REPRODUCTIONREVIEW

Causes and consequences of chromosome segregation error in preimplantation embryos

Cayetana Vázquez-Diez1 and Greg FitzHarris1,2 1Centre de Recherche du Centre Hospitalier de l’Université de Montréal, Montréal, Québec, Canada and 2Département d’Obstétrique-Gynécologie, Faculté de Médecine, Université de Montréal, Montréal, Québec, Canada Correspondence should be addressed to G FitzHarris; Email: [email protected]

Abstract

Errors in chromosome segregation are common during the mitotic divisions of preimplantation development in mammalian embryos, giving rise to so-called ‘mosaic’ embryos possessing a mixture of euploid and aneuploid cells. Mosaicism is widely considered to be detrimental to embryo quality and is frequently used as criteria to select embryos for transfer in human fertility clinics. However, despite the clear clinical importance, the underlying defects in cell division that result in mosaic aneuploidy remain elusive. In this review, we summarise recent findings from clinical and animal model studies that provide new insights into the fundamental mechanisms of chromosome segregation in the highly unusual cellular environment of early preimplantation development and consider recent clues as to why errors should commonly occur in this setting. We furthermore discuss recent evidence suggesting that mosaicism is not an irrevocable barrier to a healthy pregnancy. Understanding the causes and biological impacts of mosaic aneuploidy will be pivotal in the development and fine-tuning of clinical embryo selection methods. Reproduction (2018) 155 R63–R76

Introduction: the advent of the mosaic embryo trophectoderm which will give rise to extraembryonic structures, and the inner cell mass which will constitute Preimplantation development is initiated by the fusion all embryonic tissues (Morris & Zernicka-Goetz 2012, of highly specialised gametes, the sperm and the Niakan et al. 2012, Rossant 2016) (Fig. 1). This complex oocyte, resulting in the formation of a totipotent zygote. and coordinated series of events all take place as the Faithful execution of the first several cell divisions after embryo travels down the fallopian tubes from the ovary fertilisation is fundamental to the establishment of a en route to the implantation site, the endometrium. healthy pregnancy. Following fertilisation, the male and Thus, preimplantation development presents a highly female genomes form pronuclei in the zygote, whose unusual cellular context in which to execute such a subsequent breakdown marks the onset of the first critical succession of cell divisions. mitotic division. Preimplantation mitotic cell divisions Given that the early mitoses form the small number are ‘reductive’, meaning unaccompanied by cellular of cells from which the entire organism develops, one growth, thereby producing progressively smaller might imagine that these mitoses should be heavily cells (Fleming & Johnson 1988, Tsichlaki & FitzHarris safeguarded to ensure genetic fidelity is maintained. 2016). Simultaneously, numerous developmentally Rather, early mammalian development is synonymous important events take place. For example, zygotic with cell division errors. It has long been recognised genome activation at the 4- to 8-cell stage in humans that aneuploidy, when cells have an abnormal number and 2- to 4-cell stage in mouse means that the embryo of chromosomes, may be linked to reduced fertility no longer relies exclusively on maternally stockpiled since genetic aberrations are common in tissues from mRNAs and proteins and can synthesise these factors spontaneous miscarriages and most unisomy and from the embryonic genome (Niakan et al. 2012, Lee trisomy aneuploid karyotypes are non-viable (Hassold et al. 2014). Compaction at the 8-cell stage brings the & Hunt 2001, Jones & Lane 2013, Webster & Schuh dividing cells into close adherence with each other, 2017). Whole-embryo single chromosome copy number and cavitation at the 16- to 32-cell stage creates a aberrations are predominantly due to chromosome fluid-filled cavity marking arrival at the blastocyst stage segregation defects in oocyte meiosis, which markedly of development (Fleming & Johnson 1988). Blastocyst increase with advanced maternal age (Hassold & Hunt formation is also accompanied by the clear delineation 2001). However, the introduction and development of of the first two cell fate lineages in the embryo; the improved culture methods and assisted reproduction

© 2018 Society for Reproduction and Fertility https://doi.org/10.1530/REP-17-0569 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org Downloaded from Bioscientifica.com at 10/01/2021 11:37:30PM via free access

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Figure 1 Human and mouse preimplantation embryo development. Diagrammatic representation of the major stages in mammalian preimplantation development. Key biological processes occurring during early development and timings for human and mouse embryos are indicated below. technologies such as in vitro fertilisation (IVF) in the wide-reaching phenomenon (Dupont et al. 2010, Elaimi last four decades provided an opportunity to study et al. 2012, Hornak et al. 2012, 2016, Bolton et al. the chromosomal status of cells during early human 2016). Chromosomal mosaicism must originate from development, and led to the realisation that whilst mitotic errors during preimplantation development meiotically derived whole-embryo aneuploidies occur (Mantikou et al. 2012, Taylor et al. 2014), but why the in some embryos, ‘mosaic’ aneuploidy, where only a early mammalian embryo is inherently susceptible to subset of blastomeres within an embryo are aneuploid, mitotic errors, and precisely how these errors come is more prevalent (Taylor et al. 2014). This phenomenon about, is very poorly understood. was first reported in 1993 using simple Fluorescent in Many excellent studies have used observed situ hybridisation (FISH) to label and count the copies chromosome complements in spare embryos from the of a limited number of chromosomes, and subsequent clinical setting to attempt to extrapolate the series of studies using whole-genome hybridisation and events that lead to embryo mosaicism (Coonen et al. modern sequencing approaches have revealed single 2004, Mantikou et al. 2012, Fragouli et al. 2013, Taylor chromosome gains or losses as the predominant genetic et al. 2014). However, understanding the aetiology of anomaly in mosaic embryos, occurring in up to 90% mitotic errors requires visualisation of the events in real of embryos, depending upon the study (Delhanty et al. time, and interventional experiments to probe the role of 1993, Munné et al. 1993, 2017, Echten-Arends et al. molecular players – experiments that are hard to tackle 2011, Vera-Rodriguez & Rubio 2017). Additionally, in the clinical setting. Therefore, the underlying cellular polyploid and segmental mosaic aneuploidies are mechanisms through which chromosome segregation observed, albeit at much lower incidences (Echten- errors arise in early human development remain mostly Arends et al. 2011) (Fig. 2). Chromosomal mosaicism elusive. Since many reviews of clinical literature cover has also been reported in non-human primate, porcine, the incidence and characteristics of preimplantation bovine and murine embryos, suggesting it may be a mosaicism (Echten-Arends et al. 2011, Taylor et al. 2014,

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Figure 2 Aneuploidy in the preimplantation embryo originates from meiotic and mitotic chromosome segregation errors. Top panel: Normal fertilization of euploid gametes and error-free progression of meiosis-II and embryonic mitosis results in embryos in which all cells are euploid. Middle panel: Meiotic errors rendering gametes aneuploid (note that female meiosis-I error is represented, but may also occur during meiosis-II and male meiosis), result in embryos comprised of homogeneously aneuploidy cells. Bottom panel: Normal fertilization of euploid gametes and faithful completion of meiosis-II produce a diploid, chromosomally-balanced zygote. Errors in mitosis during embryonic cell divisions lead to a mixture of euploid and aneuploid cells. Diploid–aneuploid mosaicism is most common, with single chromosome copy number losses as the most prevalent defect, followed by single chromosome gains and complex or ‘chaotic’ mosaic aneuploidies.

Munné et al. 2017), we here discuss what is known centres (MTOCs) form, usually around two separate about the mechanisms of cell division in mammalian pairs of centrioles that nucleate and promote the embryos, highlighting recent key advances that point polymerisation of spindle microtubules. Kinetochores, towards perspectives on the aetiology and impact of complex multi-protein structures, become fully mitotic chromosome segregation errors in early embryos, assembled at centromeric regions of chromosomes and and then go on to consider their consequences. For act as a binding platform for spindle microtubules. Thus, reviews of other types of genomic errors occurring in by the stochastic and directed attachment of microtubules embryos such as segmental aneuploidies, chromosomal emanating from two MTOCs results in the formation of rearrangements, microdeletions and duplications, a fusiform spindle, along whose equator chromosomes see (Capalbo et al. 2017, Morin et al. 2017, Treff & become progressively attached and aligned (Compton Franasiak 2017). 2000, Heald & Khodjakov 2015, Petry 2016). Following the correct alignment and attachment of chromosomes, cohesin complexes affixing sister The mechanics of chromosome segregation in chromatids are cleaved, allowing replicated sister mammalian embryos chromatids to be separated and pulled towards apart in a process termed anaphase. In most cells, anaphase Form and function of the mammalian spindle comprises two components that co-ordinately separate Accurate chromosome segregation at the time of cell the chromosomes, termed anaphase A and anaphase division is essential to preserve genetic integrity and is B. Anaphase A consists of a shortening of kinetochore- achieved through a highly coordinated series of events. bound microtubules, thereby lessening the distance As cells enter mitosis, the nuclear envelope breaks down from the chromosome to the pole. Anaphase B describes and chromatin becomes further condensed into mitotic a spindle elongation that separates the spindle poles chromosomes. Simultaneously, microtubule-organising and thus further separates the chromosomes (Maiato www.reproduction-online.org Reproduction (2018) 155 R63–R76

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& Lince-Faria 2010, Asbury 2017). Key to accurate et al. 2010). The reductive divisions of early development segregation of chromosomes is that they should be are highly idiosyncratic, and understanding spindle correctly attached at the bipolar spindle, with sister behaviour in this cellular system cannot rely upon kinetochores-binding microtubules emanating from extrapolation from somatic cells. Importantly, a small opposite spindle poles. Errors in attachment in somatic number of recent studies employing live imaging cells lead to segregation error and aneuploidy (Cimini approaches to understand how spindles are assembled et al. 2001, Thompson & Compton 2011). It is thus perhaps in embryos, largely utilising mouse as a model for unsurprising that most cells possess mechanisms for mammalian embryogenesis, have begun to reveal some ensuring correct kinetochore-microtubule attachment. of the challenges faced by embryo cell divisions. Extensive work in somatic cells has elucidated two In the context of dramatically changing cell size, with major and interconnected mechanisms that operate to each division approximately halving cell size, how spindle prevent chromosome segregation errors during mitosis; structure is regulated during embryo mitosis presents an kinetochore-microtubule error correction (‘error interesting cell biological conundrum. In general, spindle correction’), and the spindle assembly checkpoint length is considered to be controlled by two classes of (SAC), both of which are outlined below. Anaphase is forces; the dynamics of the microtubules and associated followed by the partitioning of the cytoplasm (a process motor proteins as well as the physical properties of termed cytokinesis) and decondensation of mitotic chromosomes are termed ‘intrinsic’ influences, whereas chromosomes and formation of daughter nuclei (termed ‘extrinsic’ influences describe external influences on the telophase) giving rise to two daughter cells with newly spindle, such as whether the cell size limits the length formed interphase nuclei (Fig. 3). of the spindle (Dumont & Mitchison 2009, Goshima & Scholey 2010, Levy & Heald 2012). The spindle in the zygote is substantially shorter than the diameter Spindle idiosyncrasies in the early mammalian embryo of the cell, suggesting length regulation is entirely The mechanisms of mitosis have been intensely studied intrinsic. But from the second mitosis onwards, as cell in a variety of cellular contexts, demonstrating that size decreases, spindle size approaches the diameter of even slight variations in spindle dynamics may result in the cell, indicating extrinsic regulation (Courtois et al. dramatic chromosome segregation defects (Thompson 2012, Yamagata & FitzHarris 2013), as has been seen

Figure 3 Chromosome segregation during mitosis and mechanisms preventing errors. Top: Schematic of the major physical events during mitosis. Condensation of the interphase nucleus into mitotic chromosomes (blue) as the nuclear envelope breaks down and microtubule-organising centers (MTOCs) emerge (red). This is followed by MTOC clustering, bipolar spindle assembly and chromosome attachment to spindle microtubules (red). This is followed by chromosome congression and alignment at the metaphase plate as correct kinetochore-microtubule attachments are establish. Sister chromatids are pulled apart during anaphase and separation of the cytoplasm during cytokinesis ensues the formation of daughter cells. Bottom left: The error correction mechanism relies on the enrichment of Chromosomal Passenger Complex (CPC), the activity of Aurora kinases B and C and downstream effectors to destabilise incorrect kinetochore (yellow)- microtubule attachments, such as merotelic attachments. Bottom right: Spindle Assembly Checkpoint (SAC) proteins are recruited to unattached kinetochores, catalysing the formation of the mitotic checkpoint complex (MCC) which acts to prevent the metaphase-to-anaphase transition.

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Downloaded from Bioscientifica.com at 10/01/2021 11:37:30PM via free access Causes and consequences of embryo mosaicism R67 in lower vertebrate embryos (Wühr et al. 2008, Good many species, including humans, a single centriole et al. 2013). Cytoplasmic removal experiments in mouse and centrosome is thus inherited from the sperm at illustrated this point elegantly; moderate reductions in fertilisation. Pronuclear removal experiments in tri- cell size during the first few cell divisions, when the pronuclear and diploid human zygotes and somatic cell spindle is far shorter than the cell, has little affect upon nuclear transfer in mouse zygotes demonstrated that the spindle length. Contrastingly, cytoplasmic removal in sperm or donor centrosome is functional and directs later preimplantation divisions (4–8 cell and onwards), spindle assembly in the first mitotic divisions (Palermo when spindle length is similar to cell length, shortens et al. 1994, Van Thuan et al. 2006, Kalatova et al. 2015). the spindle (Courtois et al. 2012). Similarly, whereas However, little is known about how these function 1- and 2-cell embryos exhibit a pronounced anaphase during the remainder of early human preimplantation B spindle elongation, the extent and speed of spindle development, and some intriguing observations allude elongation during anaphase is decreased thereafter as that centriole dysregulation may occur in embryos. the size of the cell becomes a limiting factor to spindle Firstly, studies of fresh and vitrified human embryos elongation (Yamagata & FitzHarris 2013). Hence, have reported multipolar spindles and tri-directional reductive divisions impose ever-changing limits on anaphases (Chatzimeletiou et al. 2012, Vera-Rodriguez spindle structure and dynamics during preimplantation et al. 2015), which in somatic cells is symptomatic of development. Curiously, the spindle in the first mitotic too many centrosomes (Ganem et al. 2009). Secondly, a division is proportionally smaller than in the second recent genome-wide association study identified single- division, despite a far greater cell size (Courtois et al. nucleotide polymorphisms in the sequence of PLK4, a 2012, Yamagata & FitzHarris 2013), suggesting spindle key regulator of centriole duplication, to be associated length during the first mitosis is subject to different, with embryonic mitotic errors (McCoy et al. 2015). perhaps more meiotic-like, regulatory mechanisms, than Thus, the idea that centrosome/centriole dysregulation later divisions. Whether this is mediated by remnants contributes to human embryo mosaicism requires of cytostatic factor components that may persist in the further attention. first mitosis, remains to be seen Kubiak( & Ciemerych In the mouse embryo, the role and regulation of 2001, Maller et al. 2002). The shift in the mode of centrioles is even more intriguing. In addition to spindle length regulation in metaphase and anaphase the oocyte lacking centrioles, the mouse sperm also described above exemplifies an emerging theme of cell destroys its centrioles during the elongating spermatid division during early development; that progression phase (Sathananthan 1997), such that most of mouse from zygote to blastocysts is accompanied by gradual preimplantation development then occurs in the absence developmental shifts, rather than an abrupt switch in the of classical centrosomes until the ~64-cell stage, when way in which blastomeres approach a canonical mode centrioles mysteriously emerge (Gueth-Hallonet et al. of mitosis. Another such example is that maintenance of 1993, Courtois et al. 2012, Howe & FitzHarris 2013). spindle bipolarity in metaphase is critically dependent This unexplained series of events uncovers another on the mitotic motor kinesin-5 during the first three gradual shift in spindle microtubule behaviour in mitosis but not later divisions, reflecting a shift in embryos. Spindle assembly in the first few mitoses was the role of the motor from its oocyte role, where it is seen to rely on recruiting several cytoplasmic MTOCs, essential in metaphase, to its somatic cell role, where which form a multipolar spindle, that is later focused it is not (FitzHarris 2009). How changing cell size may and achieves bipolarity, reminiscent of meiotic spindles impact the occurrence of segregation errors is further (Schatten et al. 1986, Gueth-Hallonet et al. 1993, discussed in the context of error avoidance pathways Schuh & Ellenberg 2007), but from the eight-cell stage, below. whilst centrioles and canonical centrosomes are still absent, spindle assembly is mediated by MTOCs that arise exclusively at the nuclear periphery and along Unusual centriole behaviour in early development the spindle in multipolar intermediates (Courtois et al. Centrioles form the focal point of the spindle-organising 2012). This MTOC clustering within the spindle during centrosome in most mammalian cells. Since one mid-preimplantation development was shown to be centriole pair is inherited by each daughter cell, dependent on the Augmin complex, distinct from its role centrioles must replicate each S-phase to provide two in other systems (Watanabe et al. 2016). Additionally, pairs in the subsequent mitosis (Loncarek & Khodjakov PLK4 has been shown to be essential for bipolar 2009, Nigg & Raff 2009). Making sure that centriole spindle assembly in mouse preimplantation embryos, replication happens once-and-only-once is important, despite the fact that centrioles are not present, further since over-replication causes multipolar spindles, indicating non-canonical roles of well-characterised which cause chromosome instability as seen in cancer spindle proteins in early development (Coelho et al. cells (Kwon et al. 2008, Ganem et al. 2009, Gönczy 2013, Arquint & Nigg 2016). Although centrioles later 2015). To avoid too many centrioles after fertilisation, emerge at blastocyst stage, it remains unclear whether oocytes of most species degrade their centrioles. In they are fully functional, since they appear then to lack www.reproduction-online.org Reproduction (2018) 155 R63–R76

Downloaded from Bioscientifica.com at 10/01/2021 11:37:30PM via free access R68 C Vázquez-Diez and G FitzHarris canonical microtubule-organising ability in interphase current and emerging genetic screening techniques, (Howe & FitzHarris 2013). To summarise, multiple lines such as next-generation genome sequencing, with of evidence both from mouse and human embryos direct observation of errors to determine the temporal highlight the importance of understanding mechanisms and genetic dynamics of mosaic aneuploidy in the of centriole and centrosome function in preimplantation early embryo. Perhaps unsurprisingly, live imaging development, and multiple clues indicate that the experiments of chromosome segregation errors are way in which they function is likely distinct from yet to be presented in human embryos. However, better-studied somatic cells. Further work is needed recent live imaging of chromosome segregation errors to elucidate how centrosome function is regulated in mouse embryos suggest that micronuclei, small during development. additional nuclei containing one or few chromosomes separate from the main nucleus which, importantly, are well known to be prevalent in human embryos, may How it all goes wrong: the dynamics of errors play a critical and previously unrecognised role in the in embryos generation of mosaicism (Fragouli et al. 2013, Vázquez- Diez et al. 2016). Micronuclei as a possible catalyst for mosaicism By extensive imaging of embryonic mitoses in mouse Manifold studies of the in vitro cultured human embryos it was shown that micronuclei arise because of single have attempted to use the observed chromosome sister chromatids that remain separate from the main complements of blastomeres at various developmental group in anaphase (termed ‘lagging chromosomes’), stages to extrapolate how chromosome segregation which then form their own nuclear membrane in the errors originated in those embryos. These analyses resulting daughter cells (Fig. 4 and Video 1,). Following resulted in various potential explanations as to how encapsulation into micronuclei, the chromosomes mosaicism might arise including non-disjunction of become extensively damaged and lose centromeric sister chromatids at anaphase, disappearance of a identity indicated by the centromere marker CENP-A, sister chromatid (often termed ‘anaphase lag’ in the and therefore, fail to assemble a kinetochore and be human embryo literature) and inappropriate repeated segregated by the spindle during subsequent cells occurrence of a single chromatid (sometimes referred divisions (Vázquez-Diez et al. 2016). A similar series to as endoreplication). Whilst these studies highlight of events has been observed in somatic cells (Guerrero important principles of how errors could arise, unravelling et al. 2010, Crasta et al. 2012, Huang et al. 2012, Zhang the true nature of missegregation will require coupling et al. 2015a), and it is thought that the defective nuclear

Figure 4 Lagging chromosomes cause micronuclei in mouse preimplantation embryos. Confocal time-lapse frames of a H2BRFP and MajSatTALEmClover-expressing morula, labelling chromatin and centromeric regions, respectively. Lagging anaphase chromosomes result in the encapsulation sister chromatids into micronuclei (white arrowheads).

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Downloaded from Bioscientifica.com at 10/01/2021 11:37:30PM via free access Causes and consequences of embryo mosaicism R69 envelope on the micronucleus, leading to failed nuclear compartmentalisation, underpins the DNA damage to BOX 1. Micronucleus formation and inheritance can explain the most prevalent forms of mosaic aneuploidy in embryos the chromosome enclosed (Hatch et al. 2013). Strikingly, in embryos, the same heavily damaged chromosome Live imaging in mouse embryos revealed that after the formation re-appears as a micronucleus following each subsequent of micronuclei by a lagging chromosome (Fig. 4), the micronucleus is unilaterally inherited at each subsequent cell cell division (Vázquez-Diez et al. 2016). Although division (Vázquez-Diez et al. 2016). This can explain two of the live imaging of this type in human embryos is yet to most commonly observed features of mosaicism in the clinic. be reported, human embryo immunofluorescence Firstly, these events provide a coherent explanation for single data show the occurrence of single anaphase lagging chromosome losses, which are by far the most common genetic chromosomes, micronuclei bearing high levels of abnormality present in embryos – mosaic unisomies being up to DNA damage and lacking centromeric identity, and seven times more frequent than trisomies (Coonen et al. 2004, micronucleus-like chromosomes in mitosis devoid of Taylor et al. 2014) (Fig. 5). During cell division, a lagging chromosome causes a single chromatid to be incorporated into kinetochore staining (Kort et al. 2016). These findings a micronucleus in only one of the two daughter cells. In a strongly allude that the same sequence of events likely balanced scenario, the lagging chromosome initially forms a occurs in human embryos. Importantly, as elaborated micronucleus in the correct daughter cell, such that the in Box 1, this series of events provides a mechanism complete chromosome complement is present taking into for the high occurrence of single chromosome copy account both the main nucleus and micronucleus. However, number variation in mosaic embryos, and, provide for although both sister blastomeres are initially formally euploid, the main nucleus of the micronucleus-containing cell harbours the first time a coherent explanation as to why single a single chromosome copy loss. In the subsequent division of chromosome losses outnumber gains in early embryos that cell, the daughter cell that does not receive the MN will (Box 1, Fig. 5). This mechanism may be specific to early necessarily be hypoploid (Fig. 5, left panel). Alternatively, if the embryos, as micronuclei are typically re-incorporated lagging chromosome generates a micronucleus in the daughter back into the main nucleus in other cell types (Crasta cell whose main nucleus already contains a euploid et al. 2012, Hatch et al. 2013, Leibowitz et al. 2015, chromosome content (i.e. the initial segregation error is Zhang et al. 2015, Ly et al. 2017). unbalanced), then the micronucleus-free sister blastomere will lack a single chromosome. Subsequently, all of the progeny of this cell will display single chromosome loss (Fig. 5, right panel). Video 1 Thus, regardless of the initial ‘direction’ of lagging H2BRFP MajSatTALEmClover-expressing embryo, chromosomes, micronucleus inheritance provides a potential showing lagging anaphase chromosomes result in explanation for a high incidence of single chromosome losses. micronuclei. View video from the online version of the Secondly, micronucleus inheritance can explain single chromosome gains, the second most common genetic abnormality article available at https://doi.org/10.1530/REP-17-0569. in mosaic embryos (Echten-Arends et al. 2011, Taylor et al. 2014). Specifically, if the lagging chromosome forms a micronucleus in the daughter cell whose main nucleus already harbours a complete chromosome complement, this results in a single Chromosomal abnormalities unexplained by (micronucleus-enclosed) chromosome gain (Fig. 5, right panel). micronucleus formation Whilst such single chromosome gains are usually attributed to a classical non-disjunction event, wherein an extra sister chromatid Other chromosomal composition abnormalities have arrives within a single newly forming nucleus (Echten-Arends et al. been described in preimplantation embryos including 2011, Mantikou et al. 2012; Taylor et al. 2014), lagging ploidy defects and complex or ‘chaotic’ abnormalities, chromosomes leading to the formation of micronuclei in albeit at lower frequencies (Echten-Arends et al. 2011). non-complementary cells should have a similar impact. Furthermore, nucleation status abnormalities such as Importantly, note that in the literature, single chromosome binucleation and multinucleation are also common losses have often been attributed to ‘anaphase lag’, without the in preimplantation embryos (Royen et al. 2003). mechanism for this being clear. Within our model, the lagging anaphase chromosome acts as a trigger point by generating a Also commonly observed in human preimplantation micronucleus, but it is the clonal propagation of the blastomeres embryos, is tetraploidy, when a cell has double the following a micronucleus formation event (rather than the normal number of chromosomes. How tetraploidy lagging chromosome per se) that accounts for the high emerges is not clear but could conceivably arise incidence of cells exhibiting a chromosome loss. from cell fusion, although cytokinesis failure and Finally, note that this model unexpectedly provides a mitotic slippage – where the cells exit mitosis in the possible mechanism for aneuploid cells to ‘self-correct’. During absence of sister chromatid separation – are more cell division of a trisomic cell, if the additional chromatid is enclosed in a micronucleus, the micronucleus-free daughter of plausible explanations. In the human embryo, these that (formally aneuploid) cell will be euploid and should tetraploid conditions are likely accompanied by extra therefore have the potential to generate a clone of euploid centrosome copies, which could lead to multipolar blastomeres (Fig. 5, right panel, arrows). Whilst some spindle formation and multidirectional anaphases, both preimplantation genetic screening approaches can detect DNA scenarios resulting in aneuploidy and multinucleation. within micronuclei on at least some occasions, how reliable Whether binucleated and tetraploid embryos exhibit micronucleus detection is by such methods after several cell divisions is unclear and will be important to clarify. multipolar metaphases and tri-directional cell divisions www.reproduction-online.org Reproduction (2018) 155 R63–R76

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Figure 5 Micronucleus inheritance explains the most prevalent defects in mosaic embryos. Lagging anaphase chromosomes lead to the isolation of single chromosomes into micronuclei, which display defective nuclear structure and function, wherein chromosomes are subject to high levels of DNA damage. Likely as a consequence of DNA damage, micronuclei-enclosed chromosomes fail to assemble a kinetochore and are unilaterally inherited during subsequent divisions. Regardless of whether the micronucleus initially forms in the chromosomally-balanced sister (left) or not (right), this pattern of inheritance inevitably generates aneuploid daughters (red nuclei) during following cell cycles. Notably, this model explains the higher occurrence of single chromosome losses and the less frequent incidence of single chromosome gains observed in human embryos. has not been confirmed. However, in human somatic Aurora B, INCENP, Survivin and Borealin, which localises cells, the SAC delays mitosis in the presence of to centromeres during prometaphase (Musacchio & multipolar spindles (Kwon et al. 2008), and aneuploid Salmon 2007, Krenn & Musacchio 2015). At mis-attached human embryos show delayed first mitotic divisions, chromosomes, Aurora B kinase phosphorylates several together alluding that super-numerary centrioles may kinetochore components (Ndc80Hec1, Knl1 and Dsn1) to exist (Vera-Rodriguez et al. 2015). reduce their microtubule-binding affinity. Furthermore, Aurora B also recruits kinesin-13 family members KIF2B and MCAK/KIF2C, which have microtubule Why it all goes wrong; the embryo as a permissive depolymerising activities (Krenn & Musacchio 2015). environment for errors? The error correction mechanism disrupts improper Given the importance of maintaining genetic fidelity, kinetochore-microtubule attachments, promoting the it is perhaps unsurprising that most mammalian cells formation of new, correct, end-on attachments. Error possess multiple mechanisms for averting errors. Indeed, correction is particularly important for the detection and in somatic tissues the rate of aneuploidy is thought to be correction of syntelic (where both sister kinetochores only 2% (Knouse et al. 2014). Given the high incidence are bound to microtubules emanating from the same of mosaic aneuploidy and prevalence of mitotic errors spindle pole) and merotelic attachments (in which a in mammalian preimplantation development, it has long single kinetochore is bound to microtubules originating been suggested such mechanisms may be compromised from both spindle poles) (Fig. 3). or absent during mitotic cleavage divisions (Echten- Several recent lines of evidence reveal embryo- Arends et al. 2011, Mantikou et al. 2012, Taylor et al. specific particularities of the error correction pathway 2014, Albertini 2016). Here, we discuss the limited that could conceivably contribute to the increased rates data on whether and how these pathways function in of missegregation in early embryos. Whilst in somatic early embryos. cells CPC error correction function is mediated by Aurora B kinase (Krenn & Musacchio 2015), recent studies suggest a major role for a less-well-characterised Kinetochore-microtubule attachment error correction Aurora kinase isoform, Aurora kinase C. Human and The error correction mechanism acts locally on mis- murine preimplantation embryos exhibit higher relative attached chromosomes and involves the targeted abundance of Aurora C in early development, with recruitment of factors that will destabilise improper Aurora B abundance increasing by blastocyst stage, kinetochore-microtubule attachments. This mechanism both in mouse and human embryos (Avo Santos et al. is mediated by the chromosomal passenger complex 2011, Schindler et al. 2012, Li et al. 2017). Moreover, (CPC), a multi-protein assembly typically composed of knockout of Aurora B kinase in mouse embryos results in

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Downloaded from Bioscientifica.com at 10/01/2021 11:37:30PM via free access Causes and consequences of embryo mosaicism R71 normal developmental rates but fail to develop beyond A recent study in human embryos revealed that the blastocyst stage (Fernández-Miranda et al. 2011). On spindle poison nocodazole can arrest cell divisions in the other hand, Aurora C knockout significantly impairs mitosis, which is a first line of evidence that the SAC development, reducing blastocyst formation rates, and might in fact operate (Jacobs et al. 2017). Whether knockdown of both Aurora B and C together results these effects are a bona fide indicator of SAC function in increased rates of mitotic arrest and chromosome remains to be determined and will require direct segregation defects (Fernández-Miranda et al. 2011), investigation of SAC components. In mouse, deletion perhaps suggesting a more significant role of Aurora of SAC components Mad2 or BubR1 subtly affects C kinase in error correction in embryonic mitoses preimplantation development, but direct information (Fernández-Miranda et al. 2011, Schindler et al. of SAC activity is currently lacking (Dobles et al. 2000, 2012). Additional evidence implicating Aurora kinases Wang et al. 2004). In the meantime, there are strong in regulation of embryonic mitosis comes from the clues that, analogous to error correction, the function of identification of two Aurora B and C single-nucleotide the SAC may be non-canonical. Significantly, landmark variants in humans, one of which is associated with recent studies revealed that in the mouse oocyte, the reduced rates of aneuploidy in women advanced SAC not only surveys attachment of kinetochores to maternal age and which promotes correct chromosome spindle microtubules, but unexpectedly also specifically alignment when expressed in mouse oocytes (Nguyen arrests meiosis-I in response to DNA damage (Collins et al. 2017). Further studies using immunofluorescence et al. 2015, Marangos et al. 2015, Lane et al. 2017). and chemical inhibition approaches in human tri- Interestingly, DNA damage during mitosis has been pronuclear and diploid embryos have revealed an shown to directly alter microtubule dynamics in somatic unusual CPC component localisation in zygotes cells (Bakhoum et al. 2014). Whether DNA damage due to increased activity of Aurora-activator Haspin may influence spindle dynamics and SAC function in kinase (van de Werken et al. 2015). Determining how mammalian embryos remains elusive. Classic studies this unexpected expression pattern of Aurora kinases of SAC function in other vertebrate embryos, including regulates downstream CPC effectors such as KIF2B and Xenopus and zebrafish, demonstrated that early MCAK to modulate chromosome segregation dynamics cleavage divisions occur in the absence of a functional and microtubule interactions at the kinetochore, will be SAC, which appears later at the mid-blastula transition important to establish the role of these kinesins in the (Hara et al. 1980, Zhang et al. 2015b). Indeed, a role for early embryo. changing cell size in SAC function has been underlined by elegant recent studies. In C. elegans embryos, it was shown that early embryos possess a weak The spindle assembly checkpoint (SAC) checkpoint, and the SAC becomes increasingly robust The SAC is a near-ubiquitous signalling pathway that as development progresses and cells become smaller operates to arrest cells in metaphase of mitosis until (Galli & Morgan 2016). Analogously, in mouse oocytes, all chromosomes have been successfully attached. experimental manipulation of oocyte size found that SAC signalling is initiated at unattached kinetochores larger cells had less robust SAC function (Kyogoku & through the recruitment proteins such as MPS1, MAD1, Kitajima 2017, Lane & Jones 2017). Whether a similar MAD2, BUB1, BUB3 and BUBR1. These in turn catalyse cell size-dependent shift in SAC function operates in the the production of the mitotic checkpoint complex early mammalian embryo and might contribute to the (MCC), composed of MAD2/BUBR1/BUB3/CDC20 error-prone nature of the mammalian embryo has not (Musacchio & Salmon 2007, Lara-Gonzalez et al. 2012). yet been tested. Kinetochore-produced MCC acts as a diffusible signal in the cytoplasm that targets the APC/C to inhibit its ubiquitin ligase activity and prevent the degradation of Consequences of errors: is mosaicism such a securin and cyclin B, hence preventing cohesin cleavage bad thing? and delaying anaphase onset (Musacchio & Salmon The impact of mosaicism on embryo quality 2007, Lara-Gonzalez et al. 2012, Collin et al. 2013). The SAC coordinates achievement of correct kinetochore- In the light of the prevalence of mosaic aneuploidy microtubule attachment of all chromosomes with in mammalian preimplantation development, it is sister chromatid separation and mitotic exit (Fig. 3). essential to understand its impact on developmental It is perhaps unsurprising therefore that absent SAC potential and embryo viability, particularly in the activity has recurrently been forwarded as a candidate clinical setting. Whilst early studies of early embryo explanation for the high incidence of mitotic errors in mosaicism relied on FISH to analyse the presence embryos (Echten-Arends et al. 2011, Mantikou et al. or absence of a restricted number of chromosomes, 2012, Fragouli et al. 2013, Taylor et al. 2014). In support the advent of advanced methods such as comparative of such an idea, SAC component transcript levels are genomic hybridisation and so-called next-generation low during human cleavage stages (Wells et al. 2005). sequencing to assess all chromosomes has improved www.reproduction-online.org Reproduction (2018) 155 R63–R76

Downloaded from Bioscientifica.com at 10/01/2021 11:37:30PM via free access R72 C Vázquez-Diez and G FitzHarris our understanding of the impact of mosaicism (Munné as previously posited (Bolton et al. 2016). Increased et al. 2017). Blastocysts classified mosaic after biopsying rates of apoptosis were also seen in mosaic embryos, and analysing a moderate number of cells (often ~5 or such that the embryo cylinder from embryo chimaeras is so) are less likely to implant and more likely to miscarry largely composed of normal cells by embryonic day 7.5 than ‘euploid’, controls. Importantly, however, the same (Lightfoot et al. 2006, Bolton et al. 2016). Coherently, study also provides strong evidence indicating mosaic in human embryos, rates of mosaicism are lower in embryos can be viable (Munné et al. 2017). Specifically, blastocyst than morula stages and are further reduced diploid–aneuploid mosaic embryos had implantation post-implantation (Echten-Arends et al. 2011, Mantikou rates comparable to euploid embryos, and blastocyst et al. 2012, Taylor et al. 2014). Thus, whilst much is with up to 60% aneuploidy in biopsied cells resulted left to be learned about the consequences and fate of in successful pregnancy outcomes (Fragouli et al. 2017, aneuploid cells in the mosaic embryo, evidence to date Munné et al. 2017). These findings suggest that mosaic suggests that apoptotic pathways and other mechanisms aneuploidy does not necessarily end developmental may act in a targeted cell-type-specific manner before potential. One caveat of this conclusion is that biopsy and after implantation to remove abnormal cells from studies are naturally hampered by random selection of mosaic embryos (Albertini 2016) and that provided cells from embryos, of which a large proportion likely sufficient euploid cells are present in the inner cell mass comprise at least some aneuploid cells (Vanneste et al. to form the embryo proper, mosaic aneuploidy can be 2009). Importantly, it is becoming increasingly clear compatible with healthy, full-term development, and that preimplantation genetic screening results of a few live birth. cells is not necessarily a reliable judge of the extent of mosaicism within an embryo (Gleicher et al. 2016, An unexpected advantage: mosaicism as an 2017). Nonetheless, the emerging view is that, clinically, evolutionary benefit? embryos judged to be likely mosaic are embryos of intermediate favourability for patient transfer – less As described earlier, it appears increasingly plausible favourable than euploid, more than uniformly aneuploid that mosaic aneuploidy is, perhaps counter intuitively, embryos – but consistency of practice between clinics a normal feature of preimplantation development remains lacking (Munné et al. 2017). across multiple mammalian species (Dupont et al. Detailed and elegant experiments in mouse 2010, Hornak et al. 2012, 2016, Mizutani et al. 2012, corroborate the notion that mosaic embryos are not Bolton et al. 2016, Vázquez-Diez et al. 2016). In which necessarily doomed, and provide the beginnings of an case, it becomes reasonable to ask; could chromosome explanation. Embryo transfer experiments using two missegregation in the early embryo present some different mouse models of mosaicism demonstrated that positive biological benefit? degree of mosaicism does not affect implantation rates Unlike other animals which produce tens to hundreds (Lightfoot et al. 2006, Bolton et al. 2016). Furthermore, of embryos ex vivo, mammals produce fewer offspring. embryos with moderate but not extensive levels of Due to the physical and time investment in gestation, mosaicism can fully develop to term (Bolton et al. lactation and nursing required by their offspring, 2016), suggesting a ‘threshold’ degree of mosaicism that mammalian reproduction should be selective of the could be tolerated provided that a critical number of parental genomes, environment and maternal health healthy inner cell mass cells are preserved. Indeed, it status. One possible way to achieve such selection has been proposed that the mammalian foetus is derived could be to exploit the highly atypical environment from as little as three inner cell mass cells (Markert & of preimplantation embryo development that likely Petters 1978), leading to the hypothesis that only a poses strains on cellular resources of embryo during small portion of euploid cells are necessary to sustain development. Having mitotic divisions that are human foetal development – a notion that is supported inherently error-prone and sensitive to environmental by the observation that frozen-thawed human embryos perturbations may be physiologically advantageous that have lost almost half of blastomeres during the since upon undesired situations such as poor maternal cryopreservation procedure are still viable and result in health, low gamete quality or deleterious environment live births (Zheng et al. 2008). This may be explained (poor nutrition for example), a ‘mosaicism threshold’ by two major non–mutually exclusive mechanisms can be surpassed and chances of full-term development – the preferential proliferation of euploid cells and in an unfavourable biological context are reduced. In the negative selection of aneuploid cells in the inner other words, the mosaicism threshold could serve as a cell mass (Echten-Arends et al. 2011, Fragouli et al. sensor for whether an ongoing pregnancy is desirable. 2013). Live imaging experiments in mouse embryos Alternatively, it was recently suggested that increasing revealed that some aneuploid cells within the inner cell oocyte aneuploidy with maternal age may serve a mass undergo apoptosis, presumably to increase the positive benefit in humans by causing an extended proportion of euploid cells, though no evidence was seen period of subfertility in the 40s, prior to the complete of aneuploid cells being ‘directed’ to the trophectoderm cessation of ovulatory cycles (menopause) which occurs

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Downloaded from Bioscientifica.com at 10/01/2021 11:37:30PM via free access Causes and consequences of embryo mosaicism R73 at ~50. Since the loss of all oocytes is associated with a Funding decline in oestrogen that precipitates osteoporosis and Work in the authors' laboratory is funded by CIHR, NSERC, other changes detrimental to general health, age-related and Fondation Jeanne et Jean-Louis Levesque oocyte aneuploidy potentially provides a mechanism to allow older females to maintain a baseline level of oestrogen that beneficial to health, whilst averting Author contribution statement pregnancy (Sirard 2011). This notion is consistent with the well-known grandmother hypothesis, wherein C V-D and G F wrote the manuscript. C V-D performed the older females are considered to increase their chances live imaging experiment and produced the figures. The authors of passing genes to future generations by helping raise thank Karen Schindler and David Albertini for valuable comments and discussion of the manuscript. They apologise their children’s offspring, rather than undergoing further to any authors whose work was not cited for space reasons. G potentially hazardous pregnancies (Hawkes et al. 1998, F is Co-Editor-in-Chief of Reproduction but was not involved Alvarez 2000, Hawkes & Coxworth 2013). Within such in the review process of this paper in any way. a model one might imagine that an inherently fragile ooplasm susceptible to increased missegregation in the face of age-related ovarian changes may be crucial, References and that segregation errors in the embryo shortly after Albertini DF 2016 On becoming accepting of the imperfectionsin fertilisation are simply a manifestation of this fragility. mammalian embryogenesis. 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