How much have we learned from time-lapse in clinical IVF?

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1 Molecular Human Reproduction, Vol.22, No.10 pp , 2016 Advanced Access publication on September 21, 2016 doi: /molehr/gaw056 NEW RESEARCH HORIZON Review How much have we learned from time-lapse in clinical IVF? D. Castelló 1,*, Y. Motato 2, N. Basile 3, J. Remohí 1, M. Espejo-Catena 2, and M. Meseguer 1,* 1 IVF Laboratory, Instituto Valenciano de Infertilidad (IVI), Valencia 46015, Spain 2 Instituto FIVIR, Carcaixent 46740, Spain 3 Instituto Valenciano de Infertilidad (IVI), Madrid 28023, Spain *Correspondence address. IVF Laboratory, Instituto Valenciano de Infertilidad (IVI), Valencia 46015, Spain. damia.castello@ivi.es, marcos.meseguer@ivi.es Submitted on August 28, 2015; resubmitted on August 4, 2016; accepted on August 11, 2016 ABSTRACT: Can the time-lapse system (TLS) identify the best embryo for transfer? Although there are several studies that support this hypothesis, more research is required to improve the quality of the current evidence and also to assess live birth rate, miscarriage, stillbirth or clinical pregnancy in order to choose between a TLS or conventional incubation. In addition, although some authors report on effectiveness and safety in the use of TLS monitoring of embryo in vitro, other authors that have not found relevant differences between the two systems for the culture and subsequence embryo selection. On the other hand, TLS has emerged as a novel technology and has been introduced into clinical practice in many laboratories to perform embryo morphology evaluation and study al kinetics in ART. However, most studies only assess blastocyst formation or implantation rate as the primary end-point and additional data are required, for example, about live birth, monozygotic twinning rates and health problems. Furthermore, the features of populations studies are varied; for example, female and male age, seminal characteristics and female factor. The embryo culture conditions and culture medium used also vary. For this review, a search of PubMed was conducted to retrieve relevant studies regarding use of TLS in embryo incubation and selection, and compare them with standard embryo culture and evaluation. Key words: ART / non-invasive markers / time-lapse system / morphokinetic parameters / embryo assessment / embryo Introduction ART has resulted in over 5 million babies born worldwide. Although significant improvements have been achieved, there are still two major problems. First, the success rate is low; clinical pregnancy rates (PRs) are still around 30% per transfer as reported by Kupka et al. (2014) due, in part, to an inability to objectively determine which embryo has the highest potential for implantation. Second, there is a high multiple PR; multiple delivery rates are still around 20% (Kupka et al., 2014) due to the high number of embryos that are often transferred into the uterus. New technologies/strategies are needed to improve embryo selection and thereby increasing success rate after embryo transfer. It is here where genetic screening (improving selection based on genetic and chromosomal viability) and time-lapse embryo culture (selection based on morphokinetic markers) may play a major role. Regarding high multiple PRs, it is mandatory to reduce the number of transferred embryos, and finally to move to single embryo transfer. The embryo selection routine in IVF clinics is based on a single observation by light microscopy at set times, mainly on Days 2 or 3 (Alpha Scientists in Reproductive Medicine and ESHRE Special Interest Group of Embryology, 2011). This embryo selection strategy is currently the one that is used most often in IVF laboratories worldwide. However, nonspecific and subjective selection features may result in not choosing the best embryo/s for transfer. Extended embryo culture and transfer at blastocyst stage (Day 5 or 6) becomes an alternative which allows the selection of embryos at a more advanced stage of and minimizes the risk of multiple pregnancies. Although multiple observations have given a better understanding of embryo, it is well known that each observation also involves exposure of the embryo to suboptimal conditions outside the controlled environment of an incubator, potentially affecting the success of the treatment. Multiple pregnancies not only bring medical complications to the mother and the baby (hypertension, low birthweight and neurological damage), but also they induce social and financial problems (Strauss et al., 2008). The time-lapse system (TLS) combines three basic elements: an incubator, an optical microscope and a software program. Integrating these elements, continuous surveillance is provided while optimal The Author Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please journals.permissions@oup.com

2 720 Castelló et al. culture conditions are maintained. Therefore, TLSs give us multiple observations of the embryo al changes (Lemmen et al., 2008; Wong et al., 2010), contributing to a better understanding of morphologic mechanisms in fertilization and behavior of early human embryos (Payne et al., 1997; Mio and Maeda, 2008; Montag et al., 2011; Azzarello et al., 2012). In fact, biomarkers identified by TLS have been under investigation for their clinical value in embryo selection. For example, the time between fertilization and first cleavage is an objective parameter that can easily be determined, with certain predictive values of embryo viability (Sakkas et al., 1998) and many authors have highlighted its importance as a selection tool. Recently, new kinetic markers and their correlations with embryo quality and implantation potential have been discovered (Pribenszky et al., 2010a; Meseguer et al., 2011; Hlinka et al., 2012; Rubio et al.,2014; VerMilyea et al.,2014). In this review, we will evaluate the current status of the candidate kinetic embryo markers that have been identified and we will establish the research horizons based on this technology. Observations using time-lapse technology in the human embryo The TLS has been used for many years now, especially in research (Cole, 1967; Massip and Mulnard, 1980; Wale and Gardner, 2010). In 1997, Payne s group (Payne et al., 1997) was the first to described the events that occur between 17 and 20 h after ICSI, including second polar body extrusion, and the appearance of pronuclei (PN). Some years later, Mio and Maeda described the kinetics of events until blastocyst stage (Mio and Maeda, 2008) including the fertilization process and the from a 2-cell embryo to a hatched blastocyst. A few years later, Pribenszky et al. (2010b) reported the first live birth after time-lapse assessment of a five embryo cohort up to blastocyst stage. Nowadays, thanks to bioinformatics and the automation achieved by some equipment on the market, this technology is becoming more suitable for clinical use. Biomarkers associated with embryo An image analysis system offers several benefits that include not only the exact determination of cell divisions, but also a closer monitoring of morphological events correlated with embryo and IVF outcomes (Table I). In addition, it allows the detection of different events such as irregular divisions, the formation and reabsorption of fragments (Van Blerkom et al., 2001; Hardarson et al., 2002; Pribenszky et al., 2010a), the initiation of compaction or the appearance of the blastocoel. This technology also connects fragmentation with the meiotic and mitotic cell cycle (Stensen et al., 2015). Shoukir et al. (1997) were the first group to report that embryos transferred after completing their first cell division at 25 h after insemination using conventional IVF, showed significantly better pregnancy and implantation rates compared with those that did not complete this first cell division within 25 h after insemination. Sakkas et al. (1998) obtained the same results but with embryos derived from ICSI cycles, where fertilization times are more defined. Thereby, both studies demonstrate the utility of early cleavage as an indicator of embryo viability, and thus as a predictor of pregnancy. In another study by Lemmen et al. (2008), 102 embryos were evaluated for h, analyzing the timing and co-ordination of events during early from zygote to cleavage-stage embryo. They observed that implanted embryos had a faster and more synchronous appearance of nuclei after the first cleavage, and those embryos reaching 4-cell stage Day 2 had faster PN disappearance. However, Azzarello et al. (2012) demonstrated that PN breakdown (PNB) occurred later in those embryos that resulted in a birth. The first studies using time-lapse imaging were limited to measurements at the early stages, and pronuclear formation and fusion, such as first cleavage timing. In 2010, Wong et al. (Wong et al., 2010), combining image and gene expression technologies, proposed new kinetic markers with defined predictive ability. This study was focused on blastocyst formation of 242 supernumerary frozen human zygotes cultured until Day 5. The mean values (±SD) for these three parameters were proposed as predictors of blastocyst formation until 4-cell stage with high sensitivity and specificitywere:durationofthefirst cytokinesis (14.3 ± 6.0 min); time between the first and second mitotic division or 2 3 cell stage (11.1 ± 2.2 h); and time between second and third mitosis or 3 4 cell stage (1.0 ± 1.6 h). After this initial study from Wong et al. (2010) several groups have extended the research for predictive kinetic markers by evaluating larger data sets and proposing models of embryo selection. For example, Meseguer et al. (2011), reported data on a set of 522 embryos transferred on Day 3, with images taken during 64 h in culture. A subset of 247 embryos with known implantation data was analyzed and the following parameters were proposed to predict implantation: t5 or time between ICSI and the 5 cell stage ( h); t3 t2, representing the duration of the period as a two blastomere embryo ( 11.9h); and the synchrony from three cell stage to four-cell stage ( 0.76 h). Another aspect worth mentioning is the correlation between poor implantation potential and certain morphological events that can only be determined through time-lapse analysis (Meseguer et al., 2011): direct cleavage from zygote to three-blastomere embryo, defined as: t3 t2 <5 h; uneven blastomere size at the two-cell stage during the interphase where the nuclei are visible; and multinucleation at the four-cell stage during the interphase where the nuclei are visible. By combining standard morphological assessment plus exclusion criteria plus inclusion criteria, an algorithm (Fig. 1) for embryo selection was proposed to classify embryos into 10 categories (A+/A, B+/B, C+/C,D+/D,E and F, with different percentages of implantation: Table II). Following these findings, Rubio et al. (2012) in a retrospective study, analyzed 5225 embryos confirming that embryos with direct cleavage of 2- to 3-cells (termed DC2 3 <5 h) had a very low implantation rate (1.2%). Based on these findings, the authors suggested DC2 3 as a novel exclusion criteria for embryo selection which, together with other exclusion and selection criteria, can be used as embryonic pattern selection. Nevertheless, in a recent publication with the purpose of standardizing the nomenclature and unify its meaning, Ciray et al. (2014) have proposed modifications of the term DC, or Direct Division, by utilizing, up to this moment, t3 t2 = 0, also called Trichotomous mitosis. On the other hand, recent publications have demonstrated a decreased al and implantation potential of human embryos with anomalous not shown in

3 Table I Some early events associated with embryo. Target Population Time-lapse system Major finding End-point Reference... PB extrusion 1448 transferred embryos EmbryoScope (Unisense The timings at which second polar body extrusion ( h), Day 3 Aguilar et al. pronuclear fading ( h) and length of S-phase ( h) (2014) occurred were linked successfully to embryo implantation. Fertilization and the first appearance of male and female PN Early disappearance of PN Duration of first cytokinesis and Blastocyst formation Generate and evaluate an embryo selection tool based on timings of events and morphological patterns Analyze associations between embryo division kinetics and ability to reach blastocyst stage Describe the events associated with the blastocyst formation and implantation Direct cleavage from two to three cells (DC2 3) Fragmentation of human cleavage-stage embryos and the relation with meiotic and mitotic cell cycles Atypical embryo phenotypes identified by time-lapse microscopy: high prevalence and association with embryo PB, polar body; PN, pronuclei. 341 fertilized oocytes EmbryoScope (Unisense 50 fertilized oocytes Time-lapse using Nomarski differential interference contrast optics 102 fertilized oocytes, 29 transferred embryos Diapot 300 microscope with camera in a Closed system (Nikon) 100 embryos IX-70/71 microscopes (Olympus) with an aperture for dark-field illumination 247 transferred Embryos EmbryoScope(Unisense 834 Embryos EmbryoScope (Unisense 3215 blastocyst, 832 transferred EmbryoScope (Unisense 1659 transferred Embryos EmbryoScope_ (Unisense Total of 1943 oocytes from 297 patients and 372 embryos from 100 patients Sixty-seven women undergoing IVF treatment with 651 embryos EmbryoScope (Unisense Eeva System (Auxogyn Inc., USA) The timing of PB2 extrusion is significantly delayed when female age >38 years, but not related to subsequent embryo, and time to first cleavage is a significant predictive marker for embryo quality on Day 3. The 65% of the fertilized oocytes had extruded the second PB at 3 h postinjection and the first appearance of male and female PN occurred at 5 h post-injection in 51% of oocytes fertilized. Early disappearance of PN and onset of first cleavage was correlated with a higher number of blastomeres on day 2 and synchrony in appearance of nuclei after the first cleavage was significantly associated with pregnancy success. The success in progression to the blastocyst stage can be predicted using three dynamic imaging parameters: (i) duration of the first cytokinesis (14.3 ± 6.0 min), (ii) time interval between the end of the first mitosis and the initiation of the second (11.1 ± 2.2 h ) and (iii) the time interval between the second and third mitoses (1.0 ± 1.6 h). The most predictive parameters for implantation were: (i) time of division to 5 cells, t5 ( h after ICSI); (ii) time between division to 3 cells and subsequent division to 4 cells, s2 ( 0.76 h) and (iii) duration of cell cycle two, i.e. time between division to 2 cells and division to 3 cells, cc 2 ( 11.9 h). Embryos with a t5 of h not only have a higher implantation potential, but also a higher probability of developing into a good morphology blastocyst. The most predictive parameters for blastocyst formation were time of morula formation, tm ( h after ICSI), and t8 t5 ( 8.78 h) and the time for expansion blastocyst, teb ( h after ICSI), and t8 t5 ( 5.67 h after ICSI) predict blastocyst implantation. Embryos with DC2 3 had a statistically significantly lower implantation rate than embryos with a normal cleavage pattern. The process of fragmentation of in vitro-derived embryos was related to the progress of the meiotic and the mitotic cell cycles. A high percentage of embryos with atypical phenotype(s) had good quality on day 3 but the blastocyst formation rates for these embryos were significantly lower compared with their respective control group. Day 3 20 h postinjection Day 2 D5-6 Day 3 Development to blastocyst Development to blastocyst Implantation rate Day 3 Day 3 Liu et al. (2015) Payne et al. (1997) Lemmen et al. (2008) Wong et al. (2010) Meseguer et al. (2011) Cruz et al (2012) Motato et al Rubio et al. (2012) Stensen et al. (2015) Athayde Wirka et al. (2014) Learning from time-lapse in clinical IVF 721

4 722 Castelló et al. Figure 1 Hierarchical classification of embryos based on timing of cell division to 5 cells (t5), synchrony of divisions from 2 cell to 4 cell stage (s 2 ) and duration of second cell cycle (cc 2 )(Meseguer et al., 2011). Table II Human embryo implantation according to categories of the hierarchical classification tree model applied in the time-lapse monitoring system Embryo n n Implantation Embryo Implantation category Total Implanted (%) category (%)... A A 52 A B B 27 B C C 19 C D D 14 D E E 8 The implantation potential is listed for all categories A+ to E, as well as for the combined categories: A (52%), B (27%), C (19%), D (14%) and E (8%) (Meseguer et al.,2011). conventional culture, including abnormal syngamy, first cytokinesis and anomalous cleavage patterns (Athayde Wirka et al., 2014) and blastomere fusion (Hickman et al., 2012; Montag et al., 2014).This abnormal blastomere fusion, termed reverse cleavage (RC), was reported by Liu et al. (2014) in a total of 789 human embryo cultured in a TLS until Day 3, and they concluded, first, that growth and of embryos with RC was significantly compromised, culminating in poor implantation potential. Second, that RC can occur at any stage during the first 3 days of embryo culture, and third that embryos can show RC on more than one occasion during. RC is not restricted to those affecting the oocyte, as sperm factors, such as poor progressive sperm motility, might have an impact on this abnormal blastomere fusion. Concerning early embryonic and its potential to reach the blastocyst stage, several groups, such as Cruz et al. (2012) demonstrated that t3 t2 and t4 t3 were statistically significant indicators of blastocyst in 834 embryos analyzed. In the same way, Hashimoto et al. (2012) reported that better quality blastocysts presented significantly shorter times for synchrony between the 3 and 4 cell stages. In addition, Dal Canto et al. (2012), after analyzing 151 embryos, concluded that cleavage from the 2 to 8 cell stage occurs progressively earlier in embryos with the ability to develop to blastocyst stage, expand and implant. Despite the increasing number of studies reporting models and algorithms for embryo selection through TLS in the IVF laboratory, several authors believe that embryo selection based on time-lapse imaging should remain an experimental strategy, basically because these studies do not consider that other variables could also be as important as culture conditions; for example the use of ICSI, type of ovarian stimulation protocol and intrinsic patient characteristics (Racowsky et al., 2015). Authors like Polanski (Polanski et al., 2014), also argue that there are no data reporting the effect of TLSs on rate of live births, congenital abnormalities, miscarriage, stillbirth or clinical pregnancy in order to choose between TLS or continue using conventional incubation (Armstrong et al., 2015). In summary, new markers based on timings, and the presence or absence of abnormal morphological events are only detected through time-lapse technology. The majority of these biomarkers are detected during the early stages of embryo (before the 5-cell stage) allowing early and more informed decisions about selection (Fig. 2). In addition, kinetic markers related to later stages of embryo have been studied providing additional information (Campbell et al., 2013a; Desai et al., 2014).

5 Learning from time-lapse in clinical IVF 723 Figure 2 Nomenclature proposed for kinetic markers in human embryo. Duration of second cell cycle defined as the time from division to a 2-blastomere embryo until division to a 3-blastomere embryo or t3 t2; duration of third cell cycle defined as the time from division to a 3-blastomere embryo until division to a 5-blastomere embryo or t5 t3; the duration of the second transition from a 3-blastomere embryo to a 4-blastomere embryo or t4 t3 and the duration of the third transition from a 5-blastomere embryo to a 8-blastomere embryo or t8 t5. Direct cleavage (DC2 3) from zygote to 3-blastomere embryo, defined as: t3 t2 < 5h. Aneuploidy and morphokinetic analysis Aneuploidy plays a major role in implantation failure and early miscarriage, affecting the live birth rates in assisted reproduction. Franasiak et al. (2014) found that there is a predictable aneuploidy increase after the age of 26 years although a higher prevalence (more than 40%) among women less than 23 years old was also identified. Thus, the no euploid embryos rate lies between 2% and 6% in women years of age, while in 44-year-old women it is 53%. In addition, good prognosis patients are also affected; Yang et al. (2012) reported a 44.9% aneuploidy rate for blastocysts of patients who were less than 35 years old with no prior miscarriage. In this sense, PGS has been used to improve embryo selection and outcomes. Nevertheless, embryo biopsy is an invasive procedure that may not be readily available at every laboratory. In addition, PGS is not always allowed owing to legal restrictions in some countries and sometimes is avoided owing to ethical beliefs. Taking these reasons into consideration, some groups have focused on analyzing the morphokinetic behavior of chromosomally normal and abnormal embryos to propose its use in patients that do not choose PGD/PGS for ethical, economical or legal reasons. Correlating embryo with chromosomal abnormality is not a new concept in embryology. However, this relation was established in the past purely based on static observations. Magli et al. (2007) observed a high proportion of abnormalities in arrested and slow cleavage embryos, as well as in those presenting highly accelerated cleavage. In an attempt to introduce time-lapse technology into PGS of embryos, Chavez et al. (2012) analyzed blastomeres from 75 human embryos by the 4-cell stage (before genome activation). The ploidy analysis was performed on each individual blastomere via 24 chromosome array comparative genomic hybridization (acgh) and showed aneuploid cells in over 75% of the embryos analyzed. Interestingly, the authors discovered that different cell cycle parameters in conjunction with dynamic fragmentation analysis largely reflected the underlying ploidy of the four-cell stage embryo. A calculation of the mean values and SDs for the cycles in euploid embryos resulted in the following values: 14.4 ± 4.2 min for the first cytokinesis where two blastomeres were clearly distinguishable; 11.8 ± 0.71 h from 2 blastomeres to 3 blastomeres; 0.96 ± 0.84 h from 3 to 4 blastomeres. Their results suggest that the cell cycles of chromosomally euploid embryos are less variable than those of chromosomally abnormal embryos. In the same way Campbell et al. (2013a), applied time-lapse technology to 98 blastocysts to identify embryos at risk of presenting a single or multiple aneuploid chromosome constitution. In their first study, they observed that multiple aneuploid embryos presented a delay in the initiation of compaction and in the time to reach the full blastocyst stage. Based on these results, a predictive algorithm was developed, classifying embryos as having high, medium or low risk of chromosomal abnormality. The overall aneuploidy rate in this study was 60%. Subsequently, the same group (Campbell et al., 2013b) performed a retrospective analysis to evaluate the effectiveness and potential impact of this model for unselected IVF patients (88 transferred blastocysts) without biopsy and PGS. Although the results showed that the algorithm appeared to offer a good prediction of PRs and outcomes, it has not been validated prospectively. On the other hand, Kramer et al. (2014) retrospectively applied this classification model and it failed to segregate euploid embryos from aneuploid embryos maintained in their laboratory. In a more recent study (Chawla et al., 2015), a total of 460 embryos cultured in TLSs were selected for blastomere biopsy on Day 3 of. These embryos belonged to patients undergoing IVF treatment and genetic screening (acgh) for sex selection. They found a significant difference for mean time of pronuclear fading (PNf) t2, t5, t5 t3, t5 t2 between normal and abnormal embryos. Similarly, Basile et al. (2014) identified the variables t5 t2, followed by t5 t3, as the most relevant variables related to normal chromosomal content. On the basis of these results, Basile group proposed an algorithm for embryo selection to classify embryos from A to D. Each category exhibited significant differences in the percentage of normal embryos (A, 35.9%; B, 26.4%; C, 12.1% and D, 9.8%). Besides these studies, in the one performed by Rienzi et al. (2015), no correlation was found between morphokinetic parameters and aneuploidy, and this group argues that this may happen because of the following factors: inter laboratory variations, different embryo culture

6 724 Castelló et al. systems, different populations studied, day of embryonic biopsy and female age. Therefore, it is essential to consider these types of variables when analyzing the results as they could have a considerable effect on al competence (Cohen et al., 2012), implantation potential (Scott et al., 2012) and blastocyst formation (Porter et al., 2002). Finally we have to take into account the variety of tests and screenings we have done so far. For example, comprehensive methods such as acgh (Chavez et al., 2012; Yang et al., 2012), single nucleotide polymorphism microarray or next-generation sequencing have been used to analyze the euploid and aneuploid state of embryos (Campbell et al., 2013a; Franasiak et al., 2014). Considering this fact, we might come upon the reasons behind the origins of the variation in findings, and why they are so different among the different groups. As a consequence, the results require cautious investigation. Nevertheless, the use of this technology is relevant because it allows us to analyze and compare the of an abnormal embryo versus a normal one. Blastocyst formation prediction based on morphokinetic analysis The main reason for long culture of embryos to blastocyst stage is to improve the morphological selection and synchronization with the patient s endometrium. Although in most IVF clinics the transfer at blastocyst stage is associated with higher implantation rates, outcome must be balanced with the possible disadvantages of extended culture such as higher economic costs, risk of canceled cycles and the possible epigenetics effects (Miles et al., 2007; Kirkegaard et al., 2012). Wong et al. (2010) were the first to describe an association between of human embryos to blastocyst stage and early kinetic markers (first cytokinesis, the time between first and second mitoses, and the time between second and third mitosis) proposing this information to avoid the need of long culture. However, embryos in this study were not finally transferred. Therefore it was confirmed, once again, that observations on Day 2 and Day 3 are useful for the accurate evaluation of embryo (Kirkegaard et al., 2013; Desai et al., 2014). One of the biggest studies including 9530 embryos evaluated through TLS was reported by Herrero et al. (2013). In this study, the authors described that early parameters (t2, t3, t4) were able to predict short-term but late parameters (t5, t8) were significantly better predictors of embryo viability to later stages, such as blastocyst stage. More recently, a study by Milewski et al. (2015) based on 432 embryos, used the time to reach 2, 3, 4 and 5 cell stage (t2, t3, t4 and t5) and the intervals between the second and third division (t3 t2 and t4 t3). They proposed a predictive model of blastocyst and in so doing, a new parameter (Sc) was obtained using a multivariate logistic regression model. More specifically, the Sc variable represents the sum of the products of the three parameters of the model multiplied by the corresponding odds ratios (OR). This parameter is described by the formula: Sc= s_ t2* ORs_ t2 + s_ t5* ORs_ t5 + s_ cc2* ORs_ cc2. Recently, Motato et al. (2016) analyzed the kinetic markers in 7483 embryos. They identified predictive parameters for blastocyst formation: the time of morula formation (tm) h after ICSI, and t8 t5 ( 8.78 h) or time of transition from five-blastomere embryos to eight-blastomere embryos. Using the two parameters proposed by them, a new model for blastocyst formation prediction was developed. Although these models present statistically significant differences among embryos that developed to blastocyst stage, their application to different populations is still necessary in order to strengthen their results. Consequently, once again, it is emphasized that morphokinetic parameters can help us to provide more accurate information about the quality and potential for embryo transfer. New horizons for embryo analysis by the TLS Despite intensive research in different fields of human reproduction (genomics, proteomics and metabolomics), most embryologists still depend on day by day static assessments using standard variables; which include al rate based on cell count on Day 2, Day 3 and blastulation on Day 5 and Day 6, and morphological features such as fragmentation, degree of symmetry in cleavage-stage embryos, and quality of the inner cell mass or trophectoderm in blastocysts (Alpha Scientists in Reproductive Medicine and ESHRE Special Interest Group of Embryology, 2011). Although, the time-lapse technique has made it possible to monitor embryos over many days and thereby, collect large amounts of information, this analysis is still performed manually, and images are mostly used as a qualitative reference. Therefore, to make full use of this increased number of microscopic images, (semi)automated computeraided tools are needed. These tools also allow the possibility to collect and analyze the embryos from multiple clinics, increasing our knowledge of early human embryo (Molder et al., 2015). Another advantage is the ability of monitoring embryos remotely, via the Internet, allowing the of tele embryology: In this way, a fully qualified embryologist would assess the embryos from any location worldwide. It would be extremely motivating if these new studies could be incorporated with the ones that we have already been using. The embryo selection process could be improved with the use of new options, and of new variables. For example, the timing of PNB (Azzarello et al., 2012), PNF as the reference starting time point (Liu et al., 2016), the timings of second polar body extrusion, PNF, length of S-phase (Aguilar et al., 2014), the DC embryos, and the RC embryos that have fewer than six intercellular contact points at the four-cell stage (Liu et al., 2015), should be incorporated in the analysis for selecting the best embryo to transfer and, therefore, deselecting poor quality embryos with low implantation potential. Discussion and perspectives Embryo selection criteria based on routine morphological assessment do not always associate with a high implantation rate or PR (Rijnders and Jansen, 1998; Milki et al., 2002). Therefore, new objective criteria should be considered in the identification of embryos with a good prognosis for implantation or pregnancy. During the past 5 6 years, different studies based on a TLS have provided new knowledge on

7 Learning from time-lapse in clinical IVF 725 embryo offering the embryologist the possibility to improve embryo evaluation and selection. Recently, an improvement of implantation potential prediction has been reported through analysis of the morphokinetics of human embryos at early cleavage stages, using different TLSs (Wong et al., 2010; Meseguer et al., 2011; Dal Canto et al., 2012; Hlinka et al., 2012; Kirkegaard et al., 2012; Chen et al., 2013; Conaghan et al., 2013; Herrero et al., 2013). In addition, kinetic markers may aid in the prediction of which cleavage-stage embryo has the potential to reach the blastocyst stage (Wong et al., 2010; Conaghan et al., 2013). This is particularly important when prolonged culture of Day 5 or 6 is not feasible or desirable, especially considering some author s suggestions that there could be a detrimental effect of extended culture on epigenetic modification, monozygotic twinning rates, preterm births and long-term health problems in the offspring (Niemitz and Feinberg, 2004; Horsthemke and Ludwig, 2005; Kallen et al., 2010; Kalra et al., 2012). On the other hand, the extra information acquired on aberrant cleavage patterns using TLSs is useful for the identification and exclusion of embryos that would otherwise be considered as viable (Kirkegaard et al., 2013). Moreover, identification of euploid embryos may also represent an advantage of this technology although it should never be taken as a replacement of genetic screening (Campbell et al., 2013a; Basile et al., 2014). Finally, a large quantity of data is being generated representing a great resource for future investigations. Nonetheless, the drawback of this approach is the limited ability to view the embryo in multiple planes, and the embryo cannot be clearly viewed in a single plane. Thus, manipulation under a microscope and contemporaneous rotation of the embryo allow for a detailed morphological assessment of all its aspects, which cannot as yet be performed with the current time-lapse embryo-imaging equipment (Herrero and Meseguer, 2013). This general overview of TLSs has shown that the designed algorithms for embryo selection differ among the different research groups, basically because the parameters defined as the most relevant ones in each center are not exactly the same. Therefore it is important to use a uniformly defined nomenclature to help unify time-lapse monitoring practice. Additionally, the selection of different study populations with different features, such as advanced maternal age, and history of unsuccessful IVF treatments, or both, as well as different stages of embryonic for transfer, among other aspects, can affect the results. However, it is important to know that the TLS is a powerful technology for the study of embryo allowing information on a wide range of morphological and dynamic parameters to be obtained from individual embryos. Moreover, these markers can also be used as predictive markers for healthy embryo. Acknowledgements The authors thank Nicolas Garrido, Mª José de los Santos and Josep Lluis Romero from IVI Valencia, for their clinical support in this study. Authors roles D.C. and Y.M. played a role in the analysis and interpretation of data, drafting of the manuscript and final approval. M.E. was involved in the acquisition of data and final approval of the manuscript. M.M. was involved in conception and design, acquisition and analysis of data, drafting manuscript and its final approval. Funding This work has not received any financial support from any commercial entity but the instrumentation, disposables and utensils were provided by IVI. Conflict of interest None of the authors have any economic affiliation with Vitrolife. The authors do not have any conflict of interest. References Aguilar J, Motato Y, Escriba MJ, Ojeda M, Munoz E, Meseguer M. The human first cell cycle: impact on implantation. RBM Online 2014;28: Alpha Scientists in Reproductive Medicine and ESHRE Special Interest Group of Embryology. The Istanbul consensus workshop on embryo assessment: proceedings of an expert meeting. Hum Reprod 2011;26: Armstrong S, Arroll N, Cree LM, Jordan V, Farquhar C. Time-lapse systems for embryo incubation and assessment in assisted reproduction. 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