleavage rate with 300 ×g 10 min was slightly higher than 300 ×g 20 min， the difference was not statistically significant （P > 0.05）. The total fertilization rate and 2PN fertilization rate were slightly higher with the single-layer density gradient method compared to the double-layer density gradient method， but the difference was not statistically significant （P > 0.05）. The cleavage rate with the single-layer density gradient method was higher than the double-layer density gradient method， and the blastocyst formation rate is lower than that of the double-layer density gradient method， and the differences are statistically significant （P < 0.05）. ConclusionThe 300 ×g 10 min double-layer density gradient method successfully improved total fertilization rates， 2PN fertilization rates， and cleavage rates compared to the existing 300 ×g 20 min method， while reducing the time required for semen optimization processing. Although the single-layer density gradient method improves the cleavage rate， and saves reagent costs and operation time， its blastocyst formation rate has decreased. These findings provide valuable guidance and insights for semen processing methods in the field of reproductive andrology.