Revealing the secrets of a successful embryo

It is estimated that one in seven couples will experience some difficulty conceiving and for one third the reason will be classified as ‘unknown’ which can be highly frustrating for those involved.  Although there is still much we do not know about human reproduction some recent developments, such as time lapse imaging of the developing embryo introduced in 2013, are offering new insights Human reproduction is a relatively recent field of medicine. In 1968, when Professor Robert Edwards became the first person to successfully fertilise a human egg outside the body and take it to blastocyst stage (the point at which the embryo differentiates) embryology was not even considered a worthwhile field of study. In fact the enormity of this breakthrough was only fully recognised over 40 years later when he was awarded the Nobel Prize for Physiology. It was after the successful birth of Louise Brown, the first baby to be conceived as a result of In Vitro Fertilisation (IVF), that Edwards and his co-pioneer, the gynaecologist Patrick Steptoe, established Bourn Hall Clinic in Cambridge. It was here they went on to perfect the techniques that would lead to the births of over five million IVF babies worldwide. After initial studies in the mouse focused on developing drug regimes that could control the hormonal cycle responsible for oocyte maturation, similar regimes were attempted in the human. These took many years to perfect and Louise Brown was actually conceived after a natural cycle. It was thought that each month only one follicle begins to grow in preparation for the release of a single egg. It is now known, that follicles are ‘recruited’ from the resting supply nearly a year before and then undergo about three months of growth and development before ovulation. At the start of each month, there are around 15-20 follicles which have made it to the final stage of this process. Under normal circumstances only one of these follicles will become dominant and finally release an egg.  By day eight of the menstrual cycle the follicle that has responded to naturally produced follicle stimulating hormone (FSH) can be seen within the ovary using an ultrasound scan. This is the follicle that is likely to continue development and ovulate as it will be exactly the right size at the right time. During IVF treatment the natural hormones are suppressed and follicle development is controlled artificially. FSH injections are given to maintain higher levels of the hormone for longer, resulting in the recruitment of multiple follicles which mature at the same time in preparation for egg collection. There are many factors which affect the way the ovary responds to this stimulation, therefore regimes are patient specific and aim to try and retrieve around 10 eggs. [caption id="attachment_40255" align="alignright" width="200"] ICSI procedure at Bourn Hall Clinic Cambridge.[/caption] Hyper stimulation of the ovaries can be an issue for patients undergoing IVF treatment, but recent developments in our understanding are allowing greater control. The first is a better understanding of a common cause of infertility known a Polycystic Ovarian Syndrome (PCOS). It is now known that this is associated with a metabolic disturbance that can result in insulin resistance and high levels of insulin in the blood. In some women, raised insulin levels can affect the ovaries, preventing them from releasing mature eggs. This anovulation can lead to infertility. This knowledge has improved diagnosis and awareness that treatment with FSH in this group of patients needs to be carefully monitored, as conventional doses can cause high rates of ovarian hyperstimulation syndrome (OHSS) and multiple pregnancy. Another development has been the wider use of the AMH (Anti-Mullerian Hormone) test to measure a woman’s ovarian reserve. This gives an indication of the number of eggs left in the woman’s ovaries and offers a guide as to whether the ovaries will respond to stimulation, and if so at what level. Contrary to many media reports, AMH cannot provide a reliable prediction of future fertility. After trials, Bourn Hall now uses the AMH test to predict the response to FSH. This allows the dosage to be tailored to the individual and reduces the risk of over stimulation. Once the eggs are collected it is essential that their environment is carefully maintained to ensure it remains within physiological ranges, particularly temperature, pH and osmolality, as well as ensuring their nutritional requirements are met. The intention is to replicate as closely as possible the conditions in the fallopian tubes, where natural fertilisation and early embryo development occurs. In the early days the culture media was not designed specifically for human embryos but improvements in its formulation have meant that embryos can be maintained in culture for longer allowing us to transfer more advanced embryos, typically blastocysts to the uterus. Unfortunately, there is no reliably accurate predictor of an embryo’s potential to achieve a pregnancy. As a result it was common practice to place two or more embryos into the uterus. This gave an acceptable pregnancy rate but as a result one in four IVF pregnancies was a twin birth. This not only increases the risk of complications for both the mother and the unborn child, but also places a strain on NHS neonatal care units, because multiple pregnancies are commonly premature. To reduce the number of multiple births resulting from IVF, the government introduced a ‘One at a time’ initiative and implemented a ‘multiples quota’ for each clinic. This requires clinics to have a documented strategy to achieve the current target of 10%. The most obvious way to reduce the multiple pregnancy rate is to reduce the number of embryos transferred, however IVF specialists were concerned that doing this without being able to identify the optimum embryo would adversely affect the pregnancy rate. Improvements in culture media and conditions coupled with rigorous quality control in the laboratory now enable us to confidently maintain embryos in culture for five to six days. By which point only the strongest embryos will have overcome two crucial hurdles in their development, embryonic genome activation and cell differentiation. Although not all embryos will come through this natural selection process, those which do achieve a far higher implantation rate. [caption id="attachment_40254" align="alignleft" width="200"] Bourn Hall Clinic Cambridge (the world’s first IVF clinic).[/caption] Bourn Hall is the largest provider of NHS funded IVF treatment based in the East of England and its five year contract enabled it to develop and fine tune an algorithm which has not only maintained, but actually improved success rates whilst minimising multiple births. This has been achieved by the routine use of AMH to tailor the drug regime, combined with transferring a single blastocyst wherever possible. This protocol has increased the live birth rate (LBR) per cycle of treatment to 38% (2012-13) compared to 30% in 2010-11. Nationally, the LBR for IVF was 25.6% in 2010¹. Furthermore by following the full adoption of the National Institute for Health and Care Excellence (NICE) guidelines, seven out of ten couples treated at Bourn Hall now take home a baby after three cycles of IVF. Infertility is classified as a disease by the World Health Authority but fertility treatment is considered a life-style choice by some commissioners. It is to be hoped that the evidence-based data resulting from five years of consistent clinical practice will be taken into consideration when deciding on future funding of fertility treatment. As already discussed, maintaining a stable culture environment is of paramount importance for successful IVF. For this reason embryo observations, which require them to be removed from the incubator for microscopic examination, are kept to the absolute minimum, typically just once or twice a day. Such infrequent snap-shot views of a dynamic system greatly limit the amount of information about an embryo’s development. This is why ‘Time Lapse Imaging’ of the embryo’s progress is causing such excitement among embryologists, as it records previously unseen information about embryo development and is likely to bring new insights to the field. Embryos that divide in a precise and even pattern appear to be the ones most likely to implant normally and result in a live birth. So when the embryologist selects which embryos to transfer to the woman’s womb, she or he is looking for one that meets these criteria. Early Embryo Viability Assessment (Eeva) builds upon work from Stanford University in 2008, which found a correlation between cell division timings and developmental outcome of embryos². The findings suggest that by the 4-cell stage the fate of the embryo may already be determined. This research lead to the development of a time lapse imaging system coupled with intelligent computer vision software. This is able to measure and analyse key scientifically and clinically proven cell-division markers from video images. It then provides predictive information on which embryos are likely to form blastocysts at 5-6 days. [caption id="attachment_40257" align="alignright" width="200"] Sperm Freezing at Bourn Hall Clinic Cambridge.[/caption] Eeva was validated in a multi-centre, 160 patient clinical trial, with results presented at the European Society of Human Embryology and Embryology (ESHRE) Annual meeting in July 2012. The study showed that embryologists using Eeva were able to predict which embryos were able to form blastocysts with 85% specificity and also increased the consistency of embryo assessment across embryologists³. Traditionally in IVF, selection is made by a skilled embryologist who assesses the embryo under a microscope and assigns a grade based on the number and evenness of the cells, as well as the degree of acellular fragmentation. The Eeva microscopes are located inside an incubator, so the environment remains completely undisturbed. The embryos are imaged every five minutes, so their development is seen in its entirety and can be used to predict those that will develop to blastocyst. This is thought to be a significant benefit by embryologists at Bourn Hall, which is one of the first UK clinics to trial the system. Additionally, the embryologists are looking to Eeva to provide new information about early stage development. Although it is thought that fragmentation is an indication of a poor quality embryo there is also evidence that embryos can self-regulate and even ‘poor’ embryos can produce healthy babies. Furthermore, regular division is considered essential and yet at the 2014 ACE conference there was a discussion about an embryo that had divided unevenly (two cell to three cell) and had a successful outcome, which indicates there is still much to understand. Successful implantation requires cooperation from both embryo and uterus alike, yet much of the research that has been carried out to date has been focussed solely on the embryo, due in part to its availability. The process of implantation is known, yet the control mechanisms and dialogue between embryo and endometrium, in success or failure is poorly understood. The next big challenge for reproductive medicine is improving implantation and reducing recurrent miscarriage.  Recent retrospective evidence⁴ has shown that embryos cleaving from two to four cells at the correct time post fertilisation have a significantly higher chance of implantation (49% vs 21%) than those outside the range. It will be interesting to evaluate this at Bourn Hall. In 2013, 55.8% of patients aged under 38 having a blastocyst transfer at Bourn Hall Clinic achieved a clinical pregnancy.  A 50% success rate was Steptoe and Edward’s dream when they first set up the clinic. We are looking forward to seeing what can be achieved in the next 35 years. References 1. Human Fertilisation and Embryology Authority, 2013. Latest UK IVF figures: 2010 and 2011. http://www.hfea.gov.uk/ivf-figures-2006.html#1279 2. Wong, C., Loewke, K., Bossert, N., Behr, B., De Jonge, C., Baer, T. and Reijo Pera, R. 2010. Non-invasive imaging of human embryos before embryonic genome activation predicts development to the blastocyst stage. Nature Biotechnology, 28, pp.115-1121. 3. Conaghan, J., Chen, A., Willma, S., Ivani, K., Chenette, P., Boostanfar, R., Baker, V., Adamson, D., Abusief, M., Gvakharia, M., Loewke, K. and Shen, S. 2013. Improving embryo selection using a computer-automated time-lapse image analysis test plus day 3 morphology: results from a prospective multicentre trial. Fertility and Sterility, 100, pp412-419. 4. Chen, A., Tan, L., Suraj, V., Reijo Pera, R. and Shen, S. 2013. Biomarkers identified with time-lapse imaging: discovery, validation and practical application. Fertility and Sterility, 99, pp.1035-1044. Author Martyn Blayney, Head of Science at Bourn Hall Clinic