Characterisation of the physical environment of embryos throughout in vitro culture : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Massey University, Palmerston North, New Zealand
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Date
2011
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Massey University
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Abstract
Characterisation of the physical environment embryos are exposed to throughout in
vitro culture for treatments involving in vitro fertilisation (IVF) has been limited due
to measurement difficulties (since the position of an embryo is the point of interest)
and the lack of a theoretical framework. Temperature, oxygen concentrations and pH
are all important factors in an oocyte’s and an embryo’s environment which, if away
from desired levels, may impact on embryo viability. The development of
mathematical models provides a structured approach which helps to overcome
measurement difficulties.
The IVF process was broken down into 70 discrete back-to-back steps, from oocyte
aspiration to embryo transfer, which could be modelled. Models of heat transfer were
developed for a Petri dish, 4-well dish, Pasteur pipette (un-pulled and pulled), plastic
pipette tip (two sizes), denuding pipette and transfer catheter. Models of oxygen and
carbon dioxide mass transfer were developed for the Petri dish, in which oocytes and
embryos spend the majority of their time in culture. The models were solved by the
finite element method in the software package COMSOL Multiphysics 3.3a used in
conjunction with MATLAB R2006a. Models were then validated against
experimental data.
There is considerable variation in the embryology culture process, with respect to the
number and timing of steps, both between and within laboratories. Of all the factors in
an embryo’s environment embryology practice has the greatest impact on
temperature. Embryos are cultured in dishes in incubators which maintain the required
gaseous and thermal environment. While paraffin oil, which overlays culture media in
a dish, successfully buffers embryos from great changes in oxygen and pH when
dishes are removed from an incubator, maintenance of embryo temperature is
dependent on numerous factors including the setting of the surface temperature of
microscope stages, whether the lid is on or off the dish, the embryo position across the
floor of the dish, the dish’s foot height, the time out of incubator and the depth of
liquid in the dish. For a period of 5 minutes out of an incubator in a standard Petri
dish set up, the pH an embryo is exposed to will not likely rise from pH 7.33, as in an
incubator, to above pH 7.38. However, the temperature an embryo is exposed to may
change by ± < 0.5 °C or may change by ± 1 to 3 °C, depending on embryology
practice. Importantly, an increase of 1 °C in embryo temperature may adversely affect
embryo viability while a decrease of 1 °C will likely have little impact.
Transfer of an embryo in a pipette is the step identified which subjects embryos to the
greatest rate and magnitude of temperature change. While temperature in a dish may
change by 1 to 3 °C during 5 minutes out of an incubator, the temperatures within a
pulled glass Pasteur pipette can fall by > 10 °C in 10 seconds. Use of plastic pipette
tips instead of glass pipettes is beneficial for maintaining embryo temperature as the
temperature will fall by approximately 3 °C in 10 seconds, 7 °C less than in the glass
pipette under the same conditions. This work identified many simple practical steps,
such as the use of plastic pipette tips instead of glass, which minimise temperature
changes embryos are exposed to throughout the culture process.
Applying the Model of mass transfer of O2 in a Petri dish disproved the belief that
equilibration of gas in the dish occurs significantly faster without a lid. The model of
O2 transport in a Petri dish demonstrated that it takes ˜1 hour to reach 67 % and ˜4
hours to reach 95 % full equilibration of oxygen between atmospheric and 5 vol % O2
at 37 C. Modelling mass transport of CO2 provided a means to predict pH changes
within a media drop in a Petri dish. In equilibration from atmospheric to 6 vol % CO2,
the pH reached within 0.1 unit of the final value in ˜1.5 hours. An important finding
of this work was that sufficient equilibration of gas may be achieved in ˜2 hours and
therefore the pre-equilibration time for dishes (currently overnight) may be shortened,
reducing the degradation of amino acids, which occurs at 37 °C, to ammonium
(embryo toxic).
There is considerable variation in embryology practice. This work successfully
utilised engineering knowledge and mathematical modelling to describe the physical
environment of temperature, oxygen and pH that oocytes and embryos may be
exposed to throughout an open embryo culture system, used by the majority of IVF
clinics worldwide. The findings here provide a basis for establishing best practice.
Further work is needed to quantify the effects on the embryo of fluctuation in the
embryo’s environment but this work demonstrates that mathematical modelling of the
embryo’s environment in IVF is a viable tool for improving laboratory practice.
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Keywords
Fertilisation in vitro, In vitro fertilisation, Embryos, Mathematical models, Research Subject Categories::MEDICINE::Surgery::Obstetrics and women's diseases::Obstetrics and gynaecology