AbstractThe ejaculate is a heterogeneous pool of spermatozoa containing only a small physiologically adequate subpopulation for fertilization. As there is no method to isolate this subpopulation, its specific characteristics are unknown. This is one of the main reasons why we lack effective tools to identify male infertility and for the low efficiency of assisted reproductive technologies. The aim of this study was to improve ICSI outcome by sperm selection through thermotaxis. Here we show that a specific subpopulation of mouse and human spermatozoa can be selected in vitro by thermotaxis and that this subpopulation is the one that enters the fallopian tube in mice. Further, we confirm that these selected spermatozoa in mice and humans show a much higher DNA integrity and lower chromatin compaction than unselected sperm, and in mice, they give rise to more and better embryos through intracytoplasmic sperm injection, doubling the number of successful pregnancies. Collectively, our results indicate that a high quality sperm subpopulation is selected in vitro by thermotaxis and that this subpopulation is also selected in vivo within the fallopian tube possibly by thermotaxis.