Investigating a novel in vitro embryo culture system – The Walking Egg Affordable Assisted Reproductive Technology

Abstract

Introduction: The desire to have a biological child transcends race, religion and socio-economic status. However for those faced with infertility, the financial resources needed to conceive are often not available. Current research in assisted reproduction has gravitated towards cost reduction to address restricting financial factors, without compromising quality of treatment. One such initiative is the development of a low-cost embryo culture method by The Walking Egg foundation. This method utilizes a standard chemical reaction and simple equipment to equilibrate culture media pH and to regulate temperature; both aspects were investigated in this study. An exploration into the insemination concentration to achieve oocyte fertilization was also undertaken. Methods: Quality control of temperature regulation on six different heating devices, including a comparison of inter- and intra-variations was carried out. The utilization of citric acid and bicarbonate of soda for carbon dioxide production, which subsequently facilitate setting of pH values, was tested by injecting increasing citric acid volumes (1.2 ml – 3.0 ml in 0.2 ml increments) into set volumes of bicarbonate of soda. Further investigation evaluated gas production at various temperatures (37°C, 25°C and 15°C), at increasing intervals (16 – 30 hours) of equilibration and these were compared by measuring pH of the culture media. The influence of altitude on pH was explored by repeating the chemical reaction experiment at five different locations in South Africa. Furthermore, the addition of water to citric acid before gas generation was explored. The minimal insemination concentration needed for fertilization was determined by the addition of decreasing numbers of spermatozoa to non-fertilized bisected oocytes. The experiment was repeated with a selected sperm insemination number in 1 ml or 50 μl culture media to compare the tested culture system with conventional culture. Spermatozoa bound to the hemi-zonae were counted with the aid of an inverted phase contrast microscope. Hemi-zonae with bound sperm were also stained with ethidium homodimer and evaluated using a confocal laser-scanning microscopy system. After removal of hemi-zonae, the spermatozoa in culture were isolated for deoxyribonucleic acid fragmentation analyses and reactive oxygen species presence in the culture media was measured. Additionally, reactive oxygen species generation in simulated culture was measured over time. Results: All the equipment tested bar one, the warming oven, proved useable with the simplified Walking Egg in vitro fertilization culture system. By decreasing the citric acid volumes, it was indicated that 1.8 ml citric acid, diluted with 1.2 ml water, is the optimal volume to facilitate the required culture media pH. Omitting the water dilution from citric acid volumes affected the culture media pH adversely, however reducing the temperature during gas equilibration did not. A change in altitude had no effect on culture media pH. Lower insemination numbers resulted in decreased sperm binding, with 2 x 103 motile sperm insemination providing the lowest number to still obtain sufficient sperm–zona binding (≥20 sperm bound). Incubation in 1 ml vs. 200 μl culture media indicated decrease in sperm bound. Sperm deoxyribonucleic acid fragmentation and the presence of reactive oxygen species in the culture media were similar in both the test and control groups. A comparison over time revealed less reactive oxygen species in 1 ml culture media, from the simplified Walking Egg in vitro fertilization culture system after three days of culture, than 200 μl culture media drops under oil, from conventional culture after 18 hours, however the results were not statistically significant. Discussion: Purpose-made heating devices provide superior stabilization of culture media temperature. When selecting a heating device, intra-variations should be considered. Culture media can be manipulated to the required pH by carbon dioxide production, with meticulous attention paid to the citric acid volumes used. However, if gas generation is performed at room temperature, equilibration time must be increased. In conventional culture, the minimum insemination number can be reduced to 2 x 103 motile sperm. Due to lower binding of sperm in large volumes of culture media, 2 – 5 x 103 motile sperm should be considered for the simplified culture system, depending on a holistic consideration of all sperm parameters. Extended culture for at least three days with the simplified culture system can be performed without increasing reactive oxygen species present in culture media. Further research of this novel culture method should include the application of the culture method in a South African environment.