Molecular Aspects of Medicine 34 (2013) 903–918

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Molecular Aspects of Medicine

journal homepage: www.elsevier .com/locate /mam

Review

Quality control of embryo development

0098-2997/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.mam.2013.03.001

⇑ Corresponding authors.E-mail addresses: aajduk@biol.uw.edu.pl (A. Ajduk), mz205@cam.ac.uk (M. Zernicka-Goetz).

Anna Ajduk a,⇑, Magdalena Zernicka-Goetz b,⇑a Department of Embryology, University of Warsaw, Miecznikowa 1, 02-096 Warsaw, Polandb Wellcome Trust / Cancer Research UK Gurdon Institute, University of Cambridge, Tennis Court Road, CB2 1QN Cambridge, UK

a r t i c l e i n f o a b s t r a c t

Article history:Available online 4 April 2013

Keywords:Mammalian embryoAssisted reproduction technology (ART)Morphology gradingPreimplantation genetic diagnosis/screening(PGD/PGS)MetabolomicsTime-lapse imaging

Although in recent years we have seen an undeniable improvement in the field of repro-ductive biology and medicine, the efficiency of in vitro fertilization (IVF) proceduresremains relatively low, ranging from 4% to 40% depending on the patient’s age. It isbelieved that this is in a large part caused by inaccurate assessment of embryo quality priorto transfer to mothers-to-be. Thus there is a strong need for further refinement of existingselection methods and development of novel, robust and, if only possible, non-invasiveprocedures to ensure that only embryos with the highest developmental potential are cho-sen for transfer. In the present review we compare various methods for assessing the qual-ity of preimplantation embryos either currently used in IVF clinics or still to be tested.These methods include assessment of embryonic morphology, the genetic material, thetranscriptomes of the oocyte and its accompanying follicular cells, and the embryo’smetabolism. We discuss what information these parameters actually provide about theprocesses occurring in the embryo itself. We also present novel methods for selectinghealthy embryos based on most recent advanced time-lapse imaging techniques, whichshow great promise and are likely to lead to increased in vitro fertilization efficiency.

� 2013 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9032. Static morphological grading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9043. Preimplantation genetic diagnosis and screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9064. Transcriptomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9095. Metabolomics and proteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9106. Assessment of development by a time-lapse imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9117. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 913

Competing interests. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 914Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 914References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 914

1. Introduction

Since 1978, when the first in vitro conceived baby was born (Steptoe and Edwards, 1978), in vitro fertilization (IVF) hasbecome one of the most important methods for treating infertility. In the UK the number of women that underwent the

904 A. Ajduk, M. Zernicka-Goetz / Molecular Aspects of Medicine 34 (2013) 903–918

procedure has increased threefold in last two decades from 14,057 in 1992 to 45,264 in 2010. The number of IVF treatments(including fertilization by co-incubation of gametes (traditional IVF) and intracytoplasmic sperm injection (ICSI)), has in-creased in similarly astonishing way, from 18,338 in 1992 to 57,652 in 2010 (according to the Human Fertility and Embry-ology Authority (HFEA), <www.hfea.gov.uk>). A similar tendency has been also observed in the USA, where the number ofIVF and ICSI treatments increased from 64,681 in 1996 to 146,693 in 2010 (according to the Society of Assisted ReproductiveTechnologies (SART), <www.sart.org>). Although IVF procedures have been greatly improved over the years, their efficiency,measured as the live birth rate, is usually well below 50%. It ranges from 5.1% (UK)/4.1% (US) for patients older than 42 to32.3% (UK)/41.7% (US) for women younger than 35 (data from 2010, HFEA and SART respectively). In consequence, manycouples have to undergo treatment several times before they succeed and this increases the emotional and financial costs,and also brings additional health risks for women. The efficiency of IVF can be elevated by transferring multiple (usually nomore than 3) embryos in a single cycle, but this often results in multiple-pregnancies, and in consequence serious healthcomplications for mothers and their babies. Premature delivery and morbidity related to a low birth weight are amongthe most common complications (Ombelet et al., 2005). Even if children survive the early post-natal period, they are stillat an increased risk of long-term developmental and physical disability, including cerebral palsy (Petterson et al., 1993; Pha-roah and Cooke, 1996; Yokoyama et al., 1995). In the case of mothers, multiple pregnancies correlate with a higher incidenceof pre-eclampsia, myocardial infarction, heart failure, thrombosis and pulmonary oedema (Walker et al., 2004). Therefore,there is a strong need to decrease the number of embryos transferred in a single cycle (Gerris, 2009, 2005). In the UK in2001 the HFEA introduced a two-embryo transfer policy for women under the age of 40 years and in 2009 it introduced apolicy that aims to reduce the UK IVF multiple birth rate to 10%. In the first year of the policy (2009) the maximum multiplebirth rate for IVF clinics was set at 24% (the national average at the time), meaning that no more than 24% of a clinic’s annuallive birth rate should be multiple births. All clinics were obliged to have a strategy in place to identify patients suitable forelective single embryo transfer (eSET) in order to reduce the clinic’s multiple birth rate. eSET involves a careful assessment ofembryonic developmental potential prior to transfer in order to select and transplant to mothers only the single most viableembryo. The multiple birth rate for IVF centres was decreased by the HFEA to 20% in 2010, 15% in 2011 and finally to 10% in2012. Similar tendencies in IVF practice have been also observed in many other countries. Increasing awareness of healthrisks resulting from multiple pregnancies and in consequence the increasing popularity of eSET puts further pressure on sci-entists and the medical industry to develop novel, reliable methods to predict embryonic quality and live birth. Here we re-view techniques for embryo assessment that have been already used in a clinical setting or are still being trialled. Asbiologists, not clinicians, we would like to focus on the biological meaning of the embryo grading criteria rather than onreviewing the exact clinical protocols and procedures that are presented elsewhere (Cutting et al., 2008; Harton et al.,2011a,b,c). We would also like to put forward some ideas how, based on current biological knowledge, embryo selection pro-cedures could be improved.

2. Static morphological grading

Visual assessment of oocyte and embryo morphology is the most traditional method of embryo selection. Before fertil-ization it can help to distinguish between oocytes with high and low developmental potential (Fig. 1). Healthy metaphaseII oocytes have clear, moderately granulated cytoplasm, a small perivitelline space, an intact first polar body, and a colourlesszona pellucida (Ebner et al., 2003). It is thought that these parameters ensure that both nuclear and cytoplasmic maturationof the oocytes occurred in an undisturbed manner. Indeed, oocytes with such characteristics were shown to give rise to highquality embryos and correlate with successful pregnancies (Alikani et al., 1995; Ebner et al., 1999, 2000, 2002; Kahramanet al., 2000; Loutradis et al., 1999; Serhal et al., 1997). Nowadays, polarized light microscopy also enables the evaluationof the structure of metaphase spindle in the oocyte (reviewed in Shen et al., 2008). This is important as abnormalities in spin-dle structure could lead to (and often also to result from) incorrect meiotic divisions. This in turn may cause aneuploidy anddecrease the developmental competence of the resulting embryo.

After fertilization, embryos can be assessed at different developmental stages according to several criteria (Fig. 1) (Ebneret al., 2003). On day 1 of development, 1-cell embryos (zygotes) are graded according to the morphology of their pronuclei(interphase zygotic nuclei). Every zygote has two pronuclei – one with genetic material inherited from the oocyte (femalepronucleus) and the other with chromosomes coming from the sperm (male pronucleus). They remain separate until theend of the interphase of the first embryonic cell cycle. Only upon the entry to M-phase do maternal and paternal chromo-somes form a single metaphase plate that enables equal separation of genetic material into two daughter cells (called blas-tomeres at this stage). One of the criteria in assessing the quality of the pronuclei is the number and distribution of nucleoli,structures inside pronuclei responsible for rRNA synthesis (so called Z-scoring; Scott et al., 2000). Localization of nucleoli inthe area of pronuclei apposition as well as an equal number (between 3 and 7) of nucleoli in both pronuclei (typical for thehighest, Z1 score) were shown to correlate with positive IVF outcome (Scott et al., 2000; Scott and Smith, 1998; Tesarik andGreco, 1999; Ebner et al., 2003). However, not all groups agree that embryo scoring based on nucleoli is indeed predictive ofthe pregnancy rate (Salumets et al., 2001). Another important factor is the size of the pronuclei. It is thought that in healthyembryos mature pronuclei are evenly sized as any significant difference in pronuclear sizes may indicate aneuploidy (Sad-owy et al., 1998). Zygotes can be also scored based on their cytoplasmic morphology. It has been reported that good qualityembryos usually display a characteristic cytoplasmic ‘halo’ – a layer of clear cytoplasm in the cortical area formed by redis-

Morphology assessment:

PS, ZP, PB

cytoplasm and

spindle grading

PN grading

Cleavage rate, assessment of fragmentation ICM size

PGD / PGS: Blastomere biopsy TE biopsy

Transcript-omics:

mRNAs in follicular

cells

Analysis of metabolism:

Amino acid turnover

HLA-G levels Leptin levels

Metabo-lome

Secrotome and

metabo-lome

Time-lapse imaging:

Cyto-plasmic

flows

Rate and synchrony of the firstcleavage divisions

Day 1 Day 2 Day 3 Day 4 Day 5-6

Puryvate and glucose uptake

Oxygen consumption

Day 0

Polar body biopsy

Fig. 1. Methods of embryonic quality assessment. Contemporary methods of embryo scoring vary from morphological grading, through genetic andbiochemical approaches to the analysis of cellular dynamics by time-lapse imaging. These procedures can be applied at different stages of preimplantationembryonic development. It is important to notice that not all presented methods are routinely used in IVF clinics, some of them (e.g. analysis oftranscriptome and cytoplasmic movements or secretome and metabolome assessment) still require clinical testing. PS, perivitelline space; ZP, zonapellucida; PB, polar body.

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tribution of organelles (mitochondria, endoplasmic reticulum and Golgi cisterns) towards the centre of the zygote (towardspronuclei) (Payne et al., 1997; Salumets et al., 2001; Stalf et al., 2002). This organelle movement is microtubule-mediatedand can reflect the quality of this part of the embryonic cytoskeleton (Kabashima et al., 2007; Sun and Schatten, 2006;Sun et al., 2001). It has been suggested that microtubule-dependent organelle translocation towards nuclei is opposed incells by a microfilament-dependent process (Kabashima et al., 2007). This could account for why too extensive a halomay actually be an indicator of low embryonic viability: it indicates a dysfunctional actin cytoskeleton (Zollner et al., 2002).

An embryo’s morphology can be also assessed during the cleavage stages. The first cell division (from 1- to 2-cell stage) isthought to be an especially good indicator of embryonic quality. Zygotes that divide early (25–27 h post insemination) tendto develop more frequently to the blastocyst stage and achieve better pregnancy rates (Fenwick et al., 2002; Sakkas et al.,1998; Shoukir et al., 1997). It seems likely that a timely first division reflects the good quality of both cytoplasmic and nu-clear components of the embryo. It may mean that activation events accompanying fertilization, such as Ca2+ oscillations,occurred in a correct way and that mitochondria provided sufficient energy to conduct all cellular processes in a timely man-ner. It also suggests lack of DNA damage or chromosomal aberrations that otherwise could activate one of the cell cyclecheckpoints, i.e.biochemical pathways that inhibit or delay cell cycle progression if the integrity of the genetic material iscompromised. The G1/S checkpoint for example prevents cells from entering S phase in the presence of DNA damage byinhibiting the initiation of replication. The intra-S-phase checkpoint, on the other hand, leads to a block in replication andis activated by damage encountered during the S phase or by unrepaired damage that escapes the G1/S checkpoint. Finally,cell cycle progression can be also halted by the G2/M checkpoint that prevents an entry to mitosis (Sancar et al., 2004). Thereis also an M-phase checkpoint (the spindle assembly checkpoint or SAC) that ensures equal segregation of genetic material to

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daughter cells. It is activated if sister chromatids are incorrectly attached to spindle microtubules, i.e. one or both of themremain unattached or both are attached to the same spindle pole (Musacchio and Salmon, 2007; Nezi and Musacchio, 2009).Activation of the checkpoints delays cell cycle progression to give the cell time to either repair the DNA damage (G1/S, intra-S and G2/M) or align chromosomes properly at the metaphase plate (SAC). However, sometimes the checkpoints fail givingrise to cells with compromised genetic integrity. Indeed, it has been observed that checkpoints detecting DNA damage (Mar-angos and Carroll, 2012; Yuen et al., 2012) and incorrectly attached chromosomes (Hassold and Hunt, 2001; Lane et al., 2012)are significantly less effective during female meiosis than in somatic cells. Their effectiveness in early embryos is also dis-puted. The SAC has been reported to be active in zygotes and cleavage-stage embryos, however its sensitivity has not beenthoroughly examined (Wei et al., 2011). It has been also shown that both pathways of DNA double-strand break repair, non-homologous end joining and homologous recombination, function in mouse zygotes (Derijck et al., 2008). However, it seemsthat the DNA repair machinery in 1- and 2-cell mouse embryos may not work as effectively as at later embryonic stages, andthat DNA damage does not inhibit cleavage cell divisions (although it may slow them down) indicating that DNA damagecheckpoints are at these stages also error-prone (Adiga et al., 2007; Derijck et al., 2008; Shimura et al., 2002).

Embryos can be also scored according to the cleavage rate on day 2 and 3 of the development (at 2–8 cell stages). It hasbeen proposed that the best quality embryos should achieve the four-five blastomere stage on day 2 and have seven or moreblastomeres on day 3 (Giorgetti et al., 1995; Van Royen et al., 1999; Ziebe et al., 1997). Healthy embryos also have evenlysized blastomeres as cells of different sizes suggest uneven segregation of the genetic material during division, an activatedcheckpoint, or failure of cytokinesis (when one blastomere is significantly, and persistently, bigger than the rest). In bothcases blastomeres will contain an incorrect number of chromosomes and this may diminish developmental potential ofthe embryo (Giorgetti et al., 1995; Hardarson et al., 2001; Ziebe et al., 1997). Incorrect segregation of the genetic materialmay be also reflected by fragmentation, as fragments often form around micronuclei. It has been suggested that fragmenta-tion, as a part of programmed cell death, is indeed a way of eliminating blastomeres with severe genetic aberrations (Jurisi-cova et al., 1996b; Warner et al., 1998), but whether this is always the case remains still undertermined. However,fragmentation does not necessarily have to be a bad prognostic. If fragments consist only of cytoplasm, the embryo remainseuploid. Moreover, minor fragmentation may disappear with time, either by lysis of the fragments or by their resorption(Hardarson et al., 2001; Van Blerkom et al., 2001). Therefore, embryos with a low degree of fragmentation may be still con-sidered as of good quality, if other criteria such as cleavage rate and blastomere shape, are fulfilled (Van Royen et al., 1999).

Finally, embryos can be scored by their morphology on day 5 or 6, at the blastocyst stage. At this time the embryonicgenome is already fully activated (it activates on day 3, until this moment embryo uses mRNAs and proteins stored bythe oocyte during oogenesis (Dobson et al., 2004; Zhang et al., 2009)), which allows for more reliable assessment of the em-bryo quality. A high quality blastocyst has a distinct blastocoele, a trophectoderm layer (TE) and an inner cell mass (ICM). TheTE will give rise to extraembryonic ectoderm after implantation and will provide the bulk of the embryonic part of the pla-centa (Cockburn and Rossant, 2010; Zernicka-Goetz et al., 2009). In a blastocyst it should form a cohesive epithelium (Gard-ner et al., 2000). ICM, on the other hand, consists of cells that will give rise to the future embryo body and someextraembryonic tissues (Cockburn and Rossant, 2010; Zernicka-Goetz et al., 2009). Therefore, it is not surprising that the sizeof ICM measured either as the cell number or as the area has been found to be the most important factor correlating with theimplantation rate (Balaban et al., 2000; Gardner et al., 2000; Gardner and Schoolcraft, 1999; Richter et al., 2001). Morris et al.(2012) have recently reported that in mice a minimum of 4 epiblast cells (epiblast being a part of ICM that does not havecontact with the blastocoele and gives rise to the future body) has to be established for the full term embryonicdevelopment.

Although morphological assessment is inexpensive and easy to implement in the clinical environment, it is also a verysubjective method. To maximize its reliability an analysis of a full history of embryo development combining grading of zy-gotes, cleavage stages and, if possible, blastocysts is performed rather than an assessment based on a single parameter. Ex-tended culture of embryos, until the blastocyst stage, increases significantly the reliability of embryo assessment based onmorphology, but unfortunately it is not applicable for all patients (e.g. patients with low quality or low number of embryos(<3)). Moreover, extended in vitro culture of embryos has been reported to increase the risk of incorrect epigenetic alterna-tions (Market-Velker et al., 2010; Rinaudo and Schultz, 2004; Rinaudo et al., 2006). This may negatively affect IVF outcomeand health of the future babies. Therefore, it would be highly desirable to have an alternative method of embryo evaluationthat would provide predictive information about the quality of the embryo at as early developmental stage as possible, and,most importantly, would be quantitative and objective.

3. Preimplantation genetic diagnosis and screening

Reliable assessment of nuclear quality is especially important in embryos derived from couples diagnosed with inheritedgenetic disorders. This need led to the development of preimplantation genetic diagnosis (PGD) in the late 1980s. The firstprocedures were performed by Alan Handyside in 1989 in a group of couples at risk of transmitting to their children a sex-linked disease (Handyside et al., 1990). Nowadays, PGD is routinely applied to diagnose recessive sex-linked disorders (e.g.hemophilia, fragile X syndrome, neuromuscular dystrophies), dominant sex-linked disorders (e.g. Rett syndrome, inconti-nentia pigmenti, pseudohypererparathydroidism, vitamin D-resistant rickets), single gene disorders (e.g. cystic fibrosis,Tay-Sachs, Huntington disease, sickle cell anemia) and chromosomal rearrangements (translocation, inversion, deletion

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and aneuploidy). In the 1990s PGD gave rise to preimplantation genetic screening (PGS), a procedure that identifies embryoswith chromosomal aberrations in women of advanced age, as well as in women with repeated miscarriages or implantationfailure, or in case of severe male factor infertility. In contrast to PGD, PGS parents do not present an inherited geneticdisorder.

Although PGS is not limited to patients of an advanced reproductive age, it is most commonly used in this patient group,because aging increases the chances of aneuploidies (Hassold and Chiu, 1985; Hassold and Hunt, 2009). This phenomenon iscaused by an age-dependent decrease in levels of cohesins attached to the centromeric regions of the sister chromatids. Inhealthy young oocytes, during meiosis I cohesins glue together sister chromatids in a bivalent chromosome, ensuring thattheir kinetochores are attached to the same spindle pole. However, the arms and centromeric regions of oocytes from agedfemales display a reduced amount of the cohesin subunit, Rec8, and the protein shugoshin (Sgo2) that protects cohesin frompremature removal from centromeres (Chiang et al., 2010; Lister et al., 2010). This premature cohesin loss leads to preco-cious separation of sister kinetochores (in the 1st instead of the 2nd meiotic division), promoting their attachment to micro-tubules emanating from the opposite spindle poles and resulting in aneuploidy (reviewed in Handyside, 2012).

The material necessary for genetic analysis (1–2 cells for PGD and 1 cell for PGS) is usually obtained by embryo biopsy atthe cleavage stage (day 3) (Harper et al., 2010; Harton et al., 2011c) (Fig. 1). In the original method acidic Tyrode’s solutionwas used to drill a hole in the zona pellucida enabling the aspiration of blastomeres (Hardy et al., 1990), however recently ithas been gradually replaced by laser drilling (Harper et al., 2008; Veiga et al., 1997). Moreover, Ca2+ and Mg2+-free mediumhas been introduced to facilitate blastomere’s separation (Dumoulin et al., 1998). The discovery that embryos often showhigh level of chromosomal mosaicism (some blastomeres in the same embryo can be aneuploid, while other euploid) indi-cates that cleavage stage biopsy may not be representative for the whole embryo (Bielanska et al., 2002; Harper et al., 1995;Munne et al., 1995). Importantly, it still remains unknown whether existence of aneuploid cells among normal euploid cellsat the cleavage stage has serious developmental consequences.

Although removal of 1–2 blastomeres from an 8-cell stage embryo is an invasive procedure, mammalian embryos recoverfrom most of such manipulations because blastomeres can adjust their behaviour in response to changing conditions (Ros-

Animal pole

Vegetal pole

H3 R 2/17/26 methylation ↓Cdx2 ↑(pluripotency ↓)

1st division: M

2nd division: E

1st division: E

2nd division: M

1st division: E

2nd division: E

1st division: M

2nd division: M

Live birth rate:ME embryo

EM embryo

EE embryo

MM embryo

91 %

35 %

84 %

Fig. 2. Pattern of 2nd embryonic cleavage divisions affects live birth ratios. In mouse embryos 2-cell stage blastomeres divide either meridionally (i.e.parallel to AV axis) or equatorially (i.e. perpendicular to AV axis) giving rise to 4 types of 4-cell embryos: ME, EM, EE and MM. Blastomeres in ME embryostend to have more predictable fate than blastomeres in other embryo types. For example, the E-blastomere of the ME embryo containing mostly vegetalmaterial has been shown to display lower levels of histone H3 arginine 2, 17 and 26 methylation and higher levels of Cdx2 expression (Jedrusik et al., 2008;Torres-Padilla et al., 2007). The pattern of the 2nd cleavage divisions affects also the live birth ratio. EE embryos develop to term significantly less frequentlythan other types of embryos (Piotrowska-Nitsche and Zernicka-Goetz, 2005).

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sant, 1976; Tarkowski, 1959, 1961; Tarkowski and Wroblewska, 1967; Tsunoda and McLaren, 1983). Interestingly however,recent observations performed in IVF clinics indicate that biopsy of more than one blastomere negatively affects develop-ment of the human embryo and live birth output (De Vos et al., 2009; Goossens et al., 2008). If the possibility that lowerdevelopmental potential of embryos lacking two blastomeres is due to more extensive damage caused by the procedureis excluded, these results may indicate that the regulative properties of mammalian embryos are not as universal or robustas previously thought. This notion is supported by recent studies on mouse embryos indicating that although the 4- and 8-cell stage blastomeres show clear regulative properties, they are also to some extent developmentally biased (Piotrowska-Nitsche et al., 2005). This developmental bias might be at least in part related to the spatial organisation of the zygote, forexample its animal-vegetal (AV) axis that relates to earlier asymmetric meiotic divisions of the oocyte (discussed below), orthe site of sperm entry (Peng et al., 2012; Piotrowska and Zernicka-Goetz, 2001). It has been found that the first cleavageplane in most zygotes is meridional, which means it occurs along the AV axis with the animal pole marked by the secondpolar body and the vegetal pole at the opposite side of the embryo (Gardner, 1997; Howlett and Bolton, 1985; Pio-trowska-Nitsche and Zernicka-Goetz, 2005; Plusa et al., 2002). As a consequence, cytoplasm from animal and vegetal zygoticregions is distributed relatively equally between both blastomeres in the 2-cell stage embryo. However, the divisions of thesecond cleavage can be orientated in two alternative ways: either meridionally (M; i.e. parallel to the AV axis), when daugh-ter blastomeres inherit both animal and vegetal material, or equatorially (E; i.e. perpendicular to the AV axis), when at leastsome animal and vegetal material becomes separated and inherited differentially by different daughter cells (Gardner, 2002;Piotrowska-Nitsche and Zernicka-Goetz, 2005). Therefore, after the second division four types of embryos can be distin-guished: ME, EM, MM and EE (Fig. 2). It has been found that in a significant group of embryos individual 4-cell stage blas-tomeres differ in the extent of specific epigenetic modifications such as histone H3 arginine 2, 17 and 26 methylation.Specifically, in ME embryos, the E-blastomere, containing mainly vegetal cytoplasm, shows a significantly lower level of thismethylation when compared to the remaining blastomeres and a decreased expression of pluripotency transcription factorssuch as Nanog and Sox2 (Torres-Padilla et al., 2007). Progeny of both E-blastomeres of ME embryos also show significantlyhigher expression levels of transcription factor Cdx2 driving cell differentiation into trophectoderm (Bischoff et al., 2008;Jedrusik et al., 2008). In agreement with these early differences between 4 and 8-cell stage blastomeres it has been foundthat the kinetics of nuclear import/export of another key pluripotency factor Oct4 also differs among blastomeres at thisstage. These differences in Oct4 nuclear kinetics are predictive of subsequent cell fate, so cells in which Oct4 persists longerin nucleus take more asymmetric divisions and preferentially develop to ICM, whereas cells with more ‘mobile’ nuclear Oct4tend to divide symmetrically and differentiate into trophectoderm (Plachta et al., 2011). It is plausible that such distinct nu-clear kinetics of Oct4 originate from the asymmetries formed initially within the oocyte before or after fertilization. Takingall of this together, acknowledging developmental bias of blastomeres may improve protocols of a cleavage stage biopsy. Dueto recent advancements in time-lapse imaging technology (see Section 6) it is now possible to follow the developmental his-tory of blastomeres (i.e. the geometry and timing of cleavage divisions that lead to their formation) and consequently avoidaspirating blastomeres whose progeny will have a tendency to generate ICM and build foetus’ body.

Other types of embryo biopsy are also available (Fig. 1). A polar body biopsy (Verlinsky et al., 1992) can determine thegenetic status of the mother (as polar bodies contain maternal chromatin) but when father is a carrier of a genetic disorder,this technique cannot be applied. A blastocyst biopsy on the other hand enables analysis of genetic composition of TE cells(Kokkali et al., 2005; McArthur et al., 2005). Since a blastocyst biopsy does not interfere with the ICM cell number, it can beconsidered as less invasive than a biopsy at the cleavage stage, in which progenitors of ICM can be unintentionally removed.However, similar to cleavage stage embryos, blastocysts are highly mosaic, and therefore results regarding potential aneu-ploidy may not be representative for the whole embryo (Bielanska et al., 2002). It is also noteworthy that when the embryo isbiopsied on day 5, the time required for genetic analysis precludes the transfer of fresh embryos and cryopreservation withtransfer in a subsequent cycle becomes necessary.

After the biopsy, the next step in PGD and PGS is an actual genetic analysis. In PGD a polymerase chain reaction (PCR)technique is often utilized to diagnose single gene disorders. In PCR selected sequences of the chromosome are amplifiedin order to be detected. This method is not, however, always conclusive, as an absence of the amplification of the mutatedsequence does not necessarily mean that embryo is free of the mutation. It has been recently reported that in approximately10% of analysed blastomeres amplification does not work, regardless of the genotype (Harper and Sengupta, 2012).

In the case of sex-linked diseases requiring embryo sexing PCR analysis is usually replaced with fluorescent in situ hybrid-ization (FISH) using fluorescently-tagged DNA probes specific for certain regions in sex chromosomes (Griffin et al., 1994).FISH is also used to diagnose inherited chromosome abnormalities, e.g. translocations (Fridstrom et al., 2001; Mackie Ogilvieand Scriven, 2002). In this case several DNA probes are used for chromosomes involved in the examined aberration as thisensures a relatively high efficiency of the procedure (Ruangvutilert et al., 2000). FISH of a limited number of chromosomes isalso used in PGS, however, thus far it has failed to increase live birth rates (Blockeel et al., 2008; Jansen et al., 2008; Merse-reau et al., 2008; Meyer et al., 2009; Staessen et al., 2004, 2008). This may be in part, because of technical difficulties relatedto multi-probe protocols and in part because of a high level of mosaicism in the cleavage stage embryo.

In recent years novel array-based techniques have been introduced in PGD and PGS, including comparative genetichybridisation (CGH) and single nucleotide polymorphism (SNP) arrays (Harper and Harton, 2010; Harper and Sengupta,2012). In CGH, DNA from the analysed embryo and normal control DNA are amplified separately using a whole genomeamplification approach. Next, the embryonic and control DNA samples are fluorescently labelled in different colours (e.g.embryonic DNA in green and control in red). Then both DNAs are mixed together in equal proportions and are allowed to

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compete for hybridisation to metaphase spreads prepared from normal male cells (m-CGH) or to an array platform contain-ing small pieces of chromosome (a-CGH). A computer analysis calculates the ratio of green-to-red fluorescence, which re-veals in which chromosome regions one of the colours is under- or over-represented. Gains in red indicate that theembryonic DNA is deficient in that region of the chromosome and gains in green indicate an extra copy of that region inthe embryonic sample. Although these results are very promising, CGH is a time-consuming and technically complex methodthat has its limitations. CGH cannot detect polyploidies, such as triploidies, balanced translocations or inversions, changes inDNA sequence (point mutations intragenic insertions or deletions, triplet repeats) or gains and losses in regions of genomenot covered by the array. Therefore, CGH-based technology is now used mostly to diagnose chromosomal aneuploidies andimbalanced translocations.

Another novel way of analysing the embryonic genotype is with a single nucleotide polymorphism (SNP) array. SNPs areregions of the genome in which a single nucleotide in the sequence varies within the population. SNP arrays can distinguishone person from another and one chromosome from another in any person. In case of PGD and PGS SNP arrays assess howmany copies of each chromosome have been inherited by an embryo. Similar to CGH, DNA from biopsed blastomeres (orpolar bodies or TE cells) has to be first amplified by a whole genome amplification procedure. DNA is labelled with fluores-cent molecules, for example one version of SNP in red and the other in green, and the intensity of red and green signals arecompared to diagnose the inheritance of chromosomes. SNP arrays allow for testing for genetic diseases (by the haplotypingof SNPs surrounding and embedded in disease-causing genes) and aneuploidies. Both CGH and SNP arrays have been alreadyused in PGD and PGS to select healthy embryos leading to successful pregnancies and improving an IVF outcome (Brezinaet al., 2011; Hellani et al., 2008; Schoolcraft et al., 2010, 2011; Scott et al.,2012; Treff et al., 2011a,b; Wilton et al., 2001).

4. Transcriptomics

Transcriptomics, i.e. the analysis of cellular mRNA content, reflects a cell’s phenotype in a better way than analysis of itsgenome. It depends on both DNA sequence that determines the sequence of transcribed RNA, but also on epigenetic mod-ifications that define how each DNA sequence is expressed in the cell. RNA is much more prone to degradation than DNA,and therefore it has to be extracted from cells quickly and under sterile and biochemically clean conditions. If RNA is isolatedfrom a low number of cells, as it is usually the case in embryo quality assessment, it needs to be amplified prior to analysis.The most popular strategy to analyze the resulting sample involves microarray platforms that contain thousands of humangene targets. RNA/cDNA from the sample is fluorescently labelled and hybridised with gene targets immobilised on the arraychip. Computer analysis of the fluorescence provides information about the RNA expression pattern in the examined cells.Deep-sequencing is a novel alternative for transcriptome profiling. Instead of using hybridization to ‘capture’ RNA moleculeson pre-prepared arrays of target genes, deep sequencing directly sequences transcripts present in the examined sample andthen map back to a reference genome. Reads that map back to the reference are then counted to assess the level of geneexpression. More details on procedures of RNA amplification and transcriptome analysis, as well as on the advantagesand pitfalls of various methods can be found elsewhere (Malone and Oliver, 2011; Seli et al., 2010).

The use of transcriptome profiling in the assessment of embryo quality is in its very early stage. There is still not enoughdata on gene expression patterns in oocytes and blastomeres to establish how to distinguish high and low quality embryos.Several groups work on collecting such information in humans and animal models (Hamatani et al., 2004; Kakourou et al.,2013; Robert et al., 2011; Vassena et al., 2011; Wang et al., 2004; Zhang et al., 2009), providing a necessary starting point fordesigning a proper transcriptome-based oocyte/embryo selection procedure. One of the main difficulties here is the minis-cule amount of material available to assess RNA levels in oocytes or embryos. However, technology allowing for transcrip-tome analysis of single cells is improving (Guo et al., 2010; Tang et al., 2010) and therefore, in future, sampling of singlebiopsied blastomeres could be enough to determine which embryo has the greatest developmental potential. Recently, Reichet al. (2011) have also successfully conducted a transcriptome analysis of the first polar body and shown that its gene expres-sion reflects the gene expression in the whole oocyte. This provides a potential non-invasive method of a transcriptome-based assessment of the oocyte quality.

Currently, most of the transcriptome-based data that could be used to assess the developmental potential of the oocyte/embryo comes from the analysis of cumulus and mural granulosa cells isolated from ovaries together with oocytes (Fig. 1). Itis known that crosstalk between oocytes and the surrounding somatic cells through gap junctions and paracrine signalling iscrucial for maintaining the growth and development of both cell types during folliculogenesis (Eppig, 2001; Gilchrist et al.,2008). Recent research indicates that oocyte-secreted factors, such as growth differentiation factor 9 (GDF9) and bone mor-phogenetic protein 15 (BMP15) play an important role in specifying the phenotype of adjacent cumulus cells, which in turn iscritical for the oocyte growth and maturation (Gilchrist et al., 2008; Peng et al., 2013). Therefore, it is likely that analysis ofthe granulosa cell transcriptome will also be able to provide information about the developmental potential of oocytes and,in consequence, embryos. The quality of human oocytes is correlated with high expression of the GDF9 targets, hyaluronansynthase 2 (HAS2), prostaglandin-endoperoxide synthase 2 (PTGS2), pentraxin 3 (PTX3) and gremlin 1 (GREM1), in thecumulus cells (Cillo et al., 2007; McKenzie et al., 2004; Zhang et al., 2005). Surprisingly, none of these GDF9 target geneswas indicated as a positive oocyte/embryo quality marker by the study of van Montfoort et al. (2008) regarding the transcrip-tome of cumulus cells associated with oocytes that developed or failed to develop to cleavage stage embryos. The same studyshowed that the abundance of RNA encoding glutathione peroxidase 3 (GPX3), chemokine receptor 4 (CXCR4), stress-in-

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duced apoptosis inhibitor (HSPB1), cyclin D2 (CCND2) or 7-dehydrocholesterol reductase (DHCR7) in cumulus cells is neg-atively correlated with developmental potential of the oocyte. The authors suggested that expression of these genes may re-flect the hypoxic state of the cumulus cell niche and delayed maturation of the oocytes. Moreover, genes associated withsteroidogenesis, apoptosis and lutenization have been indicated as potential follicular markers of the oocyte/embryo quality(Assou et al., 2008; Hamel et al., 2008, 2010).

As presented above, although recent research provides numerous candidate genes as cumulus/granulosa cell markers ofoocyte/embryo competence, the data is still inconsistent. This is in part due to the lack of a common standard used for em-bryo quality (achieving cleavage stage or successful pregnancy), differences in sampling or distinct methods used for tran-scriptome analysis. Therefore, although the abovementioned results are very promising, they need further testing.Transcriptome analysis is an expensive procedure that requires advanced equipment and skilled staff, so its use on daily-ba-sis in IVF clinics is not likely. It will rather provide the necessary starting information to pick up the most reliable markers ofoocyte/embryo quality that could be used in a simpler embryo selection procedure. Since cumulus/granulosa cell markerscould provide an early and totally non-invasive way of embryonic assessment, there is undoubtedly a great need for furtheradvancement of this technology.

5. Metabolomics and proteomics

Another factor that has to be taken into consideration when embryonic quality is examined is embryonic metabolism.One way of accessing embryonic metabolism is through correlating embryo developmental potential and levels of sub-stances picked up or secreted by the embryo from/to the external environment, i.e. culture medium. Such approach is usuallynon-invasive, as it does not require any embryo manipulation and relays on chemical analysis of the medium (Nel-Themaatand Nagy, 2011). One of the protocols for metabolic analysis involves fluorometric measurements of pyruvate and glucoseuptake. Both these substances are substrates for energy production: pre-compaction embryos use pyruvate and lactate tofuel carboxylic acid-based metabolism (Krebs cycle) and post-compaction embryos switch to the Embden–Meyerhof glycol-ysis pathway and consume glucose. The fact that mammalian embryos switch to the anaerobic Embden–Meyerhof glycolysispathway is probably a part of their survival strategy, as during implantation blastocysts will be exposed to hypoxia (Leese,2012). As expected, a decline in puryvate levels in the medium up to the morula stage and in glucose levels at later stages hasbeen shown for well-developing embryos (Gardner et al., 2001; Gott et al., 1990; Hardy et al., 1989). Whether assessment ofpuryvate and glucose would be able to improve clinical pregnancy rates is, however, still unclear (Conaghan et al., 1993a,b;Turner et al., 1994).

Another method for assessing the energy metabolism of embryos is based on measurements of oxygen consumption as ithas been shown to correlate with quality of the embryo morphology and pregnancy rate (Agung et al., 2005; Lopes et al.,2005, 2007a,b; Magnusson et al., 1986; Shiku et al., 2001). In mammalian embryos oxygen consumption is low in cleavagestages and increases with blastocyst formation, reflecting elevated requirement for ATP caused by cavitation (it utilizes Na+/K+ ATPases) and embryo growth (and therefore increased protein synthesis) (Leese, 2012). Oxygen measurements are tech-nically challenging and often invasive (Agung et al., 2005; Magnusson et al., 1986; Shiku et al., 2001), however, an ultra sen-sitive respirometer has been recently constructed that measures oxygen concentration in non-invasive way using anelectrode submerged in the medium. Until now it has been tested only on animal embryos (Lopes et al., 2005, 2007a,b;Nel-Themaat and Nagy, 2011).

An embryo’s metabolism can be also examined by analyses of the consumption and secretion of amino acids throughmethods such as reverse-phase high performance liquid chromatography (HPLC) and proton nuclear magnetic resonance(Brison et al., 2004; Houghton et al., 2002; Seli et al., 2008). Turnover of several amino acids has been already correlated withdevelopmental potential of the embryos. For example, a decrease in glutamine, arginine and methionine uptake and alanineand aspargine release on day 2–3 and a decrease in serine uptake and alanine and glycine release at morula stage are foundto be good prognostics for a blastocyst’s development (Houghton et al., 2002). Also embryos with decreased glycine and leu-cine and increased aspargine levels in the medium (Brison et al. (2004) and higher levels of glutamate in the culture media(Seli et al., 2008) showed increased pregnancy and live birth rates. Moreover, the amino acid turnover (aspargine, glycine andvaline on day 2–3 and serine, leucine and lysine on day 3–4) has been reported to differ between euploid and aneuploid em-bryos (Picton et al., 2010). Interestingly embryos with greater viability are generally believed to have lower (quieter) aminoacid metabolism (Houghton et al., 2002; Stokes et al., 2007). This agrees with the quiet embryo hypothesis, stating that pre-implantation embryos maintain their metabolism at low levels in order to minimize energy consumption and production ofreactive oxygen species that could damage their cytoplasmic and nuclear components. Taken together, analysis of aminoacid turnover provides valuable information regarding potential IVF outcome, but it also requires expensive equipmentand is time-consuming. Therefore it has not been yet routinely applied in a clinical environment.

Embryonic quality can also be assessed by examining levels of secreted proteins such as soluble human leukocyte antigen(sHLA-G) or leptin. HLA-G is believed to play a role in immune tolerance during pregnancy (Hunt et al., 2006; Sargent, 2005).Although expression of HLA-G mRNA has been found to positively correlate with pregnancy rates (Jurisicova et al., 1996a),data regarding the predictive value of sHLA-G levels are more questionable. Some groups have found that levels of sHLA-G inthe culture medium can indeed be predictive of cleavage rate and implantation potential (Fuzzi et al., 2002; Heidari et al.,2011; Warner et al., 2008). However, other studies have presented opposing results (Sargent, 2005; Tabiasco et al., 2009).

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Leptin, on the other hand, is a small peptide involved in energy metabolism (Friedman, 2002). It has been shown that em-bryos arrested in their development secrete significantly less leptin than those that achieved blastocyst stage (Gonzalezet al., 2000). However, before leptin can be acknowledged as a reliable selection marker, more testing in clinical environmentis required.

Recently, the analysis of single secreted proteins has been gradually replaced by a more general approach, protein secro-tome profiling. This has been possible because of the development of highly sensitive techniques based on mass spectrom-etry (MS) and protein microarrays (Beardsley et al., 2010; Katz-Jaffe et al., 2006, 2005, 2009). MS operates by ionization ofmetabolites, separation of these ions according to their mass-to-charge ratio and then their detection. Protein microarrays,on the other hand, use a chip on which selected antibodies and antigens are arrayed. A fluorescently labelled sample is al-lowed to bind to the antibodies and then fluorescent signal is analysed in order to assess proteins present in the sample. Bothof these methods are technically complex and require very sensitive detector systems, due to a little amount of materialavailable for the examination. However, the development of proteomic analysis is likely to be beneficial as it appears thatthe stage-specific single embryo protein profile does relate to the IVF outcome (Katz-Jaffe et al., 2006). Moreover, analysisof the embryonic secretome has recently identified a potential protein marker (lipocalin-1) distinguishing euploid and aneu-ploid embryos (McReynolds et al., 2011).

Another ‘omics’ technique that has been recently developed and applied for embryo selection is metabolomics, i.e. thesimultaneous profiling of various secreted metabolites, including among others carbohydrates, amino acids, fatty acids ornucleotides. The fact that these metabolites vary greatly in chemical nature and are present in the medium in a wide rangeof concentrations is the main technical challenge of this approach. Currently, assessment of metabolite profile is based onsperctroscopic/sperctrometric and chromatographic techniques such as nuclear magnetic resonance (NMR) spectroscopy,mass spectrometry (often combined with chromatography) and optical spectroscopy (Botros et al., 2008). NMR spectroscopyis based on magnetic properties of atomic nuclei and provides high quality structural and quantitative information of eventhe most complex metabolite mixes. However, it can detect only relatively abundant substances (minimum concentrationabout 1 mM) and requires expensive equipment; therefore it is not frequently used in clinics. Mass spectroscopy can detectsubstances at micromolar concentrations and is therefore significantly more sensitive than NMR spectroscopy. It is very of-ten coupled with different chromatography techniques that pre-prepare samples for MS reducing the complexity of the re-sults obtained (i.e. mass spectrum). MS, similarly to NMR, is an expensive technique, requiring highly skilled staff. Arelatively inexpensive and simpler alternative and thus better suited for routine clinical use is optical spectroscopy, whichmeasures the electromagnetic radiation absorbed, emitted and scattered by the sample. The most popular types of opticalspectroscopy used in metabolic analysis are infrared (IR), near infrared (NIR) and Raman spectroscopy. They are collectivelycalled ‘vibrational spectroscopy’, as they measure molecular vibration modes, like stretching and bending of chemical bondswithin functional groups of molecules (Botros et al., 2008). In the first proof-of-concept study by Seli et al. (2011) NIR andRaman spectroscopy analyses have shown that samples of media in which embryos that resulted in live births or embryosthat failed to implant were cultured differ in their metabolite composition (Seli et al., 2007). Excitingly, the metabolites thatdistinguished these two populations and were the most predictive of pregnancy rate were identified as markers of oxidativestress (Seli et al., 2007). In following studies, metabolite analysis obtained by vibrational spectroscopy was also successfullycorrelated with the developmental potential of embryos (Ahlstrom et al., 2011; Scott et al., 2008) and it has been claimedthat a combination of morphological grading and metabolomic analysis increases the accuracy of embryonic assessment(Seli et al., 2011). However, opposing results have been also reported (Hardarson et al., 2012; Vergouw et al., 2012), suggest-ing that usefulness of spectroscopy-based metabolomic analysis needs to be carefully evaluated before it can be introducedinto clinical practice.

6. Assessment of development by a time-lapse imaging

Microscopic visualisation has been used for scoring embryo quality for decades. However, in most cases only static ‘snap-shot’ observations have been thus far available. Embryos have been screened for specific morphological features at certaintime-points of their culture, but information about the dynamics and continuity of the embryonic processes has been inac-cessible. This has been changed by recent advancements in a non-invasive time-lapse imaging that permits entirely normaldevelopment of embryos imaged in 3D for more than 3 days (Bischoff et al., 2008). In 2010 a team from Stanford Universityused such time-lapse imagining to follow the development of human embryos for 2 days, from the zygote to the 4-cell stage(Wong et al., 2010). This allowed the authors to establish that embryos with an atypically long first cytokinesis, with a pro-longed or an atypically short interval between the divisions from the 1- to 2 and 2 to 3-cell stage, and with an atypically longinterval between the divisions from the 2- to 3 and 3- to 4-cell stage fail to reach the blastocyst stage (Fig. 1). Indeed, pro-gression of cytokinesis reflects functionality of microtubule and actin cytoskeleton that is, indisputably, critical for properdevelopment. Microtubules build metaphase spindles and thus are essential for segregation of chromosomes to daughtercells while actin is required for formation of contraction ring that divides ‘mother cell’ into daughters. Therefore abnormallyprolonged cytokinesis is likely to suggest that one or both components of the cytoskeleton are dysfunctional. Also an abnor-mally short or long duration of the cell cycle indicates that some cellular processes may not be occurring correctly. Prolongedcell cycles are likely to reflect DNA damage or a chromosomal abnormality that activates cell cycle checkpoints and arrests orslow down the cell cycle progression. They may also indicate that the embryo does not have a sufficient amount of energy to

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maintain the dynamic progression of cellular processes. This may, for example, be caused either by a diminished number ofmitochondria or their dysfunction.

The results of live embryo imaging of Wong et al. (2010) accord with previous observations, in which timely pronuclearformation and the first cleavage division were also shown to correlate with quality of human embryos (Fenwick et al., 2002;Lemmen et al., 2008; Lundin et al., 2001). This correlation has been recently confirmed by a time-lapse imaging of embryoswith the EmbryoScope, a relatively simple imaging system enclosed inside an incubator (Meseguer et al., 2011) that enablesimaging of embryos from zygote to blastocyst stage, without negatively affecting their development (Cruz et al., 2011). Inter-estingly, these recent observations of embryos by the EmbryoScope have revealed that although the timing of the first threecleavage divisions may have a predictive value, the kinetics of later embryonic events, particularly the time of division to the5-cell stage and the time of morula formation are more reliable as markers of developmental potential (Cruz et al., 2012;Meseguer et al., 2011) (Fig. 1). The EmbryoScope is currently used by a number of IVF clinics throughout the world.

It would be extremely interesting to use time-lapse technology to assess not only the rate of cleavage divisions, but alsotheir spatial pattern, which has been shown to affect developmental potential of the mammalian embryo (Piotrowska-Nit-sche et al., 2005). As described above (Section 3) division of 2-cell stage blastomeres can be orientated in two alternativeways: either meridionally (M) or equatorially (E). In the former case, daughter blastomeres inherit both animal and vegetalmaterial of the zygote, in the latter – animal and vegetal material is separated and inherited differentially by daughter cells(Gardner, 2002; Piotrowska-Nitsche and Zernicka-Goetz, 2005). In mice, the segregation pattern of the animal and vegetalmaterial correlates with the developmental potential of the embryos. The majority of embryos in which at least two 4-cellstage blastomeres inherit both animal and vegetal material (embryos that have at least one meridional division) develop suc-cessfully to term (91% of ME and EM embryos, 84% of MM embryos). In comparison, only 35% of embryos in which all 4-cellstage blastomeres have exclusively either animal or vegetal material (EE embryos) give rise to viable pups (Piotrowska-Nit-sche and Zernicka-Goetz, 2005) (Fig. 2). Interestingly, it has been recently reported that human embryos with planar mor-phology (corresponding probably to EE and MM embryos) have significantly reduced rates of blastocyst formation andimplantation (Ebner et al., 2012).

In 2011 a new very promising method for assessing the quality of embryos was developed by a team from the Uni-versity of Cambridge working collaboratively with teams in Oxford and Cardiff Universities (Ajduk et al., 2011). This newmethod allows quality control of embryos as early as the zygote stage (Fig. 1). It is based on the discovery that thedynamics of cytoplasmic movements triggered by fertilization is predictive of the mouse embryo’s ability to developto term. These cytoplasmic movements last only for several hours until the pronuclei are formed and to detect themembryos have to be subjected to rapid time-lapse imaging (1 frame every 10–20 s). The series of images undergoes a

mea

n sp

eed

time

mean interval between fast movements =frequency of Ca2+ transients

mean basal speed= functionality of cytoskeleton

1

2

Displacement vector

Time-lapse imaging

Particle Image Velocimetry analysis

Vector map

•

•

Fig. 3. Analysis of fertilization-triggered cytoplasmic movements in mouse zygotes. Mouse eggs are subjected to time-lapse imaging (1 frame every 10 s)immediately after fertilization (Ajduk et al., 2011). Acquired images are analysed by the Particle Image Velocimetry method that follows patterns of contrastbetween subsequent images and calculates how they move. The sum of all displacement vectors calculated for the zygote in a given time-point (i.e. meancytoplasmic speed) is plotted over time. The graph shows when fast cytoplasmic movements occurred in the embryo. The mean interval between the fastmovements (in red) and the mean speed in periods in between the fast movements (mean basal speed, in blue) are indicative of developmental potential ofthe embryo. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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computer analysis based on Particle Image Velocimetry (PIV) method, developed to follow movements of amorphous ob-jects, such as clouds, and frequently used in fluid dynamics. PIV follows patterns of contrasts (white and black pixels)between sequential pairs of images and calculates displacement vectors for them. This creates a vector map that coversall the zygote showing how cytoplasm moves over time and allows quantification of the movement (Fig. 3). PIV analysisof fertilized eggs exposed to different experimental conditions revealed that these cytoplasmic movements depend onfunctionality of the actin cytoskeleton, as they are completely abolished when actin is depolymerised. Moreover, themovements coincide with Ca2+ oscillations (also triggered by sperm) and therefore they provide valuable informationabout the number and frequency of Ca2+ transients. It is important to notice, that this is the first, and to our knowledgethe only, non-invasive and quantitative way of assessing these two parameters in the cell at the same time. Ca2+ oscil-lations have been shown to be essential for proper embryonic development, as they not only trigger completion of mei-osis, establishment of the block to polispermy or recruitment of maternal mRNAs necessary for activation of the embryogenome, but they also influence postimplantation development (Ducibella et al., 2006). It has been suggested that Ca2+

has to be elevated for a certain time to induce each of these processes properly (Ducibella et al., 2002; Ozil et al., 2006,2005; Ozil and Huneau, 2001; Toth et al., 2006). If the number of Ca2+ transients is too low (total duration of elevatedCa2+ is too short), embryo implantation is impaired (Ozil et al., 2006; Toth et al., 2006). On the other hand, if the numberof Ca2+ transients is too high (Ca2+ is elevated for too long) postimplantation development of the embryo becomesaffected (Ozil et al., 2006). In agreement with this, Ajduk et al. (2011) have shown that embryos displaying highlyfrequent increases in cytoplasmic movements after fertilization (very frequent Ca2+ transients) and low cytoplasmicspeeds in the intervening periods (reflective of a poor quality actin cytoskeleton) developed to term three times less suc-cessfully than embryos displaying more average values of these parameters. Although these observations have beenmade upon mouse embryos, cytoplasmic movements very similar in character have been also discovered in activatedhuman eggs (Swann et al., 2012), and the predictive potential of cytoplasmic movements is currently being tested inhuman embryos.

7. Conclusions

The past three decades have provided a broad repertoire of powerful embryo selection methods. Some of them allow thedevelopmental potential of the embryo to be assessed as early as the zygote stage (pronuclear grading, PGD and PGS of polarbodies, analysis of the cytoplasmic movements) while others require embryos at cleavage stages (analysis of cleavage rate,metabolic measurements, PGD/PGS of day 3 embryo) or even at the blastocyst stage (some types of morphology grading andmetabolic analysis, blastocyst PGD/PGS) (Fig. 1). It is important to note that since the human embryonic genome activatesonly at the 4–8 cell stage (Dobson et al., 2004; Zhang et al., 2009), methods assessing zygotes or early cleavage embryos pro-vide information regarding predominantly a maternal component of the embryo. At these stages the only paternal influencethat can be assessed is the general integrity of paternal DNA, the presence of sperm-derived asters and the introduction ofother sperm proteins, such as phospholipase C zeta that triggers Ca2+ oscillations in the egg (Cox et al., 2002; Saunders et al.,2002). Therefore it can be argued that it is beneficial to delay embryo quality assessment until the blastocyst stage, when theembryonic genome is already activated and paternal influence is stronger. On the other hand, delay in assessment of the em-bryo’s viability means that embryos cannot be transferred to mothers-to-be as soon as possible but have to remain in in vitroconditions for longer and be either transferred or cryo-preserved at the blastocyst stage. Although culture conditions haveimproved over recent years, several reports describe the negative effects of prolonged periods of in vitro culture on epigeneticmodifications and gene expression in embryos (Market-Velker et al., 2010; Rinaudo and Schultz, 2004; Rinaudo et al., 2006).Taken together, the benefit of a more accurate assessment of embryonic quality at the blastocyst stage does not necessarilyoutweigh the negative effects of prolonged in vitro culture. This seems to be especially true when IVF is used as a treatmentfor the decreased fertility caused by an advanced reproductive age of mothers. It has been shown that oocyte quality de-creases significantly with age, and therefore it is believed that abnormalities in the development of embryos from oldermothers are caused predominantly by the maternal component, in which case early embryonic selection is sufficiently accu-rate and prolonged in vitro culture can be omitted.

Embryo selection methods can be also grouped according to the cellular compartment they focus upon. Some of themassess nuclear quality, looking for potential genetic abnormalities (e.g. pronuclear grading, PGD or PGS) while others concen-trate on cytoplasmic components, analysing the quality of embryonic metabolism, the cytoskeleton or Ca2+ homeostasis(analysis of metabolites, secreted proteins or cytoplasmic flows). In our opinion the most comprehensive selection methodshould combine these approaches. This can be achieved, for instance, by following the timing of embryonic cell divisions, astheir duration and synchrony can be affected by improper chromosome segregation, cytoskeleton properties and energy lev-els. An alternative approach could combine pronuclear grading or PGS (in case of patients with high number of embryos (>3))with the analysis of embryo’s metabolism or cytoplasmic movements.

In conclusion it is clear that a great deal of scientific effort has recently been put into the development of reliable and non-invasive procedures for selecting high quality embryos. Both scientists and clinicians believe that an accurate scoring systemis a necessary step towards a maximally effective IVF procedure. Therefore, we hope that recent advances in molecular biol-ogy and spectroscopic and microscopic techniques, combined with increasing knowledge about cellular processes occurringin embryos, will allow us to significantly improve the outcome of infertility treatments.

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Competing interests

A U.S. patent has been filed by MZG and AA on the method of assessing mammalian embryo viability based on analysis ofthe cytoplasmic flows (US application No. 61/503827).

Acknowledgements

We would like to express our thanks to the Wellcome Trust for supporting the work in MZG group. AA was also supportedby the Homing-Plus grant from the Foundation for Polish Science. We also thank Chris Graham for providing lots of inspi-ration for our work presented here.

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