Abstract
The pioneers of in vitro fertilisation (IVF) were Robert Edwards, a scientist working at Cambridge University, and Patrick Steptoe, a gynaecologic surgeon from Oldham, who collaborated together to develop the techniques required to collect human eggs from the ovary and fertilise them in the laboratory. The birth of Louise Brown in 1978 is regarded as the single most important milestone in the world of assisted conception as it revealed that babies could be born as a result of eggs and sperm being mixed together in a laboratory to create embryos which were then transferred back into the patient’s uterus to create a pregnancy; a process known as IVF [1].
1 How It All Started
The pioneers of in vitro fertilisation (IVF) were Robert Edwards, a scientist working at Cambridge University, and Patrick Steptoe, a gynaecologic surgeon from Oldham, who collaborated together to develop the techniques required to collect human eggs from the ovary and fertilise them in the laboratory. The birth of Louise Brown in 1978 is regarded as the single most important milestone in the world of assisted conception as it revealed that babies could be born as a result of eggs and sperm being mixed together in a laboratory to create embryos which were then transferred back into the patient’s uterus to create a pregnancy; a process known as IVF [1].
Robert Edwards’ interest in human fertilisation began in the 1960s; as a physiologist he had the theoretical background to develop culture media and appropriate culture condition, and in 1968 achieved fertilisation of human eggs.
2 Culture Media
Although the modified Tyrode’s medium (T6) was one of the original culture media used, the first major breakthrough in the world of assisted conception was the development of human tubal fluid (HTF) medium in 1985 [2]. This medium was formulated based on the composition of the fluid in the fallopian tubes, and this resulted in higher pregnancy rates than embryos cultured in T6. Overnight, this became the medium of choice for virtually every clinic; they used a basic recipe to make the medium in-house and then tested its suitability for use with a mouse embryo assay. If there was >80% blastocyst rate, the medium was deemed suitable for use.
If we fast forward to the current day, the compositions of media are more complex (Table 19.1) and are now commercially available. Although each brand is similar in terms of its basic composition there are various supplements that are unique to some companies, some of which are considered confidential and the exact recipe not shared with the customers. Culture media can be purchased as a series of different media that meet the requirements of the embryo at different stages in its development known as sequential media, or, the more recently introduced, single-step media which avoids the embryo having to be removed from the incubator during its culture to have the medium replaced.
Table 19.1 Examples of composition of modern culture media
3 Mimic in vivo Conditions
For obvious reasons it is essential that the environment that the gametes and/or embryos are exposed to mimics the in vivo conditions as best as it can in terms of sterility, temperature, pH and osmolality.
4 Sterility
In 2007 the EU Tissues and Cells Directive (EUTD) [3] was introduced, covering all procedures involving donation, procurement, testing, processing, preservation, storage and distribution of human tissues and cells, for human application. This Directive also extended the Human Fertilisation and Embryology Authority’s remit to include the licensing of services involving fresh gametes and meant that all IVF labs had to raise the bar to ensure they met certain air quality requirements to minimise the chance of contamination between gametes and embryos and the ambient environment. Prior to this, embryology laboratories were effectively a room with the lights dimmed down and/or blinds on the windows. Many laboratories introduced mobile air purifiers to upgrade the air quality but this was not a requirement. The EUTD was interpreted differently in different EU member states. In the United Kingdom, the requirement is for laboratories to carry out procedures involving the manipulation of gametes and/or embryos in an environment where the air quality is at least Grade C in the critical work area supported by a background environment of at least Grade D. The Directive, however, does not require micromanipulation procedures such as intracytoplasmic sperm injection (ICSI) or embryo biopsy to be carried out within a hood. However, as with all handling of gametes or embryos, documented procedures should be in place at all times to keep the risk of contamination minimal.
5 Temperature
We all understand the importance of keeping gametes and embryos at the correct physiological temperature, but this does not only apply to the temperature within the incubators but also to everything the gametes and embryos come into contact with i.e. culture media, hot blocks, work surfaces and consumables (such as Petri dishes, test tubes, catheters).
It has been well documented that the meiotic spindle which segregates chromosomes during cell division is highly temperature dependant. In order for the chromosome segregation to be successful it is crucial that eggs and embryos remain as close to the physiological temperature as possible. We know that the microtubular organisation in oocytes held at lower temperature is disrupted, and with extended exposure the spindle is irreversibly disrupted [4]. In order to avoid this damage to the spindle it is critical that laboratories ensure that everything that comes into contact with the gametes and embryos is kept at 370C. This process however, is not as simple as setting all the equipment to 370C; all equipment needs to be validated to ensure that the temperature where the gametes and embryos will be situated is set correctly to 370C (Figure 19.1).
Validation not only applies to the equipment but also to the processes; for example the temperature within the egg collection dish throughout an egg collection procedure. This temperature mapping allows the temperature changes within the dish during the egg collection procedure to be assessed and allows the process to be optimised to ensure that the temperature changes were minimised.
6 pH
All culture media are made up of a balanced salt solution backbone, and this salt solution has many purposes including helping to maintain the physiological pH of media. In mammals the optimal range is 7.2–7.4, and therefore an appropriate buffer should be used to achieve the required pH. Sodium bicarbonate is the most commonly used buffer for maintaining pH in culture media but in order to maintain the physiological pH it relies on a constant supply of carbon dioxide (CO2). This buffering system is ideal for culturing gametes and embryos in a carbon dioxide incubator for long periods of time. In order to determine the pH of a medium based on the amount of bicarbonate and CO2 present, the Henderson–Hasselbalch equation (Figure 19.2) is applied. The bicarbonate concentration in culture media varies but is approximately 25 mmol and, as such, you will usually see incubators set at 5–6% CO2 to achieve the correct pH. If there is not enough CO2 present, the pH increases and the medium turns a pinky/purple colour and cell growth is inhibited.
Certain procedures such as egg collection, ICSI and embryo biopsy require gametes and embryos to be in an atmospheric environment for fairly long periods of time thus potentially affecting the pH of the medium. Bicarbonate buffered media are not suitable for holding gametes and embryos during these procedures for the reasons described earlier and therefore alternatives to a bicarbonate buffer are used. HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) and more recently MOPS (93-(N-morpholino) propanesulfonic acid) are two examples of zwitterions (organic chemical buffering agents) that do not require a CO2-enriched atmosphere to maintain the required physiological pH despite changes in the CO2 concentration, and therefore are suitable for culture of gametes and embryos for short periods of time.
7 Osmolality
Another area to consider for successful culture is the osmolality of the culture medium that gametes and embryos will be exposed to. The physiological osmolality of human plasma is 275–295 mOsm/kg and although mammals can tolerate small variations in their osmolality, maintaining the appropriate level is essential for successful culture. Areas to consider when maintaining osmolality are minimising medium evaporation by ensuring that the correct volume of medium or oil overlay is used, the use of humidified incubators and wherever possible using dish lids to minimise evaporation effects. These again would require validating as part of the laboratory set-up.
8 Gamete Preparation
8.1 Sperm Prep
Various studies have described that sperm quality is affected by the duration and type of arousal the male is exposed to whilst producing a sample [5,6,7]. Gone are the days when the male partner would be handed a magazine and sent off to use a hospital toilet cubicle to produce a sample. Nowadays there are designated rooms; fully equipped with reclining chair, sample hatch directly through to the laboratory and, more frequently, a video system including headphones to enhance privacy (Figure 19.3). Magazines pose significant infection control issues and feedback from patients describes how unpleasant it is to be given the same magazine that the man before was handed!
