Reproductive BioMedicine Online (2013) 27, 710– 721

www.sciencedirect.com www.rbmonline.com

SYMPOSIUM: FUTURES IN REPRODUCTION REVIEW

How should we assess the safety of IVF technologies? Daniel R Brison

a,b,*

, Stephen A Roberts c, Susan J Kimber

d

a

Department of Reproductive Medicine, Old St Mary’s Hospital, Central Manchester University Hospitals NHS Foundation Trust, Manchester Academic Health Sciences Centre, Manchester M13 9WL, United Kingdom; b Faculty of Medical and Human Sciences, University of Manchester, Manchester M13 9PT, United Kingdom; c Centre for Biostatistics, Institute of Population Health, 1st Floor, Jean McFarlane Building, University of Manchester, Manchester M13 9PL, United Kingdom; d Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United Kingdom * Corresponding author. E-mail address: [email protected] (DR Brison). Professor Daniel Brison is a Consultant Embryologist and Scientific Director of the clinical Department of Reproductive Medicine, St Mary’s Hospital, Manchester, and Honorary Professor of Clinical Embryology and Stem Cell biology at the University of Manchester. He is Person Responsible to the UK Human Fertilisation and Embryology Authority for licences in embryo research and embryonic stem cells, and Co-Director of the NW Embryonic Stem Cell Centre. He has worked in reproductive medicine/biology and stem cell biology for nearly 30 years. His clinical and research interests include: the characterization of early human development at the molecular level, the impact of environmental factors, statistical modeling of embryo selection, markers of embryonic health, the regulation of pluripotency in embryos and embryonic stem cells, the impact of technology in ART on embryo and child health, and the derivation and characterization of clinical grade stem cells for the treatment of disease. Abstract Clinical IVF treatment was established over 30 years ago through pioneering work by Edwards and Steptoe and other teams

around the world and is now considered routine treatment. However, the pace of scientific and technological advances means that IVF practitioners can now access an increasing array of new and invasive technologies. The examples are many but include: extended embryo culture, development of media to include growth factors, developments in genetic screening, use of time-lapse technology and the advent of vitrification of embryos and oocytes. In parallel, wider scientific and medical advances are raising our awareness of the potential impact of assisted reproduction technology on areas such as embryonic development, gene expression and genomic imprinting and the developmental origins of health and disease. A recently suggested paradigm for assessing new technologies in IVF includes development in animal models such as rodents and large animals, preclinical research with human gametes and embryos donated to research, prospective clinical trials in IVF and, finally, follow-up studies of IVF children. In this paper, we describe efforts to address key areas of this pathway, namely preclinical research using human gametes/embryos and long-term, follow-up studies of the health of assisted reproduction children. RBMOnline ª 2013, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: assisted reproduction, embryos, gametes, human, IVF, gene expression VIDEO LINK: http://sms.cam.ac.uk/media/1401011

1472-6483/$ - see front matter ª 2013, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.rbmo.2013.09.006

How should we assess the safety of IVF technologies?

Introduction In 1978, the first test-tube baby, Louise Brown, was born at Oldham General Hospital in Greater Manchester (Figure 1) following the pioneering work carried out at Kershaw’s Cottage Hospital, Oldham by Robert Edwards and Patrick Steptoe (Edwards and Steptoe, 1980; Johnson, 2011). Thirty-five years later, in 2013, assisted reproduction treatment is now considered mainstream medical practice, with an estimated 5 million babies born to date (ESHRE, 2012; Higgins, 2013) and now representing 2% of all live births in the UK. During this time, only limited investigations into the health of assisted reproduction children have been performed. There is a number of reasons for this, including: (i) the absence of centralized national data registers; (ii) legislation which did not allow follow-up data linkage studies, e.g. in the UK, restrictions in the Human Fertilisation and Embryology Act 1990 (which covers assisted conception and embryo research in the UK) did not support such linkage studies prior to the issue of regulations in 2010; and (iii) lack of grant funding for such studies, driven in part by a lack of awareness of potential health issues. In the last few years a number of developments have occurred which have put the health of children born of assisted reproduction treatments in the spotlight. New Techological Developments assisted reproduction follow-up studies with sufficient scale and power to detect specific health problems are still relatively rare; however, a number of publications over the years has raised generalized low-level concerns. Increased perinatal risks for singletons born after assisted reproduction treatment have been well documented and are summarized in a recent systematic review showing an increase in preterm birth, low

711 birthweight, small-for-gestational-age babies, congenital abnormalities and perinatal mortality (Pandey et al., 2012). Low birthweight and an increased risk of hypertension in assisted reproduction children (Ceelen et al., 2008; 2009) are both markers of early onset adult diseases such as cardiovascular problems and type 2 diabetes, according to the Barker hypothesis and the development origins of health and disease hypothesis (Barker, 1994; see also next section). Other risks identified include disruptions to genetic imprinting, both in its own right as the cause of specific, very rare diseases such as Angleman or Beckwith–Wiedemann syndromes and also as a possible marker of more generalized epigenetic disruption (Bowdin et al., 2007; Chason et al., 2011; Dı´az-Garcı´a et al., 2011; Lidegaard et al., 2005; Maher et al., 2003; Nelissen et al., 2013; Rancourt et al., 2012; Turan et al., 2010). However, few of these studies have been able to distinguish the effect of assisted reproduction treatment from the consequences of parental subfertility, and fewer still have been able to address the role of specific technologies (Pandey et al., 2012; Reddy et al., 2007; Schieve et al., 2004). Some studies have attempted to investigate the use of intracytoplasmic sperm injection (ICSI), oocyte in-vitro maturation, stage at embryo transfer and cryopreservation (Fadini et al., 2012; Finnstro ¨m et al., 2011; Henningsen et al., 2011; Kalra et al., 2012; Maheshwari et al., 2012; Romundstad et al., 2008). Results from these studies are often conflicting: for example, a recent cohort study (Davies et al., 2012) from Australia described an increased risk of congenital malformation in children born after ICSI, but not after IVF, which contrasts with the majority of previous studies which show no difference between IVF and ICSI. Interestingly, birthweight may alter in association with

Figure 1 The hospital building and operating theatre in which Louise Brown was born on 25 July 1978 is still standing and was operational as of late 2012. Lower right hand photograph courtesy of Dr Zul Anjum.

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specific practices, including altered embryo culture conditions (DuMoulin et al., 2010; Nelissen et al., 2012), transfer of embryos after freeze–thawing (Kalra et al., 2011; Vergouw et al., 2012; Wennerholm et al., 2009), or the impact of ovarian stimulation on the endometrium (Imudia et al., 2012, 2013). These studies have led to increased awareness of the need for larger-scale, follow-up studies with sufficient power to detect potential health issues. As an example, in recognition of the importance of such studies the Human Fertilisation and Embryology (Disclosure of Information for Research Purposes) Regulations issued in 2010 now allow the Human Fertilisation and Embryology Authority (HFEA) to disclose data for use in linkage studies between the central register of IVF treatments and external child health databases. Advances in Science and Medicine advances in science and medicine have led to increased understanding of the basic mechanisms of disease and how these might arise early in development. This includes the role of epigenetic mechanisms and the developmental origins of health and disease hypothesis (www.mrc.soton.ac.uk/dohad; Barker, 1994). Allied to this awareness, there has been an increase in understanding of the stress to which animal and human embryos may be exposed in vitro and therefore of the risk that manipulations carried out during treatment may potentially predispose children to increased health risks. Preimplantation embryos are both resilient and highly sensitive to environmental stress in vitro. This combination of properties means that stress has the capacity to reprogramme later development, or even adult health, via an impact on the key developmental events occurring during the period of manipulations, such as the reorganization and activation of the embryonic genome, epigenetic regulation including genetic imprinting and the establishment of the first cell lineages in development (Johnson, 2005; Leese, 2012, Table 1; Reik, 2007; Watkins et al., 2008). Evidence of this reprogramming can be found in animal models in which

embryo culture has been associated with epigenetic disorders in both fetal and placental tissues, carried into adulthood and associated with disease (Morgan et al., 2008; Rivera et al., 2008; Young et al., 2001). Advances in science and technology and changes in practice have led to a recent influx of new technologies into the assisted reproduction field: for example, extended embryo culture and changes in media, such as the use of sequential media and supplementation with growth factors. Added to these are preimplantation genetic screening/diagnosis, changes in cryopreservation practice including the widespread use of vitrification (Brison et al., 2012), in-vitro maturation, oocyte chemical activation, time-lapse microscopy of embryos in vitro, electronic witnessing using radio frequency identification tagging and a number of other procedures. The increasing use of these new technologies may further increase, or in some circumstances possibly reduce, risk in the treatment (Harper et al., 2012). Advances in technology have increased the potential for the analysis of the impact of treatment on embryonic development and child health: for example, DNA methylation studies are now possible in tissues (Katari et al., 2009; Turan et al., 2010) and even individual live embryos (Yamagata, 2010), while whole-genome transcriptomic studies are now possible in individual embryos (Shaw et al., 2013; Vassena et al., 2011). The advent of human embryonic stem cell (ESC) research has stimulated interest in early embryogenesis and in particular the regulation of pluripotency early in development (Bruce, 2013; Trouson, 2013 and Torres-Padilla, 2013) and has also provided a useful model for studying the early embryo in the shape of human ESC lines (e.g. cell signalling in embryos and ESC; O’Leary et al., 2012). The Impact of Technology on ART child health arising from all of the above, there has been an increased awareness of the potential impacts of assisted reproduction treatment on embryo development and child health. In the UK, the recently updated NICE guidelines highlight both the

Table 1 The risk/benefit assessment of assisted reproduction treatment, determined by the theoretical basis behind a technology (should it work?), the evidence for effectiveness (does it work?), an estimate of risk and, finally, the likely costeffectiveness of the procedure.

x x

Should it work?

Non-invasive technologies Metabolomics Amino acids Time lapse

xxx

x Invasive preimplantation genetic screening Polar body 8-cell Blastocyst

Does it work?

Risk

Is it worth it? Risk/benefit ratio

Cost-effectiveness

? Yes Yes xxxx xxx

No Yes Yes?

– Low Low

– Yes? Yes

– ? ?

? Yes ?

? No ?

Moderate High Moderate

? – ?

? – ?

? = Uncertain; – = not applicable. Adapted from Brison et al. (2007).

How should we assess the safety of IVF technologies?

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Novel processes in IVF

Figure 2 Human Fertilisation and Embryology Authority (HFEA) Chair’s letter CH(11)06, September 2011, containing guidance on the requirements for assessing novel technologies in assisted reproduction. Note that clinics who wish to use a novel process are obliged to provide evidence that it is safe and effective.

paucity and the importance of follow-up studies and research into health outcomes (NICE, 2013). Assisted reproduction technologies have always been validated for efficacy and safety in the UK; however, the system has changed over the last few years. The requirement was formalized by the transposition of the European Union Cells and Tissue Directives (EUCTD) into the HFE Act in July 2007. Very recently, the HFEA has changed its approach to regulating novel technologies away from approval through the centre-based licensing process, to a more sector-wide approach that involves a greater role for scientific advice from experts in the field. Thus clinics have been required since 1 October 2011 to assume responsibility for validating novel technologies introduced into their clinical practice (Chair’s letter CH(11)06, September 2011; Figure 2). This requirement puts responsibility into the hands of professionals, particularly the clinics’ Scientific Directors, who, along with the Persons Responsible to the HFEA (in the UK), will share accountability for many of the technologies in question.

How should new technologies be validated? The need to validate new technologies and practices in assisted reproduction was recently highlighted by Van Steirteghem (2008) and Harper et al. (2012), and prior to that by Brison and colleagues (Brison, 2005; Brison et al., 2004, 2007). One important step is for new technologies to be assessed for risk as well as potential benefit (e.g. Brison et al., 2004) and hence to calculate potential risk/benefit ratios (Brison et al., 2007) (Table 1).

Harper et al. (2012) went on to describe a paradigm by which new technologies might be assessed, including through basic animal research, preclinical research using human gametes/embryos, small-scale clinical studies and larger-scale randomized controlled trials (Evers, 2013). Following such a pathway would allow new technology to be introduced in a safe and controlled manner and would then be complemented by long-term, follow-up studies of child health (Figure 3; Harper et al., 2012). Animal research has generated much of our understanding of early human embryo development and has at least partly underpinned advances in assisted reproduction treatment. The majority of IVF technologies have arisen from basic animal research, notably the IVF procedure itself (although famously, not ICSI), embryo culture, addition of growth factors to embryo culture media (Sjo ¨blom et al., 1999, 2005) and use of non-invasive metabolic assays to determine embryo health (reviewed by Leese, 2012). It should be noted that data extrapolation from animal models is not always applicable to humans, for reasons of species differences and also genetic background, since the human population is outbred and many animal models, notably mouse, are highly inbred. A full consideration of the role of animal research in assisted reproduction is outside the scope of this article, but is covered in more detail in Me ´ne ´zo et al. (2013), Harper et al. (2012) and elsewhere in this issue (Bruce, 2013; Gosden, 2013; Torres-Padilla, 2013). Similarly, consideration of the way in which new technologies are tested in clinical trials once preclinical research has been completed is also outside the scope of this article.

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When and how should new technology be introduced into the IVF laboratory?

Should it work?

Does it work?

Risks?

Is it worth it?

Follow up studies of ART children

Figure 3 The pathway for assessing new technologies in assisted reproduction, from preclinical research on animal and human gametes and embryos through to assessment of clinical and cost-effectiveness. Modified from Harper et al. (2012).

The topic is discussed thoroughly in Evers (2013), who highlights the longstanding shortcomings of the assisted reproduction field in terms of evidence-based medicine. It may be that practitioners are finally learning this lesson, as there have been a number of noteworthy attempts recently to subject new technologies to clinical trials (preimplantation genetic screening, Mastenbroek et al., 2007; metabolomics, Hardarson et al., 2012; supplementation of culture medium with growth factors, e.g. Granulocyte macrophage colony stimulating factor (GMCSF), Ziebe et al., 2013; www.clinicaltrials.gov NCT00565747), selection of spermatozoa using hyaluronate binding, HABSELECT (http:// www.controlled-trials.com/ISRCTN99214271/). Commercial companies play a key role here as the developers of new technologies and products, in preclinical testing and validation of products, not least the significant investment required in clinical trials. Once accepted into routine use, it is essential to then continue to monitor the outcomes of assisted reproduction treatment using sophisticated data analysis and statistical modelling on large-scale multicentre data, including those data held in national registers such as those held in the UK by the HFEA and in the Scandinavian countries. Recent examples include data analysis and statistical modelling aimed at determining factors which predict both live birth and multiple births, as part of the drive to refine single-embryo transfer strategies in the UK to reduce the incidence of multiple births. These include an analysis of maternal age (Lawlor and Nelson, 2012) and the use of the embryo–uterus model employed by our group as part of the TowardSET project (Roberts et al., 2010a,b). Current registry data are inevitably limited in extent and do not include data on many important treatment variables related to developing technology. Extending these registries to include such data would be challenging given the rapid pace of technological development. Additionally, as discussed in

Roberts et al. (2010a), data collected for regulatory purposes is not optimal for addressing research questions and often lacks appropriate quality control. However, given that single-centre data lack statistical power even to detect modest differences in treatment outcome rates, there is an urgent need for multicentre, preferably national or even international, initiatives to collate high-quality and detailed data to allow independent evaluation of technologies in both the short and longer term. The growing use of electronic patient records may offer an opportunity for such initiatives, but attention must be given to future monitoring and research needs at the time of inception so that data completion and accuracy can be ensured. We now go on to describe our efforts to address key neglected areas of the technology validation pathway, namely preclinical research using human gametes/embryos, and long-term, follow-up studies of assisted reproduction treatment child health.

Preclinical research using human gametes and embryos Omics: genomics, transcriptomics, proteomics, metabolomics Human embryos contain few cells and only very small amounts of genetic material, mRNA transcripts, proteins and metabolites. As a result, much of our knowledge of the impact of assisted reproduction treatment on the human embryo comes from assessments of embryo developmental progression in culture and embryo morphology, including both traditional static assessments (Alpha, 2011) and, more recently, morphokinetic assessments made using time-lapse microscopy (Aparicio et al., 2013; Wong et al., 2010). More detailed molecular and biochemical data have

How should we assess the safety of IVF technologies?

Embryos and - OMICS Genome

Phenotype

DNA sequence, SNPs Karyotyping, CGH arrays

Transcriptome

Parts of cell systems biology

Synthesis

Reductionism

RT-PCR amplification cDNA array RNAseq

Proteome

Immuno techniques 2D gels+Mass spec Post translational

Metabolome - low MW

HPLC, Mass spec IR, Raman, NMR

- metabolites

Whole system

Figure 4 Embryos and omics: the different levels of omics technologies which can be used for the assessment of single human embryos, with closeness to phenotype increasing from left to right. CGH = comparative genomic hybridization; HPLC = high-performance liquid chromatography; IR = infrared; MS = mass spectrometry; MW = molecular weight; NMR = nuclear magnetic resonance; RT = reverse transcription; SNP = single-nucleotide polymorphism. OMICs technologies are part of a Systems biology approach to understanding the function of the entire cell (embryo), rather than the traditional reductionist approach of considering only aspects of function.

had to wait for the development of technologies sufficiently sensitive to yield results from single cells. These have accumulated slowly but are now with us, and as a result we can collect omics data from individual human embryos on: metabolism (Brison et al., 2004; Leese, 2012; Leese and Barton, 1984; Sturmey et al., 2008), genomics (Handyside et al., 1989; Hens et al., 2013) and, more recently, the embryonic transcriptome at the level of individual embryos and even blastomeres (Galan et al., 2010; Shaw et al., 2013; Vassena et al., 2011; Figure 4). Of these analytic technologies, genomics technologies are discussed extensively elsewhere (Beim et al., 2013) and metabolomics are considered briefly below. However, arguably the most useful readout of embryo health following assisted reproduction treatment is provided by transcriptomics or gene expression profiling. Transcript expression is a measure of the function of the genome and, although mRNA still needs to be translated into protein products and secondarily generate metabolites and other intermediaries which function in the cell, a large amount of information can now easily be obtained from transcript patterns. In preimplantation embryos, gene expression patterns take on particular significance because, following fertilization, the embryo reorganizes and expresses both maternal and paternal genes (embryonic genome activation) (Braude et al., 1988; Niakan et al., 2012). Embryonic genome activation is essential to the development of the embryo and can thus be used as a global marker of embryonic health. Moreover, gene expression during preimplantation development is subject to epigenetic regulation (Chason et al., 2011) which can potentially respond to environmental cues (Bruce, 2013; Mann and Denomme, 2013; Padmanabhan and Watson, 2013; Torres-Padilla, 2013). As a result of these controls on gene expression, together with chromosomal mosaicism which is well documented in human embryos (Delhanty

715 and Handyside, 1995), it is clear now that human embryo development is highly heterogeneous (i.e. variable from embryo to embryo). In animal models such as inbred mice, gene expression studies commonly pool large numbers of embryos for analysis, but in the human, we and others showed many years ago that this obscures essential information by masking differences between embryos of likely differing developmental competence (Bloor et al., 2002; Metcalfe et al., 2004). As a result, our group has focused on analysis of individual embryos, using polyA-PCR amplification to provide an unbiased source of transcripts for analysis in the form of cDNA (Bloor et al., 2002; Brady and Iscove, 1993; Kimber et al., 2008). This approach has provided an archive of stored amplified cDNA from a range of embryos from oocyte to blastocyst, including separated inner cell mass and trophectoderm lineages, individual blastomeres and equivalent cells from ESC lines. These oocytes and embryos have all been donated to research following informed and written patient consent, under ethics committee approval and licensed by the HFEA. The majority of embryos were generated by conventional IVF or ICSI treatment and donated to research when surplus to requirements; however, a subset of embryos were generated from oocytes chemically activated to yield embryos specifically created for research purposes (Camarasa et al., 2010; DeSousa et al., 2009; Sneddon et al., 2011). This archive of amplified single-embryo cDNA has allowed us to establish the molecular anatomy of human preimplantation embryo development and to assess how it varies in response to assisted reproduction treatment. The approaches used to assay gene expression depend partly on the biological problem being addressed and also on continual advances in technology. However, although the approach is powerful, it is constrained by the scarcity of material and by the ethical difficulties inherent in obtaining ‘normal’ control embryos. Thus, we have access to embryos consented by couples with a variety of fertility-related problems. As such, we cannot assume the embryos developing in the culture dish represent identical molecular profiles to those in vivo or provide the optimal healthy molecular profile and time course. We do not have known healthy control embryos and must rely on surrogates for evaluation of normalcy such as morphology and occurrence of a sibling pregnancy. In utilizing these data, we must therefore look for diagnostic molecular patterns and test their predictive power thoroughly before using these patterns for making clinical choices. For an in-depth discussion of the validation of Omics technologies, see Scott and Treff (2010).

Candidate gene approach Using this approach, we have been able to examine markers, or families of markers, of embryo developmental competence and cell fate and their response to assisted reproduction treatment. We were able to show initially that oocytes and embryos express a range of apoptosisregulatory BCL2 family molecules which partly determine whether cells, including blastomeres, live or die. The expression patterns and overall balance between pro- and anti-apoptosis members altered with embryo culture and in embryos which underwent abnormal development (fragmen-

716 tation) (Metcalfe et al., 2003, 2004). We went on to look specifically at markers of embryonic cell fate, including genes which determine cell lineage (pluripotency genes and markers of inner cell mass, trophectoderm and primitive endoderm). We showed that human embryos respond to peptide growth factors added to culture medium by altering expression of some of these genes (Kimber et al., 2008). Of particular note was the finding that embryos respond to growth factors leukaemia inhibitory factor (LIF) and heparin-binding epidermal growth factor (HBEGF) in a reciprocal manner: i.e. they upregulate LIF receptor in response to HBEGF, and the HBEGF receptor ErbB4 in response to LIF (Kimber et al., 2008). The same logic was applied to gene expression patterns in fresh embryos compared with those which had been cryopreserved and thawed. In this study we found that cryopreserved embryos showed altered expression of a number of genes and notably there was some shift in expression of genes marking pluripotency versus trophectoderm differentiation (Shaw et al., 2012). Of interest, we did not see altered expression of the TSC2 gene with freezing, which had been reported previously by Tachataki et al. (2003). Most recently, we have looked at the impact of vitrification on oocyte gene expression patterns. Using vitrification, it is possible to carefully control the experimental conditions since vitrification and warming, unlike slow freezing, can be performed in a matter of minutes, with sham-treated sibling oocytes available as controls. We have further used this methodology to assess the impact of different variables in the vitrification process (S. Abdelsalam, C.T. Fitzgerald, S.J. Kimber, D.R. Brison, unpublished data). Preclinical testing of this type using oocytes donated to research may allow methods to be optimized further in the preclinical phase. Although oocytes used for research are generally only available following failed fertilization, we have shown that these can remain developmentally competent. Following chemical activation using a conventional parthenogenetic stimulation protocol, we showed that embryos arising from failed to fertilized and immature oocytes matured in vitro, showed normal patterns and levels of expression of genes marking pluripotency and trophectoderm differentiation (Sneddon et al., 2011; Figure 5).

Microarrays: the systems biology era The use of microarrays has now moved gene expression profiling into the area of systems biology. Rather than studying candidate genes or even families of related genes as described above, it is now possible to screen the entire expressed genome (transcriptome) of individual human embryos, inner cell mass and trophectoderm samples and even isolated blastomeres using the amplified cDNA archive described above. This approach has enabled great insight into early developmental biology in the human including qualitative information on the expression of a range of regulatory and signalling pathways (see Shaw et al., 2013). Furthermore, use of these datasets in comparison with equivalents from human ESC lines also affords us the opportunity to perform functional analyses using these lines. Array technology also now permits quantitative estimates to be made of embryonic genome activation in human embryos (Shaw et al., 2013; Vassena et al., 2011) and has allowed quantification of the extent of heterogeneity

DR Brison et al.

Control

Activated

Control

Activated

Figure 5 Immunofluorescence assessment of pluripotency markers Oct4 (A–D) and Sox2 (E–H) in blastocysts derived from oocytes following chemical activation (C, D, G, H; Sneddon et al., 2011) compared with conventional IVF (A, B, E, F). Green indicates proteins; blue indicates nuclei (DAPI). Bar = 100 lm.

between human embryos at the same developmental stage (Shaw et al., 2013), using bioinformatic analytical methods such as principle component analysis plots and heatmap clustering (Figure 6). These methods give global readouts of the impact of technology on the transcriptome of the embryo, allowing us to ask the question ‘does a particular assisted reproduction technology leave a gene expression fingerprint?’

ESC lines as markers of embryonic health Human ESC lines provide a model system for understanding events such as the regulation of pluripotency and cell fate decisions in early human embryos (e.g. O’Leary et al., 2012; Trouson, 2013). They can also be used as a model for understanding early development, allowing us to start to assess the role of genes and genes families in human development through differentiation of human ESC. However, they can also provide a practical readout of the development status or health of the embryo from which the line was derived, and therefore have considerable utility in assessing the impact of a new assisted reproduction technology. For example, we showed that activated failed-to-fertilize oocytes could give rise not only to normal human embryos but also to human ESC lines which were

How should we assess the safety of IVF technologies?

717

Figure 6 Principle component analysis of transcript levels in individual oocytes (purple), 4-cell embryos (orange) and blastocysts (green). A and B present the same 3D plot rotated on the horizontal plane. Courtesy of Dr Helen Smith.

biparental, genetically normal, and they showed transcript profiles similar to other lines derived from conventional IVF embryos (Camarasa et al., 2010; DeSousa et al., 2009).

Metabolomic and metabolic profiling approaches The metabolic profile of individual human embryos can be easily measured by analysing culture medium, as pioneered by Leese and colleagues (for review, see Leese, 2012) and continued into studies on the secreted metabolome of the embryo (Brison et al., 2007; Hardarson et al., 2012; Hollywood et al., 2006). These methods have the ability to predict embryo developmental capacity (Brison et al., 2004; Gardner et al., 2011) and can therefore be used not only to select embryos for transfer (Brison et al., 2004; Hardarson et al., 2012) but also to assess the impact of a new assisted reproduction technology, particularly allied to other omics such as transcriptomics.

Summary In our view, it is essential to study the impact of new technologies on human embryo development using human embryos donated to research as fresh embryos, cryopreserved embryos, fresh or cryopreserved oocytes which can be activated to generate embryos for research and human ESC lines generated from equivalent embryos. Gene expression microarray technology currently holds the most promise for assessing the impact of new technologies because it can be used on single embryos, although there will likely be a valuable role for the proteome and metabolome/metabolites as these are closer to phenotype than mRNA transcripts.

Follow-up studies of assisted reproduction children Ultimately, once a novel technology has been assessed according to the pathway described in Figure 3 (Harper et al., 2012) and enters into clinical use with sufficient evidence base to demonstrate its efficacy and cost-effective-

ness, the remaining essential step is to conduct follow-up studies to ensure the health of children born from the technology. Although there seems little question that, overall, the health of the five million and more assisted reproduction children born to date does not give cause for concern, unfortunately the history of assisted reproduction treatment is littered with the debris of small-scale, cohort follow-up studies. As already discussed, the majority of these carried out to date have arguably served to alarm or to provide false reassurance, in equal measure, and the conclusions are frequently conflicting, first because most studies are insufficiently powered to detect small-scale but potentially important changes in health parameters (Evers, 2013), and secondly because, until recently, we have not been aware of all of the significant parameters which ought to be evaluated, including consideration of the impact of assisted reproduction treatment. The accumulation of data in large national assisted reproduction registers in a number of countries in recent years has provided the opportunity to address at least the first of these issues by conducting large-scale child health screens via linkage to health outcome databases. The Scandinavian countries have led the field in using linkage between population-based health and treatment records and these valuable studies now need to be repeated in other countries, including other assisted reproduction treatment, for example in North America making use of the SART database. The UK has the largest and longest established assisted reproduction database, consisting of over 110,500 children born between August 1991 and October 2009. It contains offspring up to age 22 currently, an essential feature when planning studies of early-onset adult diseases. The UK resource is much broader in scope and data-rich compared with other similar national databases, a fact important when considering the role of particular technologies (Fauser et al., 2005; Nelson and Lawlor, 2011; Roberts et al., 2010a). As already mentioned, this invaluable resource is now available for data linkage studies following the issue of regulations permitting the disclosure of the data. The first such study has been carried out by Sutcliffe

718 and colleagues into the risk of cancer in children following assisted reproduction treatment (Williams et al., in press). Another study has been proposed by our group (S.A. Roberts, N.M. Macklon, D.R. Brison, in press), as part of the EU consortium EpiHealth, which aims to examine the impact of a number of assisted reproduction technologies on child health (http://epihealth.biotalentum.eu). These and other UK population-based studies that will follow will play a fundamental role in assuring the health of UK assisted reproduction children and facilitating risk assessment of new technologies. In order to maximize the use of such a resource for the benefit of patients and the public, it will be important to set up robust systems to link the HFEA register to health outcome records. It is equally important for the sector to collect as much detailed data as possible on assisted reproduction cycles, including the use of new technologies such as new media formulations and novel equipment, in order to facilitate long-term safety assessment. Although the HFEA has no statutory duty to collect these data, it currently provides an important service to the sector. The availability of enhanced register-based data would also be invaluable to the manufacturers of culture media, the status of which, as a CE-marked class III medical device, requires manufacturers to carry out post-market clinical follow up of products. Available guidance suggests that this process can be enhanced by use of national data registries (MHRA, section VIII).

Conclusion Technology brings challenges as well as opportunities. The opportunities are to introduce new procedures and technologies in assisted reproduction treatment which are effective in terms of improving outcome rates and thereby reducing stress to patients caused by repeated attempts at IVF. Equally important is reducing risk by producing more effective techniques and, above all, facilitating single-embryo transfer to avoid multiple births. The challenges are make sure that these technologies are indeed effective and, above all, safe, by using preclinical testing, clinical trials and follow-up studies. The responsibility for the effectiveness and safety of assisted reproduction treatment remains firmly with clinicians and scientists in the field. We now call on funding bodies to provide support for such research. The extent to which we as a community are able to carry out such studies will determine the legacy of assisted reproduction treatment and the ultimate significance of the award of the Nobel Prize to Bob Edwards in 2010.

Acknowledgements The authors would like to acknowledge CRUK, MRC, the Department of Health and the EU FP7 Health programme for funding and Cheryl Fitzgerald, Henry Leese and staff of the Human Fertilisation and Embryology Authority for helpful comments.

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