Abstracts Category : Other

Investigatinga novel in vitro embryo culture system The Walking Egg AffordableAssisted Reproductive Technology

by Gerhardus Marthinus Boshoff

Introduction: The desire to have a biological childtranscends race, religion and socio-economic status. However forthose faced with infertility, the financial resources needed toconceive are often not available. Current research in assistedreproduction has gravitated towards cost reduction to addressrestricting financial factors, without compromising quality oftreatment. One such initiative is the development of a low-costembryo culture method by The Walking Egg foundation. This methodutilizes a standard chemical reaction and simple equipment toequilibrate culture media pH and to regulate temperature; bothaspects were investigated in this study. An exploration into theinsemination concentration to achieve oocyte fertilization wasalso undertaken. Methods: Quality control of temperatureregulation on six different heating devices, including acomparison of inter- and intra-variations was carried out. Theutilization of citric acid and bicarbonate of soda for carbondioxide production, which subsequently facilitate setting of pHvalues, was tested by injecting increasing citric acid volumes(1.2 ml 3.0 ml in 0.2 ml increments) into set volumes ofbicarbonate of soda. Further investigation evaluated gasproduction at various temperatures (37C, 25C and 15C), atincreasing intervals (16 30 hours) of equilibration and thesewere compared by measuring pH of the culture media. The influenceof altitude on pH was explored by repeating the chemical reactionexperiment at five different locations in South Africa.Furthermore, the addition of water to citric acid before gasgeneration was explored. The minimal insemination concentrationneeded for fertilization was determined by the addition ofdecreasing numbers of spermatozoa to non-fertilized bisectedoocytes. The experiment was repeated with a selected sperminsemination number in 1 ml or 50 l culture media to compare thetested culture system with conventional culture. Spermatozoa boundto the hemi-zonae were counted with the aid of an inverted phasecontrast microscope. Hemi-zonae with bound sperm were also stainedwith ethidium homodimer and evaluated using a confocallaser-scanning microscopy system. After removal of hemi-zonae, thespermatozoa in culture were isolated for deoxyribonucleic acidfragmentation analyses and reactive oxygen species presence in theculture media was measured. Additionally, reactive oxygen speciesgeneration in simulated culture was measured over time. Results:All the equipment tested bar one, the warming oven, proved useablewith the simplified Walking Egg in vitro fertilization culturesystem. By decreasing the citric acid volumes, it was indicatedthat 1.8 ml citric acid, diluted with 1.2 ml water, is the optimalvolume to facilitate the required culture media pH. Omitting thewater dilution from citric acid volumes affected the culture mediapH adversely, however reducing the temperature during gasequilibration did not. A change in altitude had no effect onculture media pH. LowerAdvisors/Committee Members: Huyser, Carin (advisor), Ombelet, Willem (coadvisor).