The IVF Laboratory: Reflections on the Past
Dr Peter Hollands
The Agora Gynaecology and Fertility Centre03 October 2013
It is now 35 years since the birth of the first IVF baby and since that time several million babies have been born worldwide. The science behind IVF has been constantly evolving during this period to the point that an embryologist working in a modern laboratory would not recognise some of the earlier technology which set the foundations of their profession. New laboratory technology, whilst welcome if it improves treatment results, must of course be introduced with care to ensure that safety and efficacy of treatment is maintained (Harper et al., 2012). This review gives an historical overview of the development of IVF and the clinical laboratory services which are so essential to success.
Buildings and Facilities
When Bob Edwards and Patrick Steptoe were looking for a suitable place to set up the first IVF clinic in the World they settled on a Jacobean mansion not far from Cambridge called Bourn Hall. This was more out of necessity than choice as they famously struggled to convince the Medical Research Council and others to assist with the funding of their work. The embryology laboratory and operating theatre were then created by erecting portacabins on the front lawn of Bourn Hall! Whilst these portacabins served their purpose at the time it soon became clear that IVF requires purpose built laboratories in order to control the environment in which IVF takes place. One of the most memorable problems with the portacabins was that they became very hot in the summer and very cold in the winter. In the late 1980’s the new laboratory building at Bourn Hall was completed and it exceeded all of the current requirements. The new laboratory had positive pressure air conditioning and led the way to what is seen today in purpose built IVF laboratories in many clinics around the world. Other workers began to report the importance of laboratory air quality in IVF (Boone et al., 1999) and more recently laboratory air quality has been linked to overall pregnancy rate (Legro et al., 2010; Khoudja et al., 2013).
Bob Edwards and Clinical Embryologists
Bob Edwards, through his pioneering research, effectively created the role of Clinical Embryologist which we know today. The team he brought together at Bourn Hall were initially selected from his trusted colleagues and friends at Cambridge University. This group of pioneer Clinical Embryologists included Simon Fishel, Roger Simons, John Keith, Jacques Cohen, Carole Fehilly and myself. Simon and I had been post-graduate research students at Churchill College, Cambridge with Bob Edwards and the other members of the team, with the exception of Roger Simons, were experienced post-doctoral workers. Roger is an excellent Clinical Embryologist with exceptional skill, patience and dexterity but he did not have an academic or research background. There was of course no formal training or regulatory body at this stage of Clinical Embryology and most of us worked on a ‘see one, do one, teach one’ basis! It is also interesting to note that the Clinical Embryologists at Bourn Hall all worked closely with technicians who supported their work and also carried out the more mundane jobs in the laboratory. These people were highly skilled in their own right and were originally trained by Bobs’ dear friend and colleague Jean Purdy who was his assistant during his research and early clinical work in IVF. As the field of Clinical Embryology evolved the technicians working in the laboratory became trained as Clinical Embryologists and many of them went on to make very significant contributions to clinical embryology both at Bourn Hall and at other clinics.
These were exciting, pioneering times and each day brought new challenges and of course new ideas from Bob Edwards. In the late 1980’s I remember talking to Bob Edwards about the problems we faced when a male patient had insufficient sperm to make a suitable preparation for IVF. This was of course before the days of ICSI. Bob suggested that we should take a single sperm and inject it under the zona pellucida which is a technique later known as SUZI or sub-zonal sperm injection (Bonduelle et al., 1994). Bob challenged me to try this new idea and before I knew it I was sat at the old Zeiss micromanipulator which we had ‘borrowed’ from the research lab at Cambridge University about to inject a sperm underneath the zona of a human oocyte! I remember that Bob told me very clearly that I must not inject the sperm into the oocyte as he was very concerned that such a procedure would damage the spindle of the oocyte and result in an abnormal embryo. I carried out my task as instructed and the next day we were delighted to see normal fertilisation and subsequently the embryo cleaved. The embryo was transferred back to the patient but unfortunately the patient did not get pregnant and as a result we decided not publish this trial in the medical literature.
Laboratory Equipment and Reagents
The laboratory equipment which was used at Bourn Hall in the very early days was based entirely on the equipment used in the research laboratory at Cambridge University where Bob Edwards carried out all of his research. There were, of course, no companies dedicated to the production of IVF specific equipment which meant that the laboratory was equipped with what today would be considered as research equipment.
Equipment and Consumables
In 35 years there will inevitably have been changes to equipment in any area of medicine and IVF is no exception. Perhaps the biggest changes in the UK came as a result of regulations from the Human Fertilisation and Embryology Authority (HFEA) and the European Union Tissue Directive (EUTD) which stated amongst other things that IVF should be carried out in a laboratory with specific air quality. In the early laboratories air quality was not monitored and many clinics, including Bourn Hall, used horizontal laminar air flow hoods as work stations for IVF. Once again the use of horizontal flow came from the original research laboratory at Cambridge University and whilst it protected the material being worked on it did not protect the worker. Class II flow hoods, specifically designed for use in IVF with a built in microscope and heated surfaces, are now in routine use in all IVF labs.
Heated surfaces in the early laboratory at Bourn Hall were provided by using a hollow Perspex block, especially made in the workshops at the Physiological Laboratory at Cambridge University, through which water heated to 37°C was circulated. These blocks fitted to the dissection microscope enabling oocytes, zygotes and embryos to be maintained at a steady temperature during manipulations.
All culture at the start of IVF was carried out in drops of media under mineral oil. This mineral oil was obtained from a chemist shop and was equilibrated by putting it into a bottle with a layer of culture media and gassing the culture media with 5% CO2 in air until the media turned the classic ‘salmon pink’ (using phenol red indicator) indicating that the correct pH had been reached. The mineral oil and media was then allowed to separate prior to use and the mineral oil poured off as required. There was no batch testing or quality control of the mineral oil. This procedure worked very well until one Monday morning (known by people who were involved as ‘Black Monday’) when all of the embryos were found to be dead. A thorough investigation showed that there was a toxic batch of mineral oil which caused the death of the embryos. The actual nature of the toxicity was never discovered. This led to batch testing of all mineral oil using sperm and multinucleate embryos and the problem, or the toxicity, never arose again. Today mineral oil preparation is carried out by the same companies who produce culture media to the highest pharmaceutical grades (Sifer et al., 2009).
All gamete and embryo manipulations were carried out using long-form Pasteur pipettes and a rubber bulb. These Pasteur pipettes were purchased from a standard laboratory supplier and carefully washed and heat sterilised in the laboratory. At the point of use the long-form Pasteur pipettes were flame sterilised in the flame of a small paraffin burner which was placed inside a horizontal laminar air flow hood.
The media used in early IVF was Earle’s medium. It was manufactured and supplied for research use only and it was supplied to the laboratory in glass bottles containing 500mL of 10x concentrated media. This media was diluted in the laboratory using sterile filtered pure water which was also purchased in bulk from a scientific supplier. Once diluted the media was supplemented with sodium pyruvate as a source of energy for the developing zygote and embryo, sodium bicarbonate as a buffer and penicillin and streptomycin. A special batch had to be made for those patients with an allergy to penicillin with contained streptomycin only and latterly streptomycin and gentamycin. The osmolarity of the media was then checked using an osmometer with a target value of 280 mosmol/L. The media was the filtered through a 0.2µm filter to remove any contaminating bacteria into sterile bottles ready for use.
Culture media of course also needs protein supplementation which was originally achieved by using the serum of the woman being treated. Blood was collected on the morning before egg collection (remembering that at this stage that all patients were inpatients at Bourn Hall for the whole of their treatment) and taken immediately to the laboratory to be centrifuged before it clotted. It is interesting to note that some blood samples carried very high levels of fat which was traced back to the excellent English breakfast on offer at Bourn Hall! The serum was then removed from the red cells and allowed to clot. The clot was then removed and the remaining serum was heat inactivated in a water bath at 56°C for 30 minutes. Once heat inactived the serum was filtered through a 0.2µm filter to remove any bacteria and kept at 4°C before use. The media was supplemented with 8% serum v/v for egg collection and insemination and 15% serum v/v for embryo culture. Embryo transfer (or replacement as it was known at Bourn Hall) was carried out in 50% serum.
The final notable difference from today in terms of media was that embryos were transferred (or replaced as was the preferred terminology at Bourn Hall) in media which contained 50% serum v/v. It is also interesting to note that specific media containing hyaluron have been developed for embryo transfer (Nakagawa et al., 2012) which reflects back onto the use of 50% serum based media for embryo transfer. It soon became clear that the use of patients’ serum in all patients resulted in problems and the search began for alternatives such as Albuminar 5 (Ashwood-Smith et al., 1989) which was the forerunner of the human serum albumin used widely in IVF media today.
This description of the preparation and use of culture media for use in IVF may alarm those readers who are relatively new to IVF! Nevertheless, this technology did provide us with initial success and its’ importance in clinical embryology cannot be understated. More recent developments have seen the provision of specific IVF culture media designed for human clinical use by various large companies and resultant comparisons of these media (Pool et al., 2012). These media are produced to the highest pharmaceutical standard and have no doubt contributed to the increased live birth rate which we see in clinical practice today (Mantikou et al., 2013). Culture media continues to be refined to provide optimum culture conditions for both cleavage embryos and blastocysts and media supplements such as GM-CSF are considered important by some workers. Other growth factors such as brain-derived neurotrophic factor, colony-stimulating factor, epidermal growth factor, granulocyte macrophage colony-stimulating factor, insulin-like growth factor-1, glial cell-line derived neurotrophic factor, and artemin may be important in the enhanced development of human embryos in vitro (Kawamura et al., 2012).
The incubator of choice for early IVF was a ‘dry’ incubator with a water jacket similar to those used in microbiology laboratories to culture agar plates. These incubators maintain a steady 37°C but do not provide any carbon dioxide for buffering purposes. The culture media required and atmosphere of 5% CO2 in air in order to maintain the correct pH. For this reason, when using these early dry incubators, the embryo culture dishes were placed into a dessicator jar which was then gassed from a cylinder containing 5% CO2 in air. The dessicator was then placed into the dry incubator to warm and about 15-20 minutes later the tap on top of the dessicator had to be momentarily opened to allow the pressure of the warmed gas to be released. The tap could then be closed and the dessicator left to culture. One drawback of this system was that if the embryologist forgot to release the pressure then the lid of the dessicator could lift under the pressure of the warm gas and the CO2 in air would be released from the dessicator. This would result in a change in the pH of the media and was therefore possibly damaging to the embryos.
As workload and technology improved CO2 incubators were introduced which are fed by 100% CO2 and the incubator itself maintains a steady 5% CO2 for culture. One of the drawbacks of these large incubators is the fact that the door was opened too often to gain access to various different embryos and this led to the introduction of smaller, self-contained incubators such as the MINC and Planer BT37 which maintain both temperature and gas concentration more accurately and are in common use today (Cooke et al., 2002). The use of pre-mixed gas in these type of incubators containing 6% CO2, 89% N2 and 5% O2 (in comparison to the 20% O2 in other culture systems) results in better embryo quality and pregnancy rates (Kasterstein et al., 2013) presumably because of the reduction of reactive oxygen species (ROS) in such systems (Meintjes et al., 2009; Lee et al., 2012).
Semen Collection and Preparation
In the early days of IVF it was believed that the best semen samples could be obtained by collecting the semen into 2 different pots, this was known as a ‘split-ejaculate’. The patient was given 2 semen collection pots, held together by sellotape, and he was asked to produce semen and put the first drops of the ejaculate into the first pot and the rest of the ejaculate into the second pot. The rationale for this rather cumbersome process was that the first few drops of the ejaculate contained the best sperm. Thankfully for male patients undergoing fertility treatment this process has now been made obsolete and the whole ejaculate is now collected into just one pot.
Semen preparation at the beginning of IVF was kept deliberately simple as there was a concern that over-manipulation, or exposure to chemicals other than culture medium, would damage the sperm. The standard preparation was a simple ‘swim-up’ of sperm which was achieved by layering medium over the semen in the collection pot and then simply waiting for the sperm to swim into the medium which was then pipetted off, centrifuged very gently just once and the diluted for insemination of oocytes to a concentration of 100,000 sperm/mL. Second line semen preparations, usually where semen volume was low, was to suspend the semen in medium, gently centrifuge it and remove the supernatant, add fresh medium and allow the sperm to swim up once again. This was called swim-up from a pellet and was considered to be quite invasive but acceptable as a method of preparing sperm for IVF. The use of Percoll as a density gradient technique for semen preparation (Prakash et al., 1998) was shown to be more effective than swim up techniques and this was the basis for the routine density gradient sperm preparation used in IVF laboratories today.
Insemination using IVF
All oocytes, which were collected using laparoscopy as pioneered by Patrick Steptoe (Litynski, 1998), were left for several hours before insemination to ensure that the oocytes were suitably mature. Oocytes were inseminated, usually in the late afternoon, using a carefully diluted preparation of sperm containing approximately 100,000 motile sperm per mL. This sperm concentration was found to optimise fertilisation whilst minimising polyspermy.
Assessment of Fertilisation
In the early days of assisted reproduction all procedures used standard IVF and it was the role of the clinical embryologist to assess the presence of pronuclei on the morning following insemination. This assessment was carried out by the very careful dissection of the inseminated oocytes using two green needles. This was a highly skilled, and potentially damaging, procedure which was only carried out by the most experienced clinical embryologists. Despite this there were some instances of damage to the oocyte during this procedure. More recently this procedure has been superseded by the using of first pulled Pasteur pipettes and today by specifically designed ‘stripping’ tips which make the whole procedure much safer for everyone involved!
Assessment of Embryonic Development
Embryonic development was assessed using the least invasive methods under a dissecting microscope at 50x magnification. Exposure of embryos to light was kept to an absolute minimum and the microscope light had a green filter on it reduce exposure to high intensity light. This is in contrast to current assessment of embryonic development which often using high power magnification and relatively high light intensity or even constant monitoring of embryos using time lapse technology (Campbell et al., 2013).
Embryo Replacement (Transfer)
Embryo replacement was always carried out towards the end of the working day in the most relaxing and quiet conditions possible. The patient would be brought to the theatre and prepared for the procedure and the Clinical Embryologist would then explain the number and quality of embryos available for replacement and freezing. Both the physician (often Patrick Steptoe) and the Clinical Embryologist would scrub and wear sterile gloves, the physician also wore a sterile operating gown. Everyone wore face masks.
The embryos were then loaded into the catheter and it was handed to the physician. The Clinical Embryologist then remained at the side of the physician and when the catheter was in place the Clinical Embryologist would gently press the plunger of the syringe to expel the embryos into the uterus. It was considered to be very important that the Clinical Embryologist carried out this ‘final step’ in the care of the embryos and it is a tradition that is used in many clinics even today. The patient would then remain in bed for at least 30 minutes following transfer.
In the early days of IVF most embryos were frozen at the cleavage or blastocyst stage using propan-diol based media for cleavage stages and glycerol based media for blastocysts. These freezing media were all prepared in the laboratory using basic research reagents. All embryos were frozen using a Planer controlled rate freezer with manual seeding. This machine had a reassuring ‘clunk’ as it operated which I am sure many Clinical Embryologists today would still recognise. Embryos were initially frozen in glass vials which were heat sealed. These had a tendency to explode on thawing if the heat sealing was not perfect! In due course controlled rate freezing for pronuclate embryos was also introduced.
Controlled rate freezing has today been replaced in most clinics by vitrification (Kuc et al., 2010) with closed system vitrification systems offering excellent clinical outcomes (Desai et al., 2013) for both cleavage embryos and blastocysts. Vitrification also has the advantage of requiring very little equipment and it is a very rapid process making it very attractive in a busy modern laboratory.
The technology used in an IVF laboratory has evolved considerably over the past 35 years as new ideas and inventions are brought into day to day practice and assisted reproduction moves into the 21st Century. These changes have brought considerable benefits to patients not only in overall success rates but also in the patients it is possible to treat using innovations such as ICSI and PGD. The future will no doubt bring further innovations and possibly automation to the process of IVF which will make it even further removed from the early days. Nevertheless, it is important to understand the technological development of IVF and to appreciate the challenges which were met by Bob Edwards and his original team of Clinical Embryologists.
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