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Aneuploidy, the presence o f one or a few chromosomes in abnormal copy number, is by far the most significant type o f chromosomal defect in terms o f human cost, accounting for 70% o f the spontaneous abortion data as well as contributing substantially to early infant mortality (Hassold and Jacobs, 1984; Hassold et al.,

1986). Such an alteration in copy number o f a whole chromosome in effect alters the dosage o f each gene product produced and the phenotypic consequences o f this imbalance during early development are generally severe, unless attenuated by mosaicism with a normal cell line. This is evident from the vast majority o f aU aneuploid conceptions which spontaneously abort before term leaving only frill aneuploidies involving the sex chromosomes and trisomies (3 copies o f a homologue) o f chromosomes 21 ,18 and 13 represented in the livebom population (Hassold and Jacobs, 1984). These potentially livebom autosomal trisomies present as clinically distinct multiple malformation syndromes, but only Down syndrome (trisomy 21) shows any real viability, Edward and Patau syndromes (trisomies 18 and 13), being invariably lethal in the perinatal period (Hook, 1982; Goldstein and Nielsen, 1988). In contrast sex chromosome imbalance appears less detrimental to development, presumably buffered to a large extent by X inactivation and the heterochromatic content o f Yq. In livebirths these include essentially phenotypically normal females XXX and males XYY, mildly dysmorphic infertile females XO (Turner syndrome) and males XXY (Klinefelter syndrome) and very rarely cases o f increasing polysomy X or Y associated with increasing malformations and mental retardation (Kleczkowska et al., 1988; Nielsen and Wohlert, 1990; Kassai et al., 1991) (Table

1.1).

A consequence o f such high levels o f aneuploidy is selection for the loss o f additional chromosomes or duplication o f missing chromosomes, which can result in disomic constitutions including homologues o f the same parental origin (uniparental disomy, UPD) (Warburton, 1988; Engel, 1993). This compensatory mechanism can reveal regions o f genomic imprinting, where parental genomes contribute differentially to development mediated by DNA méthylation, the classic example being Prader-WiUi and Angelman syndromes resulting from maternal and paternal UPD 15 respectively (NichoUs, 1994). On a wider scale (uniparental diploidy) this

imprinting is apparent from the non-viability o f mammalian digynic embryos (two female pronuclei) forming ovarian teratomas o f disorganised fetal tissues and diandric embryos (two male pronuclei) forming an overgrowth o f extra-embryonic tissues known as a hydatidiform mole (Szulman and Surti, 1978a, b; Farrington et al., 1984).

Although the overall chromosome abnormality rate is 50% in spontaneous abortions, this ranges from a low o f 30% for maternal ages up to 29 years to a high o f 82% for ages 40 and above. This significant maternal age effect is caused solely by an increase in trisomies which make up about two thirds o f all karyotypic abnormalities in spontaneous abortions, most commonly o f chromosomes 16 (22%), 22 (18%) and 21 (10%), other trisomies such as 1 ,5 and 19 are rarely if ever identified (Hassold et al., 1980; 1986; Hassold and Chiu, 1985; Eiben et al., 1990). From this it has been estimated that at least a third o f clinically recognised pregnancies in women over 40 years are trisomie (Hassold and Chiu, 1985), which translates to a 1 in 25 risk o f a Down syndrome birth at 46 years compared to that o f 1 in 1000 at 30 years (Cuckle et al., 1987). Monosomy, the corresponding loss o f one homologue, would seem to disrupt development at even earlier stages prior to recognisable pregnancy loss as 45,X (10.5%) is the only monosomy represented in the spontaneous abortion data, with a conspicuous absence of autosomal monosomies (Hassold and Jacobs, 1984). Studies typing DNA polymorphic markers mapping close to the centromere show clearly that the origin o f these trisomie conceptions lies predominantly with errors in meiosis during maternal gametogenesis. Most cases (ranging from 60-100%) of trisomies 13, 16, 21 and X, originate from maternal MI errors whilst the majority (60%) o f trisomy 18 originates from maternal Mil errors (Hassold et al., 1987; 1995; Antonarakis et al., 1992; Bugge et al., 1998; Thomas et al., 2001). A paternal origin is only indicated in a few percent o f these trisomies, but is responsible for about half o f XXY cases (Jacobs et al., 1988) as well as the majority o f 45,X cases where over 80% show a missing paternal X (Hassold et al., 1988). Mitotic errors in embryogenesis are implicated in 5-20% o f cases, but this may be underestimated as errors in pre-mitotic divisions during gametogenesis may also appear as being o f meiotic origin (Antonarakis et al., 1993). Although cytogenetic techniques are more difficult to apply to gametes, with Mil oocyte chromosomes o f poor morphology and sperm nuclei requiring labour-intensive hetero specific fertilisation techniques to yield

incidence o f maternally-derived aneuploidy. Whilst frequencies vary according to study, on average sperm karyotypes show 1-4% aneuploidy compared to 15-20% seen in oocytes (Martin ei al., 1991; Pellestor et a l, 1991; Jacobs, 1992; Benkhalifa et a l,

1996). In part this reflects the higher sensitivity o f mammalian spermatogenesis to meiotic disturbances compared to oogenesis, good evidence o f which has been shown in the mouse (LeMaire-Adkins et a l, 1997; Odorisio et a l, 1998).

The precise mechanisms acting to generate aneuploidy are still not completely understood, but it is clear that aberrant patterns in chromosome or chromatid association and separation during cell division hold the key to subsequent nondisjunction (Fig 1.4). During meiosis I it is vital that the bivalents held at chiasmata remain associated until all are correctly aligned on the metaphase plate and anaphase can begin. This explains why a large number o f trisomies are associated with meiotic nondisjunction o f achiasmate or low exchange chromosomes, as shown by the reduced recombination maps (Warren et a l, 1987; Sherman et a l, 1991; 1994; Hassold et a l, 1995), which fail to physically hold bivalents together leading to random segregation o f univalents. From observations of extra or missing single chromatids in Mil oocytes, Angell (1994; 1997) has suggested that a consequence of this may be premature univalent division followed by random chromatid segregation as an important cause o f aneuploidy. It is now apparent that both the frequency o f recombination and the position o f these events along different chromosomes may effect the likelihood o f malsegregation. From studies o f nondisjoined chromosomes 21, a general correlation has been noted between reduced recombination and an enrichment for distal exchanges in MI errors and increased recombination and more proximal exchanges in Mil errors (Hawley et a l, 1984; Lamb et a l, 1996). Meiotic mutants in Drosophila and yeast provide good models for this and show that more distal exchanges may not be sufficient to ensure normal orientation and segregation of bivalents, particularly when associated with smaller chromosomes whilst in contrast more proximal and particularly pericentric exchanges may act to interlock bivalents preventing their normal disjunction (Koehler et a l, 1996a,b; Ross et a l, 1996). Lamb

et a l, (1996) proposes from this a two hit system to explain the predominance of maternal MI errors in human trisomy. The first event establishes a susceptible pairing configuration in fetal meiosis whilst the second event is an age-related impairment o f

Fig 1.4: Chromosome Malsegregation. H yperhaploid (n=24) w , , — - H yperhaploid N ondisjunction Mil . H ypohaploid (n -2 2 ) H ypohaploid % % or Mil Norm al G am ete (n=23) Norm al G am ete

M l _____________N ondU junction ' H ypohaploid (n=22) - H yperhaploid (n=24)

a Classical Nondisjunction - Meiosis ! & IL

Norm al G am ete

M I M il H yperhaploid (n=^24)

N ondisjunction ' H ypohaploid (n=22)

— Nor ma l G am ete

h Premature Division & Random Segregation - M eiosis I.

N orm al Cell (2n=46) M itosis

M onosom ie (2n=45) chrom osom e lost

c Anaphase Lag - Mitosis.

Fig 1.4: Three basic ty pes o f chromosome malsegregation can occur during either meiosis or mitosis, a Nondisjunction: homologous chromosomes or chromatids fail to disassociate and segregate together to one pole at anaphase, shown here during meiosis I (MI) or meiosis II (M il) h Premature separation, division or failure to pair: chromosomes or chromatids disassociate prematurely and randomly segregate, shown here for a univalent during MI. c

Anaphase lag: chromosomes are excluded from the segregation process or lost subsequent to delayed segregation to one pole, shown here during mitosis.

the meiotic process, such as defective spindle apparatus (Battaglia et al., 1996), which gives an increased risk o f nondisjunction. This general interpretation is broad enough to encompass other factors that may contribute to spindle disturbances linked to aneuploidy such £is hormonal imbalance and reduced intrafollicular vascularity (Gaulden, 1992; Van Blerkom, 1998).

Polyploidy, the presence o f additional chromosomes sets (n), is not uncommon in early development with 12% o f karyotypic abnormalities in spontaneous abortions showing triploidy (3n = 69) and 9% tetraploidy (4n = 92) (Hassold et al., 1986; Eiben et al., 1990). However the resulting severe chromosomal imbalance means that abortion almost always occurs in the first or second trimester and although intrauterine survival is enhanced with mosaicism for a diploid line (Carakushansky et al., 1994), progression to term is rare (Book and Santesson, 1960; Cassidy et al., 1977). An imprinting effect is evident in the triploid phenotype, particularly involving placental morphology, with those o f diandric origin characteristically large and molar (partial mole) and those o f digynic origin small, underdeveloped and nonmolar (Szulman and Surti, 1978a, b; Jacobs et al., 1982; Zaragoza et al., 2000). Polyploidy can arise like aneuploidy fi-om errors in cell division, though in this case involving the abnormal distribution o f a whole chromosome set rather than single chromosomes or alternatively fi'om polyspermy, fertilisation with more than one sperm. Dispermy the main cause o f triploidy in abortuses (Jacobs et al., 1978; Zaragoza et al., 2000) is also a fi*equent occurrence in IVF embryos, as noted by the presence o f three pronuclei (3PN), although in part this reflects the much higher (up to x500 fold) sperm concentrations used for insemination

in vitro compared to in vivo. Triploidy may also result fi*om complete nondisjimction at MI or Mil, and in digynic cases this meiotic failure can take the form o f non­ extrusion o f a polar body nucleus which then becomes incorporated into the embryo (Penrose and Delhanty, 1961; Zaragoza et al., 2000). Tetraploidy and higher orders o f ploidy result from any meiotic or post-zygotic mitotic failure in cytokinesis following DNA replication. Although tetraploid cells seen in early cleavage embryos have been considered abnormal their presence particularly in later blastocyst stages may represent early syncytiotrophoblast lineages (Angell et al., 1987).