Polymerase chain reaction enzyme-linked immunosorbent assay detection of mycoplasma consensus gene in sperm with low oocyte penetration capacity

Objective: To determine the prevalence of mycoplasmas in washed sperm and to compare the penetration of zona-free hamster oocytes by sperm with and without mycoplasmas. Design: Prospective comparative study. Setting: Clinical and academic research environment. Patient(s): Semen from 34 male patients. Intervention(s): Specimens were divided, Percoll washed, and scanned for differences in kinematic parameters. Sperm DNA was extracted and assayed for mycoplasma DNA using the polymerase chain reaction-ELISA method targeting the consensus gene of 15 mycoplasma species. Remaining sperm were processed by centrifuge, Percoll, or TEST (TES and Tris) Yolk Buffer (TYB) and assessed for penetration capacity. Main Outcome Measure(s): Detection of mycoplasma DNA. Result(s): Mycoplasma DNA was detected in 29.4% of the Percoll-washed sperm. The penetration of oocytes by mycoplasma-positive sperm (59.5% +/- 17.3%; mean +/- SEM) was less than mycoplasma-negative sperm (86.8% +/- 5.4%) in the TYB-processed group. Conclusion(s): Mycoplasma DNA is demonstrated in almost a third of the Percoll-washed sperm. Because there were no other cell types except sperm, the results suggest that the mycoplasmas were either internalized or attached to the membranes. The reduced penetration by mycoplasma-positive sperm after 48-hour TYB suggest mycoplasmas required time to affect sperm function. Similarities between hypo-osmotic swelling and between kinematic parameters suggest that the mechanism does not involve differences in membrane integrity and in motility patterns.