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. / Dissertation (MSc)--University of Pretoria, 2017. / Obstetrics and Gynaecology / MSc / Unrestricted
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:up/oai:repository.up.ac.za:2263/63049 |
| Date | January 2017 |
| Creators | Boshoff, Gerhardus Marthinus |
| Contributors | Huyser, Carin, boshoffgm@gmail.com, Ombelet, Willem |
| Publisher | University of Pretoria |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Dissertation |
| Rights | © 2017 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. |
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