Clinical sperm selection prizes the fastest sperm as “best”, presuming them most fertilisation-competent, yet this rests on motility as a proxy, with no molecular definition of superiority. Whether motile sperm differ qualitatively or quantitatively in molecular identity is unknown, leaving selection grounded in inference, not mechanism. Here, we combined motility grade-based microfluidic selection with flagellar waveform/energetics analysis and quantitative proteomics, to define the molecular identity underlying each grade. The device sorted raw semen into rapid progressive (Grade A; VSL ≥ 25 µm/s), slow progressive (Grade B), and non-progressive (Grade C; VSL < 5 µm/s) populations. High-speed dark-field analysis quantified flagellar beat frequency, amplitude, wave-speed, and dissipated power; LC-MS/MS characterised each subpopulation's proteome.
Proteomes separated cleanly by grade (PC1 = 53.7%), revealing motility-grade-specific expression profiles. Distinct profiles were identified per subgroup, with seven proteins specific to Grade A, 16 to Grade B, and eight to Grade C, over a shared core of ~1,640; protein number rose as motility declined, from ~1,800 (Grade A) to ~3,500 (Grade C). Grade A was enriched for cholesterol-efflux/capacitation-priming proteins, RHO-GTPase/cytoskeletal signalling, and antioxidant defence, with the highest protein tyrosine phosphorylation, capacitation's biochemical hallmark; Grades B and C instead accumulated oxidative-phosphorylation, proteasomal, and quality-control machinery. Grade A thus carries a lean, capacitation-primed proteome, an active gain, not passive retention, while declining motility reflects pathological accumulation, not alternative competence. As expected, this was mirrored biophysically: velocity and flagellar beating declined progressively from Grade A to C, alongside reduced DNA fragmentation and increased normal morphology versus raw semen.
This study defines the molecular profiles of sperm across motility grades, including distinct protein expression patterns and predominant functionalities of Grade A sperm linked to fertilisation success, establishing the molecular profile of the most fertilisation-competent sperm.