Objective:To investigate the effects of different semen sample collection methods on sperm DNA fragmentation index (DFI) test results, then to evaluate the accuracy of the current semen sample collection method in the assessment of male fertility.Methods:In this study, 50 semen sample obtained on the day of oocyte retrieval from patients undergoing in vitro fertilization at the Reproductive Medical Center in Maternity and Child Health Hospital of Guangxi Zhuang Autonomous Region from August 2021 to January 2022 were collected. For each semen, a small amount of samples were collected in three different retention methods for routine semen and sperm DFI testing. Three different ways of retaining samples were as follows: group A, after mixing of the semen, 50 μL sample was directly collected; group B, after density gradient centrifugation, 50 μL sample was collected at the interface between semen and gradient fluid; group C, after density gradient centrifugation, the sperm pellet was upstream, then 50 μL sample was collected from upstream liquid. After semen treatment, routine semen testing and sperm DFI testing were performed. Pearson was used to analyze the correlation between DFI and the percentage of immobile sperm and the percentage of forward sperm movement. Results:The sperm motility rate of group C [(96.83±2.28)%] was significantly higher than that of group A [(57.16±11.28)%, P<0.001] and group B [(22.54±9.35)%, P<0.001], and there was a statistical difference among the three groups. The immotile sperm rate of group B sample was (77.46±9.35)%, which was significantly higher than that of samples from group C [(3.14±2.31)%, P<0.001] and group A [(42.83±11.28)%, P<0.001]. There was also a statistical difference in DFI among the three groups ( P<0.001). The DFI of group B [37.18% (30.41%, 47.80%)] was significantly higher than that of group A [22.00% (14.75%, 29.25%), P<0.001] and group C [0.78% (0.00%, 2.07%), P<0.001]. Pearson analysis results showed that the DFI of group A and group B was positively correlated with the percentage of immobile sperm ( r=0.304, P=0.032; r=0.612, P<0.001), while the DFI of group B was negatively correlated with the percentage of sperm forward movement ( r=-0.517, P<0.001). Conclusion:For the same semen, the DFI of immotile sperm was significantly higher than that of motile sperm. Therefore, due to the interference of immotile sperm, the DFI value by the current sample retention method cannot accurately reflect the DNA status of active sperm participating in fertilization. This suggests that the samples used for DFI testing should be collected from motile sperm collected by gradient centrifugation, upstream or other methods, which can more accurately assess male fertility.

Objective:To investigate the effects of different semen sample collection methods on sperm DNA fragmentation index (DFI) test results, then to evaluate the accuracy of the current semen sample collection method in the assessment of male fertility.Methods:In this study, 50 semen sample obtained on the day of oocyte retrieval from patients undergoing in vitro fertilization at the Reproductive Medical Center in Maternity and Child Health Hospital of Guangxi Zhuang Autonomous Region from August 2021 to January 2022 were collected. For each semen, a small amount of samples were collected in three different retention methods for routine semen and sperm DFI testing. Three different ways of retaining samples were as follows: group A, after mixing of the semen, 50 μL sample was directly collected; group B, after density gradient centrifugation, 50 μL sample was collected at the interface between semen and gradient fluid; group C, after density gradient centrifugation, the sperm pellet was upstream, then 50 μL sample was collected from upstream liquid. After semen treatment, routine semen testing and sperm DFI testing were performed. Pearson was used to analyze the correlation between DFI and the percentage of immobile sperm and the percentage of forward sperm movement. Results:The sperm motility rate of group C [(96.83±2.28)%] was significantly higher than that of group A [(57.16±11.28)%, P<0.001] and group B [(22.54±9.35)%, P<0.001], and there was a statistical difference among the three groups. The immotile sperm rate of group B sample was (77.46±9.35)%, which was significantly higher than that of samples from group C [(3.14±2.31)%, P<0.001] and group A [(42.83±11.28)%, P<0.001]. There was also a statistical difference in DFI among the three groups ( P<0.001). The DFI of group B [37.18% (30.41%, 47.80%)] was significantly higher than that of group A [22.00% (14.75%, 29.25%), P<0.001] and group C [0.78% (0.00%, 2.07%), P<0.001]. Pearson analysis results showed that the DFI of group A and group B was positively correlated with the percentage of immobile sperm ( r=0.304, P=0.032; r=0.612, P<0.001), while the DFI of group B was negatively correlated with the percentage of sperm forward movement ( r=-0.517, P<0.001). Conclusion:For the same semen, the DFI of immotile sperm was significantly higher than that of motile sperm. Therefore, due to the interference of immotile sperm, the DFI value by the current sample retention method cannot accurately reflect the DNA status of active sperm participating in fertilization. This suggests that the samples used for DFI testing should be collected from motile sperm collected by gradient centrifugation, upstream or other methods, which can more accurately assess male fertility.