Human sperm rotates with a conserved direction during free-swimming in 3D.

In human spermatozoa, head rotation is essential for different swimming modes and fertilisation since it links the molecular workings of the flagellar beat with sperm motion in 3D. Determining its direction of rotation has been hindered by the sperm head symmetry and translucent nature, and by the fast three-dimensional (3D) motion driven by the helical flagellum beat. Analysis has been mostly restricted to 2D single focal plane image analysis limited to head centre position tracking, in absence of head rotations in 3D. Despite the conserved helical beating of the human sperm flagellum, its head rotating direction has been reported to be mono or bi-directional, and even intermittently changing direction of rotation. Here, we directly measure the head rotating movement of freely swimming human sperm with multi-plane 3D+t microscopy, and show that: a) 2D microscopy is unable to distinguish the head rotating direction in human spermatozoa; b) head rotating direction in non-capacitating and capacitating solutions, for both aqueous and viscous media is counter-clockwise (CCW), as seen from head-to-tail, in all rotating spermatozoa, regardless of the experimental conditions; c) head rotation was suppressed in 57% of spermatozoa swimming in non-capacitating viscous media, though CCW rotation was recovered after incubation in capacitating conditions within the same viscous medium, possibly unveiling an unexplored aspect of the essential need of capacitation for fertilisation. Our observations show that the CCW rotating direction in human sperm is conserved. It constitutes a robust and persistent helical driving mechanism that influences sperm navigation in 3D, thus of critical importance in cell motility, propulsion of flagellated microorganisms, sperm motility assessments, human reproduction research and self-organization of flagellar beating patterns and swimming in 3D.