3D Tracking Reveals Helical Motion as a Key Strategy for Human Sperm Chemotactic Navigation

BACKGROUND: Understanding human sperm chemotaxis is crucial for advancing assisted reproductive technologies. However, only 2%-12% of human sperm respond to chemical gradients, and traditional two-dimensional tracking cannot capture their real swimming motions in three-dimensional space. OBJECTIVES: To characterize the influence of progesterone gradients on the 3D swimming patterns and kinematics of human sperm during chemotactic swim-up migration. MATERIALS AND METHODS: This study used a dual-chamber chemotaxis assay with progesterone (10 pM) or control medium. Three- dimensional swimming trajectories of spermatozoa from four healthy donors were tracked at 0, 20, and 40 min using digital holographic microscopy (DHM). From approximately 54,000 trajectories, we analyzed spatial distribution, orientation angles, kinematic parameters (VCL, VSL, LIN, ALH, BCF, and RPS), and classified swimming patterns (typical, helical, hyperactivated, and hyperhelical). RESULTS: Progesterone increased swim-up proportion (23.0% ± 4.0% vs. 12.2% ± 5.8% control, p < 0.05). Sperm swimming upward showed lower velocity and linearity than non-swim-up sperm. Continuous helical motion was 4.7-fold more common in progesterone conditions (13.2% vs. 2.8% control) and maintained this pattern more stably during upward migration (38.0% vs. 12.5% control). DISCUSSION AND CONCLUSION: Spermatozoa that maintain continuous helical motion navigate more effectively within chemotactic gradients, facilitating systematic spatial exploration and stable directional alignment with progesterone. These findings reveal sophisticated 3D swimming strategies during chemotaxis, establishing helical motion as key facilitators of gradient navigation and directional persistence, with implications for understanding sperm selection mechanisms and assessing sperm functionality in clinical settings.