Oocyte insemination with the Walking Egg simplified IVF culture system – an investigation into reduced sperm numbers, sperm DNA fragmentation and reactive oxygen species formation

RESEARCH QUESTION: What is the lowest number of sperm that can be used for oocyte insemination during either conventional or the Walking Egg simplified IVF? Does the use of low numbers of sperm in high volume (1 ml) culture media have an effect on sperm DNA fragmentation and reactive oxygen species formation? Also, does the extended co-incubation of embryos with sperm and cumulus cells in the Walking Egg culture tubes induce higher levels of reactive oxygen species? DESIGN: Binding of sperm to the zona pellucida was compared using a modified hemi-zona assay. In the first part of the study, the binding capacity of decreasing concentrations of motile spermatozoa was evaluated, followed by a comparison of sperm binding after simulated insemination by conventional or the Walking Egg simplified culture protocol. Sperm DNA fragmentation was determined between test and control samples in the second part of the study and reactive oxygen species was measured in spent culture media. As a supplementary examination, reactive oxygen species formation, with the simulated co-incubation of cumulus and sperm cells, was compared between the conventional and Walking Egg IVF culture systems. RESULTS: Sperm-zona binding in 50 μl culture media, indicated mean sperm binding of more than 20 sperm per hemi-zona with as low as 1000 sperm used for insemination. Using a higher volume of culture media, as is done in the Walking Egg simplified IVF culture system, resulted in 42.8% reduced sperm-zona binding. No significant difference in DNA integrity was observed between the two test groups. The amount of ROS generated during conventional IVF in the first 18 hours of incubation was more than that produced in the simplified culture system over sixty-six hours. Only during extended culture for 114 hours in the simplified culture system, did the ROS generated slightly surpass that of conventional IVF at 18 hours. CONCLUSION: Oocyte insemination with as little as 2 x 10(3) motile sperm showed sufficient sperm-zona binding capacity to be indicative of fertilization potential, supporting the Walking Egg simplified IVF insemination protocol. No difference in DNA fragmentation was observed between conventional and the simplified IVF culture systems, while reactive oxygen species formation was indicated to be at a slower rate during incubation with the Walking Egg simplified IVF culture system than with conventional IVF.