Elective Single Embryo Transfer: an update to UK Best Practice Guidelines

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1 Human Fertility an international, multidisciplinary journal dedicated to furthering research and promoting good practice ISSN: (Print) (Online) Journal homepage: Elective Single Embryo Transfer: an update to UK Best Practice Guidelines Stephen Harbottle, CIara Hughes, Rachel Cutting, Steve Roberts, Daniel Brison & On Behalf Of The Association Of Clinical Embryologists & The (ACE) British Fertility Society (BFS) To cite this article: Stephen Harbottle, CIara Hughes, Rachel Cutting, Steve Roberts, Daniel Brison & On Behalf Of The Association Of Clinical Embryologists & The (ACE) British Fertility Society (BFS) (2015) Elective Single Embryo Transfer: an update to UK Best Practice Guidelines, Human Fertility, 18:3, To link to this article: Published online: 22 Sep Submit your article to this journal Article views: 2595 View related articles View Crossmark data Citing articles: 10 View citing articles Full Terms & Conditions of access and use can be found at Download by: [ ] Date: 21 November 2017, At: 16:17

2 Human Fertility, 2015; 18(3): The British Fertility Society ISSN print/issn online DOI: / POLICY & PRACTICE Elective Single Embryo Transfer: an update to UK Best Practice Guidelines Stephen Harbottle 1, CIara Hughes 2, Rachel Cutting 3, Steve Roberts 4 & Daniel Brison 5 On Behalf Of The Association Of Clinical Embryologists & THE (Ace) British FERTILITY Society (Bfs) 1 Cambridge IVF, Cambridge University Hospitals NHS Trust, Kefford House, Maris Lane Cambridge, UK, 2 HARI Clinic, Rotunda Hospital, Dublin, Eire, 3 Jessop Fertility, The Jessop Wing, Tree Root Walk, Sheffield, UK, 4 Center for Biostatistics, Institute of Population Health, University of Manchester, Oxford Road, Manchester, UK, and 5 Department of Reproductive Medicine, Old St Mary s Hospital, Central Manchester University Hospitals NHS Foundation Trust, Manchester Academic Health Sciences Centre, Oxford Road, Manchester, UK Abstract A significant number of multiple pregnancies and births worldwide continue to occur following treatment with Assisted Reproductive Technologies (ARTs). Whilst efforts have been made to increase the proportion of elective single embryo transfer (eset) cycles, the multiple pregnancy rate or MPR remains at a level that most consider unacceptable given the associated clinical risks to mothers and babies, and the additional costs associated with neonatal care of premature and low birth weight babies. Northern Europe, Australia and Japan have continued to lead the way in the adoption of eset. Randomised controlled trials or RCTs, metaanalyses and economic analyses support the implementation of an eset policy, particularly in light of recent advances in ARTs. This paper provides a review of current evidence and an update to the eset guidelines first published by Cutting et al. (2008) intended to assist ART clinics in the implementation of an effective eset policy. Keywords: Elective single embryo transfer (eset), multiple pregnancy, ART, IVF, ICSI, ACE, BFS, guidelines Introduction In 2009, the Human Fertilisation and Embryology Authority (HFEA) introduced a policy to encourage fertility centres to adopt elective single embryo transfer (eset) into routine use (HFEA, 2008). This policy required a phased reduction in the multiple birth rate (MBR) from 24 % in 2009, 20 % in 2010, 15 % in 2011, to 10 % in 2012 and thereafter. Initially these targets formed a license condition, but in early 2014 a legal challenge to the HFEA by a UK-licensed fertility centre resulted in the removal of the license condition for all centres (HFEA, 2013). Despite this, the HFEA, the Association of Clinical Embryologists (ACE), the British Fertility Society (BFS) and other stakeholder professional organisations remain fully supportive of the One at a Time project supporting eset and committed to the current 10 % MBR target. The most recent HFEA trends and figures report (HFEA, 2014) revealed that clinics continue to replace two embryos in the majority of fresh or frozen IVF or ICSI procedures (51.0 % down from 56 % in 2012) with 27.0 % (up from 22.2 % in 2011) receiving eset. An examination of the annual HFEA data published from 2008 to 2013 reveals an encouraging and sustained trend in reduction of multiple pregnancy rate (MPR) from 26.6 % in 2008 to 16.3 % in During this time period, the gradual improvement in live birth rate (LBR) from IVF and ICSI treatment, which has been observed since 1994, remained unaffected. What is clearly evident is that although an on-going downward trend in MBR has been observed, further adjustments to clinical practice must still be made if the 10 % recommended target is to be achieved whilst maintaining the LBR. The message from Europe is clear: countries such as Sweden, Finland and Belgium have clearly demonstrated that a MBR of less than 10 % is achievable. Sweden continues to report the highest percentage of Correspondence: Stephen Harbottle, Cambridge IVF, Cambridge University Hospitals NHS Trust, Kefford House, Maris Lane Cambridge, UK. Tel: Fax: stephen.harbottle@addenbrookes.nhs.uk (Received 10 March 2015; revised 16 April 2015; accepted 6 August 2015) 165

3 166 S. Harbottle et al. SET at 69.9 %. In 2010, Kallen et al. (2010b) examined trends in delivery and neonatal outcome from the Swedish National Heath Register dataset spanning over 25 years. The study included in excess of 25,000 women receiving IVF treatment over the 5-year period following the introduction of eset and showed that the risk of neonatal death and morbidity was significantly reduced. When the first edition of the ACE/BFS guidelines was published (Cutting et al., 2008), many of the Northern European countries had implemented eset, but only Belgium and Sweden had legislation underpinning it. In 2010, Turkey introduced new legislation mandating SET in women less than 35 years of age in the first or second cycle of treatment and limiting clinics to the transfer of two embryos in the third or subsequent treatment cycles and in women over the age of 35 years (Kultursay et al., 2015). Since the implementation of this policy, preliminary results reported from a small multi-centre study have shown a significant reduction in MPR, with a modest downward trend in clinical pregnancy rate (CPR) from 39.9 % to 34.5 %, although this was not statistically significant (Kutlu et al., 2011). In 2010 the provincial government of Quebec introduced eset legislation with the aim of performing SET in every treatment cycle (Bissonnette et al., 2011). The legislation states that multiple embryos can only be transferred in cases where it is justified by the treating physician and on a case-by-case basis. In the first 3 months of the programme, 5 centres performed 1353 IVF cycles with an overall CPR of 32 % per embryo transfer (compared with 43 % prior to the policy being introduced), with 50 % of transfers using eset. During the same time period, the MPR was reduced from 25.6 % to 3.7 %. Prior to this legislative change, eset was performed in only 1.6 % of treatment cycles. The fact that central funding was allocated to this project by the provincial health programme must be recognised as a major contributing factor to the successful implementation of the policy in such a short period of time. Velez et al. (2013) showed that the initiative in Quebec reaffirmed the policy of 1 plus 1 should equal 2, with cumulative pregnancy rates from the sequential transfer of one fresh followed by one frozen embryo close to equalling that obtained with the transfer of two fresh embryos together. In the USA only 1 % of fresh transfers prior to 2002 were eset, with a modest increasing trend to 5.6 % overall in 2010, with a 9.6 % eset rate and a 32.4 % twin rate in the 35 age group. However, in 2012 the Practice Committee of the Society for Assisted Reproductive Technology and Practice Committee of the American Society for Reproductive Medicine published eset guidance for patient selection (ASRM, 2012). These guidelines concluded that eset should be promoted and offered to patients with a good prognosis, and to recipients of embryos from donated eggs and that eset must be applied in association with an effective embryo cryopreservation programme. The most recent data from 2013 now show a more than doubling of eset in the good prognosis 35 age group to 22.5 %, and a modest reduction in the twin rate to 28.3 % (SART, 2013). In 2011, Maheshwari et al. (2011) assessed global variation in the uptake of a SET policy (Table I). They reported that it was not possible to distinguish eset from non-elective SET from national ART reports but that in over 31 countries there had been a gradual increase in the rate of SET. They suggested that public funding, availability of effective cryopreservation, and national legislation remained the main contributory factors in the establishment of effective SET policy. Whilst legislation in the UK has defined targets the worldwide picture is very variable. Few countries have specific legislation, some have guidance, whist other countries still endorse double embryo transfer (DET). There does however seem to be consensus that embryo quality and age should be taken into account during the decision process (Table I). Aim The aim of this guideline review is to provide updated evidence-based guidance, to assist UK fertility centres in the implementation of effective eset policy with the intention of reducing MPR, whilst not discounting the patients right to choice and autonomy. The revised guideline remains highly supportive of the One at a Time initiative in the UK, which continues to encourage the practice of eset. Scope The guideline revision will address the need for eset, patient and embryo selection, superovulation practices, cryopreservation strategies, the role of the HFEA and cost implications. Levels of Evidence The hierarchy of evidence and system for grading the strength of the evidence used in this paper are outlined in Table II, as developed by the National Institute for Health and Care Excellence (NICE). Safety The safety of IVF procedures will be a major consideration throughout. IVF as an intervention occurs at a point in early human development when the embryo is uniquely vulnerable, due to the complete reorganisation of the genome and epigenome immediately after fertilisation. There is general appreciation now that the peri-conceptional environment can influence long-term programming of health in offspring, via largely epigenetic mechanisms (Watkins et al., 2010; Lucas, 2013). Human Fertility

4 ACE and BFS Elective Single Embryo transfer guidelines Table I. Summary of Global legislation in relation to eset (adapted from Maheshwari et al., 2011). Country This concern is consistent with the long-standing observation that IVF singleton babies are at increased risk of low birth weight (Hansen et al., 2002). With respect to technologies used in selection of embryos for eset consideration should be given to the potential risk of the technology, set against the accepted benefit of reducing the incidence of multiple pregnancies (see above). The impact of ART and environmental stresses on embryo development and long-term disease is a priority area for research. Recommendations Safety 1. Further research is required (a) to validate the efficacy of eset policies and (b) confirm the long-term safety of the culture and cryopreservation processes that constitute such policies. (C) Is there still a role for DET? Results from randomised controlled trials (RCTs) comparing eset with DET clearly show that the chance of live birth is reduced, unless eset is combined with an effective programme for the transfer of a subsequent Legislation Australia No legislation; eset advised 35 on first fresh cycle. No more than 2 advised 40 Austria Legislation restricts the number of eggs which can be used in IVF. All fertilised embryos must be transferred Belgium Legislation; 36 first cycle must have eset Canada No overall legislation only guidelines suggesting eset in patient 35 years in a first or second cycle and consider eset in patients 36 &37 with good prognosis. Quebec introduced legislation in 2010 which states every cycle should be eset unless there are suboptimal conditions and clinical justification is given Denmark No legislation Estonia Max of 3 can be transferred Finland No legislation France Restricted to DET. If more than 2 clinical justification required Germany Legislation restricts the number of eggs which can be used in IVF. All fertilised embryos must be transferred Greece No legislation; recommended up to 3 embryos transferred for women up to age of 40 and up to Hungary State regulation limits number to transfer to 3 except in exceptional circumstances when up to 4 can be transferred India Guidelines: Max of 3 can be transferred Italy Law restricts number of eggs which can be fertilised, all resulting embryos must be replaced Japan Recommendation by Japanese Society of Obs and Gynae; max 2 embryos since 2008 Luxemburg No legislation Netherlands No legislation New Zealand Guidance related to funding. If publically funded 35, first or second cycle eset. No more than 2 embryos (fresh or frozen) 40. Norway No legislation Portugal Law contains general addict that multiple embryos should be avoided if possible Sweden State legislation states in principle only 1 embryo should be replaced apart from in exceptional circumstances Switzerland Legislation restricts the number of eggs which can be used in IVF. All fertilised embryos must be transferred Turkey Legislation in March 2010 favouring eset - limits 35 to eset and 35 to DET. USA No overall legislation. Guidelines (ASRM, 2013a) suggest eset should be offered to patients 35 and no more than 2 embryos should be transferred. Patients no more than 2 cleavage embryos in favourable prognosis patients or if blastocyst transfer. If using cleavage stage transfer in poor prognosis no more than 3 embryos should be replaced. No more than 3 embryos cleavage or 2 blastocysts in patients years with favourable prognosis. Other patients in this group no more than 4 cleavage embryos or 3 blastocysts transferred. In patients no more than 5 cleavage stage embryos or 3 blastocysts. In patients with 2 or more previous failed cycles one additional embryo can be transferred in each age category. No limit on patients over 43 years old In donor cycles if the donor is 35 eset should be strongly recommended Key: eset elective single embryo transfer; DET Double Embryo Transfer. frozen embryo (Lukassen et al., 2005; van Montfoort et al., 2006; Moustafa et al., 2008). This supports the earlier study by Thurin et al., (2004) which reported that the LBR following eset was significantly reduced compared with DET. However, the cumulative LBR following subsequent replacement of a single frozen embryo in the eset group was equivalent to the DET group (39 % vs. 43 %). A Cochrane Database System Review has supported these findings (Pandian et al., 2009). More recently, McLernon et al., (2010) published a meta-analysis of 8 RCTs of cleavage-stage eset versus DET, 5 of which had been published in peer-reviewed journals. This showed a cumulative LBR from 2 eset (fresh frozen) cycles with day 2/3 transfer slightly but not statistically significantly lower than that from a single DET cycle (38 % vs 42 %). In addition, statistical modelling of large treatment cycle datasets including the HFEA register has shown that SET would yield about a one-third lower LBR relative to DET, for any embryo transfer (Roberts et al., 2011). However, this difference in LBR can be mitigated by patient and treatment cycle selection, resulting in an increased LBR with sequential eset, compared with DET, with both fresh and frozen embryo transfers (FETs). Thus, it is undeniable 2015 The British Fertility Society

5 168 S. Harbottle et al. Table II. NICE hierarchy of evidence, and grading the strength of the evidence. 1a 1b 2a 2b Hierarchy of evidence Systematic review and meta-analysis of randomised controlled trials (RCTs) At least one randomised controlled trial At least one well-designed controlled study without randomisation At least one other type of well-designed quasi-experimental study 3 Well-designed non-experimental descriptive studies, such as comparative studies, correlation studies or case studies 4 Expert committee reports or opinions and/or clinical experience of respected authorities A B C GPP Grade strength of evidence Requires at least one RCT as part of a body of literature of overall good quality and consistency addressing the specific recommendations. (Evidence levels 1a, 1b) Requires the availability of well-controlled clinical studies but no randomised clinical trials on the topics of recommendation. (Evidence levels 2a, 2b, 3) Requires evidence obtained from expert committee reports of opinions and/or clinical experiences of respected authorities. Indicates an absence of directly applicable clinical studies of good quality. (Evidence level 4) Good practice point that when combined with an effective cryopreservation programme eset can yield cumulative success rates that at least match, and may exceed, those from DET (Roberts et al., 2009; 2010b). Given the strength of the accumulating evidence in favour of a well-constructed eset programme, why should DET still be considered, given the attendant risk of twin pregnancies? Whilst cumulative cycles with cryopreservation maintain success rates, it is important to consider that this strategy requires multiple cycles and adds an emotional, physical and financial cost to purchasers, either through state funding or (depending on the costing models) patients. Interviews and focus groups conducted with patients undertaking fertility treatment continue to indicate a clear patient preference for twins despite the potential to maintain overall LBR with an eset strategy (Leese & Denton, 2010; Roberts et al., 2010b). It is therefore vital when considering eset that clinics ensure the needs of patients are taken into account and that patient preference is not seen as irrational. However, this must be considered in context with the short- and longer-term consequences of avoidable multiple pregnancy and birth. In particular, the desire for patients to have more than one child should be taken into consideration and eset policies need to allow funding for the transfer of all embryos of transferrable quality, regardless of earlier outcome, if eset is not to be disadvantageous in this respect. Secondly, the extended treatment duration in a complete eset programme may be particularly disadvantageous to older women whose fertility is declining rapidly. Although the evidence suggests that oocyte aneuploidy is the biggest limiting factor in older women, there is also some decline in uterine receptivity (Legro et al., 1995; Stolwijk et al., 1997; Toner et al., 2002; Roberts et al., 2010a). More significantly, repeat egg collections following failure will occur at later ages in an eset programme, reducing the chances of a successful outcome. This could potentially be mitigated by offering multiple oocyte retrievals or ORs followed by cryopreservation and eset. Finally, although the economic costs from a total health service perspective are reduced with eset (Scotland et al., 2011), this is not true of the direct costs to fertility service providers, or to patients in privately funded treatments. Selection of patients for DET Despite the evidence in favour of eset it is likely that DET will remain part of clinical practice in the UK for the foreseeable future and the question arising is for which patients should DET still be considered. The available evidence suggests that the relative chance of twins is similar for all patients regardless of the usual prognostic factors (Roberts et al., 2010b) with no specific risk factors for twinning, other than embryo quality, being identified. This of course means that the absolute chance of twins is greatest in patients with the best prognosis. In an extensive analysis of the UK HFEA register data, Lawlor and Nelson (2012) concluded that the transfer of three or more embryos at any age should be avoided and that the decision to transfer one or two embryos should be based on prognostic indicators, such as age. Thus there is, at the time of writing, no clear highquality evidence for selection of patients for inclusion in an eset or DET strategy and there are no clear predictors of the likelihood of twins other than stage of embryo development at transfer, and embryo quality. Selection is therefore based on local or anecdotal evidence or a pragmatic financial decision to include those patients most likely to have twins because they are most likely to become pregnant following treatment. A number of very similar policies for selecting patients for DET have been suggested, which essentially target eset at those with the best prognosis and therefore reduce the overall twin rate, whilst allowing DET for the poor prognosis patients to maintain their single transfer success rate. In the NICE (2013) guidelines, several factors were recommended which should be considered when determining an individual patient s suitability for eset. These include maternal age, obstetric and gynaecological history, previous treatment history, ovarian response or reserve, number of embryos created and quality of embryos or blastocysts available for transfer, and cryopreservation. For women under the age of 37 years undertaking their first treatment cycle, the NICE (2013) guidelines advocate eset. These guidelines largely support the eset algorithm proposed by Cutting et al. (2008), although they are not specific in terms of the day on which embryo transfer should be performed Human Fertility

6 ACE and BFS Elective Single Embryo transfer guidelines other than to recommend eset in circumstances where at least one top-quality blastocyst has developed. The NICE guidelines propose additional recommendations for women in the age ranges of and years (Table III). The relationship between LBR and maternal age is well established (Dessolle et al., 2011; Sifer et al., 2011). In the eset algorithm published by Cutting et al., (2008), it was recommended that eset should be routinely applied to patients 37 years of age. However, a recent report has challenged whether eset should be restricted solely to younger women. Niinimaki et al., (2013) examined whether eset for older women between 40 and 44 years could be feasible. This retrospective cohort study appears to be the first to show such a large number of older women consenting for eset: 42 % of the study population. In fresh cycles, the CPRs were 23.5 % and 19.5 % in the eset and DET group, respectively, with LBRs of 22.7 % and 13.3 %, respectively (P 0.002). This study was limited by an inability to compare the two groups directly as the suitability for eset had been individually assessed by a clinician and not randomised. These findings were challenged by Gleicher (2013), who in addition to questioning the rationale for eset in older women with reduced prognosis questioned the irrational attraction of eset. He commented on the ethics of urging older women to reduce their immediate pregnancy chances and suggested that under most circumstances women of advanced age cannot be considered to have a favourable prognosis. Gleicher highlighted the point that overall, the risk associated with a twin pregnancy (one live birth event) compared with two singleton pregnancies (two separate live birth events) becomes reduced. A retrospective analysis of the outcome and feasibility of eset in the first and second transfers was reported by Gremeau et al. (2012). The study showed that from a patient group of 783 couples embarking on a first cycle of IVF or ICSI treatment, the on-going pregnancy and cumulative delivery rates from eset and DET cycles did not differ significantly (30.9 % and 34.6 %, respectively) with 67.6 % of the transfers being eset. For the second treatment attempt, the eset rate dropped to 16.9 % suggesting that eset is less well accepted by patients for their second attempt, although it should be noted that patient acceptability and clinicians advice were not measured independently. eset has also been shown to yield a good LBR in oocyte donation programmes. A retrospective study which examined delivery rates before and after the implementation of an eset policy showed a significant reduction in the twin rate, with no impact on the delivery rate (although the study was not powered to examine this) (Soderstrom-Anttila et al., 2003). This observation was supported by another retrospective study, which suggested that when three embryos are available and eset is performed, the cumulative LBR is not compromised (Clua et al., 2012). Following these studies, NICE (2013) recommended using an embryo transfer strategy based on the age of the oocyte donor. Recommendations Selection of patients for DET 1. There should be a continued focus on minimising MBRs from IVF treatment by utilising an effective, dynamic eset strategy. (GPP) 2. In situations where embryo quality is poor, clinics should consider the transfer of two embryos. An effective eset policy should accommodate this. (B) 3. Centres should audit eset rates, selection criteria, and MBRs and where their dataset is robust, consider adjusting their eset algorithm in line with their results on at least an annual basis, focussing on good practice rather than small numbers of multiple births above or below a target rate. (GPP) Superovulation strategies Traditionally, stimulation protocols have induced pituitary downregulation with a gonadotrophin-releasing hormone (GnRH) agonist followed by gonadotrophin stimulation, or the use of a GnRH antagonist to block the luteinising hormone (LH) surge during the stimulatory phase. A Cochrane review of GnRH antagonists in assisted reproduction (Al-Inany & Aboulghar, 2002) and a more recent update (Al-Inany et al., 2011) suggest that overall the antagonist protocol gave a 1.5 % lower LBR and 2 % lower CPR compared with the agonist protocol. However GnRH antagonist use is associated with a lower risk of ovarian hyperstimulation syndrome (OHSS) compared with GnRH agonist (Mathur & Tan, 2014). The need for safety is paramount and the rationale for aggressive stimulation protocols should be questioned in an effort to reduce OHSS and in association with an effective eset strategy to reduce the Table III. NICE (2013) Guidelines Embryo transfer strategy summary table (including fresh & frozen embryos). Attempt First Cycle SET 1 Top Quality Available No Top Quality Available SET Consider DET Consider DET Second Cycle 1 Top Quality Available No Top Quality Available 1 Top Quality Available No Top Quality Available SET Consider DET SET Consider DET * Third Cycle No more than 2 embryos No more than 2 embryos * Key: * NICE only recommend a single treatment cycle in women 40 years of age; SET Single Embryo Transfer; DET Double Embryo Transfer The British Fertility Society

7 170 S. Harbottle et al. incidence of multiple pregnancies. Heijnen et al. (2007) showed that three mild stimulation protocols are as affective as two conventional stimulations in terms of outcome. Baart et al. (2009) described how different ovarian stimulation regimens can affect oocyte and embryo quality. Recommendations Superovulation strategies 1. Maximising oocyte and embryo quality is an essential part of an effective eset strategy; a focus should remain on optimising superovulation regimens. (A) Selection of embryos or blastocysts for eset It is generally accepted that robust embryo selection is a key element in the implementation of an effective eset programme. This has resulted in increased pressure and expectation on Clinical Embryologists to ensure that the embryo with the best potential to deliver a healthy live birth is selected for transfer. Significant progress has been made in recent years with the various techniques employed for embryo selection. Morphological oocyte, embryo and blastocyst assessment a) Oocyte assessment In predicting embryo quality and implantation potential, it is wise to consider the assessment and quality of oocytes as predictors of embryo competence. However, historically there has been conflict in the literature as to the reliability of these observations, particularly during ICSI. Serhal et al. (1997) suggested that anomalies including granularity and the presence of cytoplasmic inclusions such as vacuoles, smooth endoplasmic reticulum clustering, and refractile bodies had a negative impact on embryo implantation rates. In contrast, Balaban et al. (1998) suggested the exact opposite: that oocyte morphology had no direct effect on fertilisation rate, embryo quality and implantation rate following ICSI. More recently, the Istanbul consensus on embryo assessment (ALPHA, 2011) published evidence suggesting oocyte assessment anomalies should be sub-divided into (i) intra-cytoplasmic (incorporations, refractile bodies, dense central granulation, vacuoles and aggregation of smooth endoplasmic reticulum); and (ii) extra-cytoplasmic dymorphisms (first polar body morphology, perivitelline space size and granularity, discolouration, zona pellucida defects, and shape anomalies). ALPHA (2011) also concluded that giant oocytes and oocytes with smooth endoplasmic clusters should be selected against because of their likely abnormal genetic constitution and association with early foetal loss and imprinting disorders, respectively (Otsuki et al., 2004; Ebner, 2008). b) Pronuclear (PN) assessment Assessment of pronuclear (PN) morphology is based on observations of features seen in zygotes hours post-fertilisation. Grading systems were developed to assess the number, position, size and alignment of the nucleolar precursor bodies (NPB) in each embryo. Zygotes which showed pronuclei of similar size centrally located, containing NPB of equal size and number aligned at the pronuclear interface, were considered to have a good PN score and thus a good prognosis (Scott et al., 2000; Machtinger & Racowsky, 2013). However, the relationship between PN score and embryo quality has not yet been fully established. Some early studies reported good correlation between the PN score and embryo development (Scott et al., 2000; Rienzi et al., 2002; Nagy et al., 2003) but more recent studies have questioned the validity of this form of assessment to predict embryo competency (James et al., 2006; Nicoli et al., 2007; Ahmad et al., 2008). A possible explanation for this conflicting data might be the highly dynamic nature of PN formation. With such rapid changes occurring within short periods of time, early assessments can change rapidly and may therefore be misleading. This has been demonstrated using time-lapse imaging (Montag et al., 2011). A retrospective review by Nicoli et al. (2013) concluded that PN evaluation was only effective if embryo transfer is performed on day one of embryo culture. c) Cleavage stage embryo assessment Indicators of embryo development including cell number, evenness of size, and incidence of fragmentation have formed the basis of embryo assessment since the inception of our scientific discipline. More recently, parameters such as multi-nucleation of blastomeres and uneven cell division have enhanced such assessments, which can be performed at the cleavage stage of embryo development. Cleavage rate There is a considerable body of evidence suggesting that delayed or rapid embryo cleavage rate has a negative impact on implantation (Edwards et al., 1980; Van Royen et al., 1999; Desai et al., 2000; Roberts et al., 2010a). Two studies have suggested embryos that complete their first cleavage to the 2-cell stage between 25 and 27 hours post-insemination show increased implantation potential (Wharf et al., 2003; Giorgetti et al., 2007). However, further recent data from a multi-centre prospective study has re-assessed the effect of early cleavage on embryo quality, implantation and LBR (de los Santos et al., 2014). This study examined 700 embryo transfers and over 1000 early-stage embryos and showed that for conventional embryo culture and morphological evaluations, the assessment of early cleavage was not considered a valuable selection tool to improve embryo implantation. The Istanbul consensus on embryo assessment (ALPHA, 2011) states that the single most important indicator of embryo viability is the timely occurrence of cell divisions (Balaban & Magli, 2011) as summarised in Table IV. This suggests that embryos that Human Fertility

8 ACE and BFS Elective Single Embryo transfer guidelines cleave more slowly than the expected rate have reduced implantation potential and those that cleave too rapidly are more likely to be abnormal and have a reduced implantation potential. This has been confirmed by large-scale analysis of UK data (Roberts et al., 2010a). Fragmentation A fragment is defined as an anuclear, membrane-bound extracellular cytoplasmic structure (ALPHA, 2011). Johansson et al. (2003) defined fragments as cells that were 45 mm in diameter for Day 2 embryos and 40 mm for Day 3 embryos. The negative effect of increased fragmentation on embryo implantation potential is well established (Alikani et al., 2008). Furthermore, the type and distribution of fragmentation may be related to implantation potential. Large fragments were found to be detrimental whilst small or scattered fragments did not have as much impact (Rienzi, 2005). However, fragments can be transient or migratory and can disappear or be reabsorbed during normal embryo development. For this reason, identification and evaluation of specific fragment patterns may not reflect the embryo s viability as part of a time-specific embryo assessment (Antezak & Van Blerkom, 1999). Fragmentation levels of less than 10 % appear to have negligible impact on implantation (Van Royen et al., 2001). Cell size For embryos at the 2-, 4- and 8-cell stages, it is expected that the blastomeres should be of equal size and shape. When blastomere size varies by more than 20 %, reduced implantation potential is observed due to an increased incidence of aneuploidy and multi-nucleation (Plachot et al., 1998; Hardarson et al. 2001; Van Royen et al., 2003). Multi-nucleation An embryo can be considered multinucleate when it contains at least one cell that has more than one interphase nucleus (Balaban & Magli, 2011). There is considerable evidence to show that the presence of multi-nucleation correlates with chromosomal abnormalities, uneven cell Table IV. Timing of observation of fertilised oocytes and embryos and expected stage of development at each time point (ALPHA, 2011). Type of Observation Timing (hours PI) Expected Stage of Development Fertilisation Check 17 1 Pronuclear Stage Syngamy Check % in syngamy (up to 20 % at 2-cell stage) Early Cleavage Check 26 1 post ICSI 2-cell stage 28 1 post IVF Day 2 Embryo Assessment cell stage Day 3 Embryo Assessment cell stage Day 4 Embryo Assessment 92 2 Morula Day 5 Embryo Assessment Blastocyst Key: PI Post Insemination 2015 The British Fertility Society size and division and lower implantation, pregnancy and LBRs (Hardarson et al., 2001; Van Royen et al., 2003). This is supported by a recent retrospective cohort analysis, which showed that when multi-nucleation is present on Day 2 of development in at least one blastomere, there is an associated increase in the rate of aneuploidy even when a sibling blastomere is biopsied which showed no signs of multi-nucleation (Ambroggio et al., 2011). There are certain factors which are known to affect the rate of multi-nucleation such as the type of culture medium (Winston et al., 1991) and temperature fluctuations (Pickering et al., 1990), the impact of which should be kept to a minimum by good laboratory process control. The optimal stage to assess for this parameter is on day 2, but evaluation does not always confirm multi-nucleation, since the nuclei are only visible during the interphase stage and could potentially be missed (Rienzi, 2005). Time-lapse technology will serve to evaluate further the impact of multi-nucleation as evidence is published on the incidence and outcome of previously undetected multi-nucleated embryos. Blastocyst assessment and transfer Advances in embryo culture media and incubation systems have enabled the reliable extended culture of embryos to the blastocyst stage of development, 5 or 6 days following insemination. This allows embryologists flexibility in deciding when to perform the embryo transfer and theoretically should provide certain advantages over the transfer of cleavage-stage embryos. According to the ASRM, (2013b) committee opinion on blastocyst culture, these advantages include: A higher implantation rate; The opportunity to select the most viable embryo; The potential decrease in the number of embryos transferred; and Better temporal synchronisation between embryo and endometrium. However, the potential downsides to blastocyst transfer can include the increased risk of couples not having a transfer when the procedure is applied in unselected patients having fewer embryos available for cryopreservation (Glujovsky et al., 2012). In addition, there are concerns that blastocyst culture results in an increased risk in monozygotic twinning (MZT), including monochorionic and monoamniotic twinning, which are extremely high-risk pregnancies (Vitthala et al., 2009; Chang et al., 2009). This reported increase in MZT does not occur in all blastocyst transfer programmes (Moayeri et al., 2007; Papanikolaou et al., 2010); however, a notable recent analysis of a large number of cycles on the Society for Assisted Reproductive Technologies (SART) database confirmed the association between blastocyst transfer and increased incidence of MZT (Luke et al., 2014). The causal factors are as of yet unknown and couples should be counselled about the possible increased risk of MZT in each particular clinic of blastocyst, compared

9 172 S. Harbottle et al. with cleavage-stage embryo transfer. Counselling should include the advice that MZT carries greatly increased risks compared with dizygotic twinning and not just a higher incidence of twins. In addition, recent reports from national data registries in the US, Canada and Sweden show that babies born from blastocyst transfer are at significantly increased risk of pre-term and very pre-term birth ( 32 weeks) (Kallen et al., 2010b). The underlying cause of this phenomenon is unknown, but may include altered allocation of cells to the placenta in blastocysts developing in vitro. A meta-analysis of these studies (Maheshwari et al., 2013) was not able to control for confounders, but patients should be counselled about the possible risk of pre- and very pre-term birth from blastocyst transfer. There is good evidence in animal models that embryos in prolonged culture are at risk of growth disorders such as Large Offspring Syndrome in sheep and cattle (Young et al., 1998) that arise from epigenetic disorders, with some data also available from human ART (Kallen et al., 2010a; Park et al., 2011). But again, the evidence on this is incomplete and requires further high-quality prospective studies. There does however appear to be consistent evidence to suggest that in good prognosis populations (defined by such factors as age, number of previous cycles, ovarian response and number and size of follicles at hcg trigger), there is an increased likelihood of live birth per embryo transfer after the transfer of fresh blastocysts compared with cleavage-stage embryos (Gardner et al., 1998; Papanikolaou et al., 2005; Papanikolaou et al., 2006; Glujovsky et al., 2012), particularly when there are supernumerary blastocysts available for cryopreservation (Mullin et al., 2012). However the most recent Cochrane meta-analysis concluded that when both early cleavage and blastocyst stage embryo freezing are taken into account, early cleavage stage transfer results in a higher cumulative pregnancy rate compared with blastocyst transfer (Glujovsky et al., 2012). It should be noted that this evidence was based on studies in which blastocysts were cryopreserved using controlled rate freezing rather than vitrification and this is an area which should be the subject of future review. Recommendations Selection of blastocysts for eset 1. Where blastocyst transfer is performed, clinics should review their eset data and criteria for extended embryo culture regularly. (GPP) 2. Prospective studies are required to determine the impact of embryo culture to the blastocyst stage on MZT, preterm birth and associated epigenetic changes and on why MZT appears to be more prevalent in some clinics. (B) 3. Patients should be counselled that current best evidence indicates that blastocyst stage transfer may result in a successful outcome sooner, but with some specific increased risks, and may ultimately lead to a reduction in the overall chance of success. (A) There are many studies that outline the importance of morphological grading of blastocysts as a predictor of outcome (Gardner et al., 2000; Balaban et al., 2000; Goto et al., 2011; Heitmann et al., 2013). Blastocysts are usually assessed using a 3-part grading scheme which was first suggested by Gardner and Schoolcraft (1999) based on the degree of blastocyst expansion and characteristics of the inner cell mass (ICM) and trophectoderm (TE). More recently, there has been a focus on the particular importance of the TE with its grading correlating more closely with implantation and live birth than that of the ICM (Ahlstrom et al., 2011; Hill et al., 2013). Following the Istanbul consensus on embryo grading (ALPHA, 2011), it was agreed that an optimal embryo at this developmental stage would be a fully expanded blastocyst with an ICM which is prominent and easily discernible, and consisting of many cells with the cells compacted tightly adhered together, and with a TE that comprises many cells forming a cohesive, even epithelium. Standardising grading of embryos and blastocysts The previous ACE/BFS guidelines for practice on eset (Cutting et al., 2008) suggested a basic grading scheme which has been adopted both by the National External Quality Assurance Scheme or NEQAS embryo grading scheme and by NICE in the 2013 Fertility Guideline Review. The three components described in the grading scheme have been evaluated as predictors of pregnancy and it has been shown that embryo growth rate and fragmentation are by far the strongest predictors of pregnancy potential and that after including these, blastomere evenness did not improve the efficacy of the model to predict pregnancy further (Stylianou et al., 2012). However, it is important to take account of the subjective nature of morphological evaluation and that a laboratory should therefore have a robust approach to external and internal quality assurance (Bjorndahl et al., 2002). The use of time-lapse imaging for embryo and blastocyst selection Since the inception of IVF, the universally accepted method to select an embryo or embryos for transfer was via a manual morphological assessment. However, in recent years technological advances have allowed the integration of cameras into incubators to allow timelapse embryo imaging of the process of morphometric evaluation in real time. This technology enables embryos to be cultured undisturbed, without the need for removal from the incubator for observation, allowing remote monitoring of development by serial image capture (Lemmen et al., 2008). There are many publications (the vast majority of which report on retrospective analyses), which suggest a benefit of morphometric evaluation for selection and de-selection of embryos at Human Fertility

10 ACE and BFS Elective Single Embryo transfer guidelines key stages in their development (see Aparicio et al., 2013; Bolton et al., 2015) as well as the benefit of having an undisturbed embryo incubation environment. However, Armstrong et al., (2015) has recently published a critical appraisal of the design of time-lapse studies concluding they do not provide robust evidence in favour of timelapse incubation as an undisturbed culture environment or as an embryo selection algorithm. They also point out the risks of introducing technology without critical appraisal. Until recently, there had been no evidence based on randomised trials to support the use of time-lapse systems. Rubio et al. (2014) then described the findings of the first randomised controlled prospective study into the efficacy of time-lapse technology. The study, based on 843 cycles, indicated that while the use of time lapse microcopy combined with time-lapse embryo selection algorithm did not result in a statistically significant increase in the pregnancy rate (61.6 % vs %), the implantation rate was increased and early pregnancy loss was reduced (16.6 % vs %). This is without doubt an area where further high-quality large-scale prospective clinical trials are urgently required. Current evidence does not discriminate between the effects of undisturbed culture and improved embryo selection afforded by time-lapse incubation systems. More recently there has been a focus on evaluating characteristics that are negative predictors for developmental competence and therefore allow the de-selection of embryos (Montag et al., 2013). Negative predictors include direct cleavage from 1 cell to 3 cells, uneven blastomeres at the 2-cell stage and multi-nucleation at the 4-cell stage. Rubio et al. (2012) reported significantly lower implantation rates when embryos exhibited abnormal cleavage patterns. Combined with the workdeveloping eset algorithms the information gained from the early cleavage observations could be used to select embryos on day 3 of culture, negating the need for blastocyst culture as a tool for selection (Hashimoto et al., 2012). Whilst the studies to date show promise that timelapse parameters have the potential to improve embryo selection beyond standard morphology, incorporation into routine practice will require the development of robust selection algorithms. It is not yet established whether a single algorithm would be appropriate for all centres or for all treatment types. Thus, the algorithms proposed have been based on surrogate endpoints (development to blastocyst stage) or on subsets of pregnancy outcome data where the embryo outcomes are known (all or none of the transferred embryos develop, which represents a biased approach) or restricted to ICSI cycles. With the exception of the work of Herrero et al. (2013), all such studies have been far too small to enable robust algorithm development and validation: when the datasets produced include 20 or more parameters, datasets with more than 400 successful and unsuccessful outcomes are needed, and larger numbers still will be required to establish reliable thresholds The British Fertility Society From data currently available it appears likely that the use of time-lapse technology may facilitate the de-selection of embryos (Bolton et al., 2015). It is also clear that before firm conclusions can be drawn on the benefits of time-lapse technology there is an urgent need for further well-designed large-scale, independent, multi-centre studies with unselected patients to develop and validate selection criteria, followed by RCTs to demonstrate their clinical utility (Bolton et al., 2015). In addition, cost benefit analyses should be performed to ascertain if benefits attributable to time-lapse incubation systems justify the significant costs involved in the procurement and implementation of the appropriate technology. Recommendations Use of time-lapse technology 1. Centres with the resources to support time-lapse morphometric analysis may use this information to improve their eset algorithm. (GPP) 2. Technological advances including time-lapse imaging and morphometric assessment show promise as embryo selection and de-selection tools, but further high-quality prospective studies are required to develop and validate robust algorithms, followed by RCTs to demonstrate their role in an eset strategy. (C) Non-invasive testing of embryo viability The correlation between metabolic activity and embryo viability was first reported by Renard et al. (1980) in a study that looked at glucose uptake in advanced bovine blastocysts. Since then several studies have reported the use of metabolic, metabolomic, proteomic, genomic and transcriptomic markers as predictors of embryo viability (Leese & Barton, 1984; Van den Bergh et al., 2001; Gardner et al., 2001; Leese, 2002; Brison et al., 2004; 2007; Leese, 2012; Bolton et al., 2015). Recent papers by Gardner and Wale (2013) and Bolton et al., (2015) have reviewed the analysis of metabolism as a means of selecting viable embryos for transfer particularly with a view to replacing a single embryo. These papers review the data on amino acid turnover along with carbohydrate utilisation and suggest that there are distinct patterns of amino acid utilisation between viable and non-viable blastocysts as initially reported by Houghton et al., (2002) and Brison et al., (2004). Amino acid profiling has an established scientific basis (Brison et al., 2004) but is yet to find a place in routine clinical practice. Refinements of the highperformance liquid chromatography (HPLC) technology to reduce the time it takes to carry out the amino acid profiling may make the process more efficient and attractive for clinical use. Gardner and Wale (2013) have shown that glucose consumption provides a further potential biomarker of the capacity of an embryo to give rise to a pregnancy. Raman and near-infrared spectroscopy have been used to analyse culture medium to examine metabolic profiles (the overall technique