Specimen procurement

Human semen specimens were obtained from the Shanghai Ninth Peoples’ Hospital, Shanghai Jiao Tong University School of Medicine. Enrollment occurred between January, 2018, and June, 2022. Use of the semen samples was approved by the Ethics Committee of these units, and all experiments were performed in accordance with relevant guidelines and regulations. All semen specimens, both normozoospermia and asthenozoospermia, were collected from participants (20–35 years old) who gave written informed consent for the use of their leftover semen samples, and then were analyzed using computer-assisted semen analyzer (CASA, Hamilton-Thorn Research, Beverly, MA, USA). Notably, individuals having a history of long-term medication, varicocele, and infection as indicated by a large number of leukocytes in the semen were excluded from the study. The semen specimens were selected according to WHO guidelines [29]. The specimens with the sperm density higher than 1.5 × 10⁷ sperm/mL, the normal morphology higher than 4%, and the total motility higher than 40% were defined as normozoospermia, whereas the specimens with the normal morphology lower than 4% associated with the total motility lower than 40% were defined as astheno-teratozoospermia. Sperm samples were purified by Percoll gradient centrifugation and the spermatozoa pellet was washed with PBS. The collected specimens were stored at − 80 °C immediately for further use.