Evidence against the role of toll-like receptors 7 and 8 in sex selection in mice, cattle, and humans.

Recent studies suggested that treating sperm with R848, a ligand for the X-linked Toll-like receptors 7 and 8 (TLR7/8) in mice, goats, and cattle, could selectively reduce the motility of X chromosome bearing sperm (X-sperm). This reduction enables the separation of X- and Y-sperm and thereby sex selection. However, through three species and multiple methods, our results challenged prior published data. We demonstrated that TLR7/8 were equally expressed in the X- and Y-sperm via western blotting (bovine), immunofluorescence staining (mouse, bovine, and human), flow cytometry (mouse, bovine) and in vitro embryo production (mouse). Approximately 90% of murine, bovine, and human sperm exhibited positive staining for TLR7/8. Meanwhile, sperm treated with R848 and swim-up did not differ in the X:Y ratio as shown by TaqMan real-time PCR. Taken together, our findings question the restriction of TLR7/8 to the X-sperm and the value of R848 treatment as a viable method for sex selection.