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**13**

*UK*

**Meiotic Behaviour of Chromosomes Involved in Structural Chromosomal**

**Preimplantation Genetic Diagnosis**

**1. Introduction**

(Pfeiffer et al., 2004).

(Gardner and Sutherland, 2004).

instability involve low copy repeats (LCRs).

breaks and DNA damage (Shaw and Lupski, 2004).

**1.1 The origin of structural chromosomal abnormalities**

Structural rearrangements can arise premeiotically, during meiosis or during postzygotic mitotic divisions. The main causes leading to chromosomal aberrations are illegitimate recombination due to chromosome misalignment and asymmetric pairing in meiosis and mitosis, defective DNA repair mechanisms and aberrant behaviour at the replication fork

Chromosome rearrangement breakpoints are not uniformly found throughout the genome (Lupski, 2004) and certain genomic regions, especially subtelomeric and pericentromeric regions (Shaw and Lupski, 2004) are more likely to be involved in a chromosomal rearrangement. This clustering of chromosomal rearrangements around hotspots creates considerable genomic instability (Shaw and Lupski, 2004). Such regions of genomic

LCRs act as substrates for non-allelic homologous recombination (NAHR), by erroneously facilitating different chromosome regions of the same or of different chromosomes to come together. Chromatin structure can also be involved in the generation of chromosomal aberrations, with areas which are less compacted being more accessible to double strand

There are also non-recurrent rearrangements, whose breakpoints are scattered around the genome and which often occur at unique sequences (Shaw and Lupski, 2004) but even in those cases the breakpoints often involve intronic motifs of smaller repetitive sequences which are usually involved in inducing susceptibility to double strand breaks (Toffolatti et al., 2002). Such rearrangements with unique breakpoints are created by non-homologous DNA end-joining (NHEJ), which is an error prone mechanism (Lieber et al., 2003) of repairing double strand breaks in multicellular organisms (Shaw and Lupski, 2005). NHEJ can result in genomic alterations by generating deletions or duplications through erroneously joining together the ends of double strand breaks from different chromosomes

**Abnormalities Determined by**

L. Xanthopoulou and H. Ghevaria

*University College London,*

### **Meiotic Behaviour of Chromosomes Involved in Structural Chromosomal Abnormalities Determined by Preimplantation Genetic Diagnosis**

L. Xanthopoulou and H. Ghevaria *University College London, UK*

#### **1. Introduction**

240 Meiosis - Molecular Mechanisms and Cytogenetic Diversity

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and regulates some target genes in common with glucocorticoid hormones.

#### **1.1 The origin of structural chromosomal abnormalities**

Structural rearrangements can arise premeiotically, during meiosis or during postzygotic mitotic divisions. The main causes leading to chromosomal aberrations are illegitimate recombination due to chromosome misalignment and asymmetric pairing in meiosis and mitosis, defective DNA repair mechanisms and aberrant behaviour at the replication fork (Gardner and Sutherland, 2004).

Chromosome rearrangement breakpoints are not uniformly found throughout the genome (Lupski, 2004) and certain genomic regions, especially subtelomeric and pericentromeric regions (Shaw and Lupski, 2004) are more likely to be involved in a chromosomal rearrangement. This clustering of chromosomal rearrangements around hotspots creates considerable genomic instability (Shaw and Lupski, 2004). Such regions of genomic instability involve low copy repeats (LCRs).

LCRs act as substrates for non-allelic homologous recombination (NAHR), by erroneously facilitating different chromosome regions of the same or of different chromosomes to come together. Chromatin structure can also be involved in the generation of chromosomal aberrations, with areas which are less compacted being more accessible to double strand breaks and DNA damage (Shaw and Lupski, 2004).

There are also non-recurrent rearrangements, whose breakpoints are scattered around the genome and which often occur at unique sequences (Shaw and Lupski, 2004) but even in those cases the breakpoints often involve intronic motifs of smaller repetitive sequences which are usually involved in inducing susceptibility to double strand breaks (Toffolatti et al., 2002). Such rearrangements with unique breakpoints are created by non-homologous DNA end-joining (NHEJ), which is an error prone mechanism (Lieber et al., 2003) of repairing double strand breaks in multicellular organisms (Shaw and Lupski, 2005). NHEJ can result in genomic alterations by generating deletions or duplications through erroneously joining together the ends of double strand breaks from different chromosomes (Pfeiffer et al., 2004).

Meiotic Behaviour of Chromosomes Involved in Structural

unbalanced offspring (Gardner and Surtherland, 2004).

**B derB**

**A derB**

**derA A**

**3:1 segregation patterns:**

**2:2 segregation patterns**:

**A derA**

i)

iii)

**A**

**Alernate**

**Adjacent-1**

**Adjacent-2**

**A**

Chromosomal Abnormalities Determined by Preimplantation Genetic Diagnosis 243

abnormal embryos that are associated with recurrent miscarriage, infertility as well as

**B derB**

**derB**

**derB**

**derA B**

**B derB**

**derA B**

**derB B**

**derA A**

**B**

**derA A**

Fig. 1. i) Chromosomes in a cell of a carrier of a reciprocal translocation, ii) Pachytene

iii) possible segregation modes seen at meiosis (4:0 segregants not shown here).

configuration of the quadrivalent cross during meiosis in a carrier of a reciprocal translocation,

**Interchange monosomy Interchange trisomy**

**Tertiary monosomy Tertiary trisomy**

**derA**

ii)

#### **2. Structural chromosomal abnormalities and meiosis: Focussing on translocations**

Structural chromosomal abnormalities involve chromosome breakage followed by the rejoining of chromosome parts into a different configuration. These structural rearrangements can be mainly categorised into translocations (reciprocal or robertsonian), insertions, inversions (pericentric or pericentric), deletions, duplications, ring chromosomes and isochromosomes. In the case of a structural rearrangement, each chromosome pair consists of a normal and a derivative chromosome.

Many structural abnormalities are associated with clinical charactteristics, such as mental retardation, characteristic dysmophic features and often other malformations and developmental delay. For example the cri-du-chat syndrome caused by a terminal deletion on chromosome 5, or the DiGeorge syndrome caused by a microdeletion on the long arm of chromosome 22. Duplication syndromes are also known to be associated with abnormal phenotypes, such as Charcot Marie Tooth disease Type I caused by a duplication in 17p12. Other structural aberrations such as inversions, translocations and ring chromosomes do not cause any abnormalities in balanced carriers when there is no gain or loss of genetic material, however these individuals are faced with reproductive problems.

The most common type of structural chromosomal abnormality in humans is translocation, most specifically reciprocal translocations that are seen in about 1 in 500 live births (Jacobs et al., 1992) and involve the exchange of genetic material between different chromosomes. In carriers of reciprocal translcoations, each chromosome pair consists of a normal and a derivative chromosome, as shown in figure 1i. Carriers are phenotypically normal since there is no loss of genetic material unless the breakpoints disrupt important genes (Gardner and Sutherland, 2004). However they are faced with unfavourable meiotic segregation patterns when the chromosomes involved in the translocation pair up at prophase forming a quadrivalent (also known as pachytene) as shown in figure 1ii. The chromosomal status of the gametes produced by a carrier of a reciprocal translocation depends on the segregation pattern that took place in meiosis. As shown in figure 1iii, the chromosomes in the quadrivalent can segregate in a 2:2 mode, whereby each daughter cell will receive 2 chromosomes, in a 3:1 segregation mode, whereby one daughter cell receives 3 chromosomes and the other daughter call receives just one or there might be complete non disjunction in a 4:0 mode of segregation.

Robertsonian translocations have a prevalence of 1 in 1000 and involve the centric fusion of two acrocentric chromosomes (in 75% of the cases chromosomes 13 and 14 are involved) and the loss of the short arms (Garner and Sutherland, 2004). As a result a derivative chromosome is formed as shown in figure 2i, which comprises of the two long arms of the chromosomes involved in the translocation and at meiosis the chromosomes pair up as shown in figure 2ii. The total chromosome number of carriers of Robertsonian translocations is 45. Although all acrocentric chromosomes have been found to be involved in Robertsonian translocations, rob(13q14q) and rob(14q21q) constitute around 85% of all Robertsonian translocations (Therman et al., 1989).

Balanced carriers of a structural chromosomal rearrangement are usually phenotypically normal since there is no loss of genetic material, unless the breakpoints are within important genes. They are however faced with unfavourable meiotic segregation patterns when the normal and derivative chromosome(s) involved in the abnormality pair up at meiosis I. Carriers therefore are at a high risk of producing unbalanced gametes and hence genetically

Structural chromosomal abnormalities involve chromosome breakage followed by the rejoining of chromosome parts into a different configuration. These structural rearrangements can be mainly categorised into translocations (reciprocal or robertsonian), insertions, inversions (pericentric or pericentric), deletions, duplications, ring chromosomes and isochromosomes. In the case of a structural rearrangement, each chromosome pair

Many structural abnormalities are associated with clinical charactteristics, such as mental retardation, characteristic dysmophic features and often other malformations and developmental delay. For example the cri-du-chat syndrome caused by a terminal deletion on chromosome 5, or the DiGeorge syndrome caused by a microdeletion on the long arm of chromosome 22. Duplication syndromes are also known to be associated with abnormal phenotypes, such as Charcot Marie Tooth disease Type I caused by a duplication in 17p12. Other structural aberrations such as inversions, translocations and ring chromosomes do not cause any abnormalities in balanced carriers when there is no gain or loss of genetic

The most common type of structural chromosomal abnormality in humans is translocation, most specifically reciprocal translocations that are seen in about 1 in 500 live births (Jacobs et al., 1992) and involve the exchange of genetic material between different chromosomes. In carriers of reciprocal translcoations, each chromosome pair consists of a normal and a derivative chromosome, as shown in figure 1i. Carriers are phenotypically normal since there is no loss of genetic material unless the breakpoints disrupt important genes (Gardner and Sutherland, 2004). However they are faced with unfavourable meiotic segregation patterns when the chromosomes involved in the translocation pair up at prophase forming a quadrivalent (also known as pachytene) as shown in figure 1ii. The chromosomal status of the gametes produced by a carrier of a reciprocal translocation depends on the segregation pattern that took place in meiosis. As shown in figure 1iii, the chromosomes in the quadrivalent can segregate in a 2:2 mode, whereby each daughter cell will receive 2 chromosomes, in a 3:1 segregation mode, whereby one daughter cell receives 3 chromosomes and the other daughter call receives just one or there might be complete non

Robertsonian translocations have a prevalence of 1 in 1000 and involve the centric fusion of two acrocentric chromosomes (in 75% of the cases chromosomes 13 and 14 are involved) and the loss of the short arms (Garner and Sutherland, 2004). As a result a derivative chromosome is formed as shown in figure 2i, which comprises of the two long arms of the chromosomes involved in the translocation and at meiosis the chromosomes pair up as shown in figure 2ii. The total chromosome number of carriers of Robertsonian translocations is 45. Although all acrocentric chromosomes have been found to be involved in Robertsonian translocations, rob(13q14q) and rob(14q21q) constitute around 85% of all

Balanced carriers of a structural chromosomal rearrangement are usually phenotypically normal since there is no loss of genetic material, unless the breakpoints are within important genes. They are however faced with unfavourable meiotic segregation patterns when the normal and derivative chromosome(s) involved in the abnormality pair up at meiosis I. Carriers therefore are at a high risk of producing unbalanced gametes and hence genetically

**2. Structural chromosomal abnormalities and meiosis: Focussing on**

material, however these individuals are faced with reproductive problems.

consists of a normal and a derivative chromosome.

disjunction in a 4:0 mode of segregation.

Robertsonian translocations (Therman et al., 1989).

**translocations**

abnormal embryos that are associated with recurrent miscarriage, infertility as well as unbalanced offspring (Gardner and Surtherland, 2004).

Fig. 1. i) Chromosomes in a cell of a carrier of a reciprocal translocation, ii) Pachytene configuration of the quadrivalent cross during meiosis in a carrier of a reciprocal translocation, iii) possible segregation modes seen at meiosis (4:0 segregants not shown here).

Meiotic Behaviour of Chromosomes Involved in Structural

**11**

11.1 11.21 11.22

11 12

12 13 14 15.1 15.2 15.3 15.4 15.5

13.2 13.3 13.1

q13.3

CE P17 (S A) CE P 11 (S G ) Tel11q (S O)

centromere of 11 and 17 and the telomere of 11q.

**P ro bes us ed for th e diagn os is :**

14.2 14.3 14.1 13.4 13.5 21

24 25

22.3 22.2 22.1 22.3 22.2 22.1

i)

ii)

Normal chromosome 17

Derivative chromosome 17

informative for both normal and boh derivative chromosomes.

**17**

p11.2

**46, XX, t(11; 17)(q13.3; p11.2)**

13 12 11.1 11.2

21.1 21.2 21.3 22 23 24 25

11.1 11.2 12

Chromosomal Abnormalities Determined by Preimplantation Genetic Diagnosis 245

used for the enumeration of that chromosome) are used, together with another probe on the other chromosome, which can either be distal or proximal to the breakpoint on the second chromosome (Scriven et al.1998). So the main two strategies involve using two centromeric and one subtelomeric probe or one centromeric and two subtelomeric probes, where the subtelomeric probes will always be on the segments that are translocated. The aim of these probe strategies is to be able to detect all possible segregation patterns, so they need to be

> 13 12 11.2

Fig. 3. i) Probe strategy for PGD for translocation 46, XX, t(11;17)(q13.3;p11.2), ii) Metaphase nucleus of the carrier of 46, XX, t(11;17)(q13.3;p11.2) after FISH with the probes for

11.1 11.21 11.22

11 12

12 13 14 15.1 15.2 15.3 15.4 15.5

13.2 13.3 13.1 13.3

11.1 11.2

11.1 11.2 12

21.1 21.2 21.3 22 23 24 25 **der 17**

14.2 14.3 14.1 13.4 13.5 21

24 25

Normal chromosome 11

Derivative chromosome 11

22.3 22.2 22.1 22.3 22.2 22.1

**der11**

Fig. 2. i) Carrier of a Robertsonian translocation, ii) Pairing up of chromosomes, forming a trivalent during meiosis in a carrier of a Robertsonian translocation.

#### **3. Preimplantation genetic diagnosis (PGD)**

Preimplantation genetic diagnosis provides an alternative to prenatal diagnosis for couples at risk of having a child with a specific genetic or chromosomal abnormality. It involves the removal and testing of material from the oocyte or the developing embryo. First and second polar bodies for instance can be analysed in order to determine the genetic status of the oocyte (Verlinsky et al., 1990) or material can be biopsied from embryos generated through *in vitro* fertilization (IVF). In the case of embryo biopsy, one or two single blastomeres can be removed during the cleavage stage or material can be biopsied from the trophectoderm at the blastocyst stage (Dokras et al., 1990).

The biopsied material is then analysed by the polymerase chain reaction (PCR) for single gene disorders (Findlay et al., 1996) or fluorescent in situ hybridisation (FISH) for structural or numerical chromosomal abnormalities (Griffin et al., 1991, Coonen et al., 1998, Conn et al., 1998) whereas recently array comparative genomic hybridization (aCGH) has also been introduced (Alfarawati *et al*., 2011). In this way unaffected embryos are selected and transferred to the uterus, aiming to establish an unaffected pregnancy.

Couples that are at risk of having a child with a specific single gene or chromosomal abnormality may opt to have PGD in order to avoid an affected pregnancy and to avoid having prenatal diagnosis which is associated with a 1% risk of miscarriage; moreover their decision to have PGD is often linked to the couple's view on pregnancy termination. At the same time other couples that seek PGD have difficulty achieving a pregnancy due to reasons of infertility or subfertility or are victims of repeated pregnancy loss.

#### **4. PGD for structural chromosomal abnormalities: strategies and outcome**

PGD provides a unique opportunity to investigate the meiotic behaviour of the chromosomes involved in a structural rearrangement. Until recently structural chromosomal abnormalities were tested for at PGD with FISH, using probes for the chromosomes involved in the translocation.

The main FISH probe strategies used in PGD for recirocal translocations, involve the use of probes flanking the breakpoint in one of the chromosomes, in conjunction with another probe on the other chromosome. In this way on one chromosome one probe distal to the breakpoint (i.e. a subtelomeric probe specific to the segment that is translocated) and one probe proximal to the breakpoint (i.e. a centromeric probe of any other probe that will be

**B**

ii)

**der(A;B)**

i) **A**

trivalent during meiosis in a carrier of a Robertsonian translocation.

transferred to the uterus, aiming to establish an unaffected pregnancy.

of infertility or subfertility or are victims of repeated pregnancy loss.

Fig. 2. i) Carrier of a Robertsonian translocation, ii) Pairing up of chromosomes, forming a

Preimplantation genetic diagnosis provides an alternative to prenatal diagnosis for couples at risk of having a child with a specific genetic or chromosomal abnormality. It involves the removal and testing of material from the oocyte or the developing embryo. First and second polar bodies for instance can be analysed in order to determine the genetic status of the oocyte (Verlinsky et al., 1990) or material can be biopsied from embryos generated through *in vitro* fertilization (IVF). In the case of embryo biopsy, one or two single blastomeres can be removed during the cleavage stage or material can be biopsied from the trophectoderm at

The biopsied material is then analysed by the polymerase chain reaction (PCR) for single gene disorders (Findlay et al., 1996) or fluorescent in situ hybridisation (FISH) for structural or numerical chromosomal abnormalities (Griffin et al., 1991, Coonen et al., 1998, Conn et al., 1998) whereas recently array comparative genomic hybridization (aCGH) has also been introduced (Alfarawati *et al*., 2011). In this way unaffected embryos are selected and

Couples that are at risk of having a child with a specific single gene or chromosomal abnormality may opt to have PGD in order to avoid an affected pregnancy and to avoid having prenatal diagnosis which is associated with a 1% risk of miscarriage; moreover their decision to have PGD is often linked to the couple's view on pregnancy termination. At the same time other couples that seek PGD have difficulty achieving a pregnancy due to reasons

**4. PGD for structural chromosomal abnormalities: strategies and outcome**  PGD provides a unique opportunity to investigate the meiotic behaviour of the chromosomes involved in a structural rearrangement. Until recently structural chromosomal abnormalities were tested for at PGD with FISH, using probes for the

The main FISH probe strategies used in PGD for recirocal translocations, involve the use of probes flanking the breakpoint in one of the chromosomes, in conjunction with another probe on the other chromosome. In this way on one chromosome one probe distal to the breakpoint (i.e. a subtelomeric probe specific to the segment that is translocated) and one probe proximal to the breakpoint (i.e. a centromeric probe of any other probe that will be

**A der(A;B) B**

the blastocyst stage (Dokras et al., 1990).

chromosomes involved in the translocation.

**3. Preimplantation genetic diagnosis (PGD)**

used for the enumeration of that chromosome) are used, together with another probe on the other chromosome, which can either be distal or proximal to the breakpoint on the second chromosome (Scriven et al.1998). So the main two strategies involve using two centromeric and one subtelomeric probe or one centromeric and two subtelomeric probes, where the subtelomeric probes will always be on the segments that are translocated. The aim of these probe strategies is to be able to detect all possible segregation patterns, so they need to be informative for both normal and boh derivative chromosomes.

Fig. 3. i) Probe strategy for PGD for translocation 46, XX, t(11;17)(q13.3;p11.2), ii) Metaphase nucleus of the carrier of 46, XX, t(11;17)(q13.3;p11.2) after FISH with the probes for centromere of 11 and 17 and the telomere of 11q.

Meiotic Behaviour of Chromosomes Involved in Structural

Chromosomal Abnormalities Determined by Preimplantation Genetic Diagnosis 247

Once it has been established that a particular probe strategy is informative for the translocation chromosomes, the conditions of the FISH protocols are optimised and the couple can commence their stimulation to undergo their IVF cycle. The biopsied samples are then tested at PGD using the optimised protocol and embryos are diagnosed as balanced or unbalanced. Due to the fact that in the majority of cases of the nuclei of the biopsied cells during cleavage stage biopsy are in the interphase stage, it is not possuble to distinguish between balanced embryos that will be balanced carriers of the structural aberration and normal embryos (Munne, 2005). By considering the FISH signals for each embryo it is then possible to determine the segregation pattern that took place in the gamete of the carreir parent, as shown in table 1, although crossing over events between the centromere and the breakpoint can complicate the situation further and affect the interpretation of the results (Hulten, 2011). Only biopsied samples with two signals for each probe used will be balanced as two signals indicate diploid status for the loci tested. Any other combination of signals is unbalanced for the

Figure 4 shows another example of a reciprocal translocation between chromosomes 10 and 11, 46,XY,t(11;19)(q12.3;q13.1), the probe strategy used at PGD (figure 4ii) and images from single blastomeres that were biopsied on day 3 from cleavage stage embryos (figure 4ii).

translocation chromosomes bue to chromosome malsegregation at meiosis.

Fig. 4. Probe strategy for translocation 46,XY,t(11;19)(q12.3;q13.1) and FISH on the

partial trisomy 10.

lymphocytes of the carrier parent and ii) images from the biopsied single cells from cleavage stage embryos at PGD. Only nucleus D is balanced for the translocation, whereas nucleus A is unbalanced and polyploid, nucleus B shows partial monosomy 10q and nucleus C shows

The probe strategy chosen for PGD for each srtuctural abnormality case is tested prior to clinical application on control lymphocyte slides from the parents of the couple seeking PGD. For example figure 3 below shows an example of a translocation between chromosomes 11 and 17, carried by the female partner: 46, XX, t(11;17)(q13.3;p11.2).

The centromeric probe for chromosome 11 in spectrum green and the centromeric probe for chromosome 17 in spectrum aqua were used together with the subtelomeric probe for 11q in psectrum orange. The probes were tested on parental lymphocytes and in figure 3ii it is clear that the probes chosen pick up the abnormality and allow us to distinuish between the normal and derivative chromosomes on the metaphase spread.

Table 1. Possible segregation patterns for the carrier of 46, XX, t(11;17)(q13.3;p11.2).

The probe strategy chosen for PGD for each srtuctural abnormality case is tested prior to clinical application on control lymphocyte slides from the parents of the couple seeking PGD. For example figure 3 below shows an example of a translocation between

The centromeric probe for chromosome 11 in spectrum green and the centromeric probe for chromosome 17 in spectrum aqua were used together with the subtelomeric probe for 11q in psectrum orange. The probes were tested on parental lymphocytes and in figure 3ii it is clear that the probes chosen pick up the abnormality and allow us to distinuish between the

**FISH signals seen in the**

Der11 and der17 2xCEP11, 2xTel11p, 2xCEP17 Balanced Adjacent-1 11 and der17 2xCEP11, 3xTel11p, 2xCEP17 Partial trisomy 11,

Adjacent-2 11 and der11 3xCEP11, 2xTel11p, 1xCEP17 Partial trisomy 11,

Der11 and 17 2xCEP11, 1xTel11p, 2xCEP17 Partial monosomy

17 and der17 1xCEP11, 2xTel11p, 3xCEP17 Partial monosomy

11, der11 and 17 3xCEP11, 2xTel11p, 2xCEP17 Partial trisomy 11

Der17 1xCEP11, 2xTel11p, 2xCEP17 Partial monosomy

11, 17, der17 2xCEP11, 3xTel11p, 3xCEP17 Partial trisomy 11,

Der11 2xCEP11, 1xTel11p, 1xCEP17 Partial monosomy

11, der11, der17 3xCEP11, 3xTel11p, 2xCEP17 Trisomy 11 17 1xCEP11, 1xTel11p, 2xCEP17 Monosomy 11

Der11, 17, der17 2xCEP11, 2xTel11p, 3xCEP17 Trisomy 17 11 2xCEP11, 2xTel11p, 1xCEP17 Monosomy 17

Table 1. Possible segregation patterns for the carrier of 46, XX, t(11;17)(q13.3;p11.2).

**Outcome**

17

17

17

17

and 17

11 partial monosomy 17

partial monosomy

11, partial trisomy

partial monosomy

11, partial trisomy

partial trisomy 17

11, partial monosomy 17

chromosomes 11 and 17, carried by the female partner: 46, XX, t(11;17)(q13.3;p11.2).

**embryo**

Alternate 11 and 17 2xCEP11, 2xTel11p, 2xCEP17 Balanced

normal and derivative chromosomes on the metaphase spread.

**Chromosomes present in the gamete of the carrier parent**

**Segregation mode in the gamete**

**2:2 se**

3

1

3 1

3 1

3

1

**3:1 segregation modes**

**gregation modes**

Once it has been established that a particular probe strategy is informative for the translocation chromosomes, the conditions of the FISH protocols are optimised and the couple can commence their stimulation to undergo their IVF cycle. The biopsied samples are then tested at PGD using the optimised protocol and embryos are diagnosed as balanced or unbalanced. Due to the fact that in the majority of cases of the nuclei of the biopsied cells during cleavage stage biopsy are in the interphase stage, it is not possuble to distinguish between balanced embryos that will be balanced carriers of the structural aberration and normal embryos (Munne, 2005). By considering the FISH signals for each embryo it is then possible to determine the segregation pattern that took place in the gamete of the carreir parent, as shown in table 1, although crossing over events between the centromere and the breakpoint can complicate the situation further and affect the interpretation of the results (Hulten, 2011). Only biopsied samples with two signals for each probe used will be balanced as two signals indicate diploid status for the loci tested. Any other combination of signals is unbalanced for the translocation chromosomes bue to chromosome malsegregation at meiosis.

Figure 4 shows another example of a reciprocal translocation between chromosomes 10 and 11, 46,XY,t(11;19)(q12.3;q13.1), the probe strategy used at PGD (figure 4ii) and images from single blastomeres that were biopsied on day 3 from cleavage stage embryos (figure 4ii).

Fig. 4. Probe strategy for translocation 46,XY,t(11;19)(q12.3;q13.1) and FISH on the lymphocytes of the carrier parent and ii) images from the biopsied single cells from cleavage stage embryos at PGD. Only nucleus D is balanced for the translocation, whereas nucleus A is unbalanced and polyploid, nucleus B shows partial monosomy 10q and nucleus C shows partial trisomy 10.

Meiotic Behaviour of Chromosomes Involved in Structural

intrachromosomal insertion.

24.1 24.2 24.3

**7** 22

22

**Direct Insertion**

22

32 31.1 31.2 31.3

11.2 11.1 12 14 13 15.3 15.2 15.1 21

11.1 11.21 11.22 11.23

21.1 21.2 21.3

23

q24.1

22

21.1 21.2 21.3

11.1 11.2 11.21 11.22 11.23 11.1 12 14 13 15.3 15.2 15.1 21

31.1 31.2 31.3

i)

13 12

21.1 21.2 21.3 22.1 22.2 22.3

12 22

p21

11.21 11.1 11.2 21.1 21.2 21.3 23.1 23.2 23.3

11.22 11.23 11.1

**8**

15 to check for ploidy.

Chromosomal Abnormalities Determined by Preimplantation Genetic Diagnosis 249

can also be used for the detection of other structural abnormalities, provided that the smallest translocated segment is detectable by the resolution of the array platform used. Alternatively when the segments are small FISH can be used. In the case of other structural aberrations, such as inversions, insertions, duplications and deletions, FISH involves the use probes that are included in the segment that is inverted, inserted, duplicated or deleted and one or two other probes on either side (proximally and distally) of that segment in order to be able to detect all different meiotic outcome combinations. Figure 6 below shows examples of strategies used in PGD for an inversion and an

> Derivative chromosome 8

> Normal chromosome 8

> > Normal chromosome

> > > Normal

15

Fig. 6. i) Probe strategy for PGD for an intrachromosomal inversion, 46, XX,

24.1 23

> 13 12

21.1 21.2 21.3 22.1 22.2 22.3

11.22 11.23 11.1

12 11.21 11.1 11.2 21.1

24.2 24.3

Normal

chromosome 15

Chromosome 7 with the direct insertion

22 21.2 21.3 23.1 23.2 23.3 **der 8**

inv(8)(p21;q24.1) and FISH on the lymphocytes of the carrier parent, ii) probe stratege for PGD for an intrachromosomal insertion, 46, XY, dir ins (7)(p22q32q31.1) FISH on the

chromosom ii)

lymphocytes of the carrier parent. An extra probe was used in this protocol for chromosome

**der(14;21)(q10;q10)**

Fig. 5. Probe strategy for PGD for the Robertsonian translocation between chromosomes 14 and 21.

For Robertsonian translocations, the expected segregation patterns can also be worked out as shown in table 2. The resulting gametes are either nullisomic or disomic for either of the chromosomes involved in the translocation, resulting in monosomic or trisomic embryos (Scriven et al., 2001).

Table 2. Different possible segregation patterns at meiosis for the carrier of the Robertsonian translocation between chromosomes 14 and 21.

Since the first clinical PGD cases for carriers of translocations and structural abnormalities FISH was the method of choice for embryo testing in order to select those embryos that were balanced. A disadvantage of FISH however is that it only gives information for the chromosomes for which probes were used. Additional probes can be included in a subsequent round of hybridization for other chromosomes in order to test for the main chromosomes at risk of being aneuploid. Recently the application of aCGH has been reported in PGD for carriers of reciprocal or Robertsonian translocations as well as inversions (Alfarawati et al., 2011.) which allows the complete enumeration of all chromosome sets in a sample.

Results from PGD cycles for structural abnormalities using array CGH, have revealed aneuploidies for chromosomes other than those involved in the translocation, which could account for the poorer PGD outcome for women of advanced maternal age. Array CGH

**21**

Fig. 5. Probe strategy for PGD for the Robertsonian translocation between chromosomes 14

For Robertsonian translocations, the expected segregation patterns can also be worked out as shown in table 2. The resulting gametes are either nullisomic or disomic for either of the chromosomes involved in the translocation, resulting in monosomic or trisomic embryos

Table 2. Different possible segregation patterns at meiosis for the carrier of the Robertsonian

Since the first clinical PGD cases for carriers of translocations and structural abnormalities FISH was the method of choice for embryo testing in order to select those embryos that were balanced. A disadvantage of FISH however is that it only gives information for the chromosomes for which probes were used. Additional probes can be included in a subsequent round of hybridization for other chromosomes in order to test for the main chromosomes at risk of being aneuploid. Recently the application of aCGH has been reported in PGD for carriers of reciprocal or Robertsonian translocations as well as inversions (Alfarawati et al., 2011.) which allows the complete enumeration of all

Results from PGD cycles for structural abnormalities using array CGH, have revealed aneuploidies for chromosomes other than those involved in the translocation, which could account for the poorer PGD outcome for women of advanced maternal age. Array CGH

24.1 24.2 24.3

32.3 32.2 32.1 31

**FISH signals seen in**

**Outcome**

**the embryo**

11.1 11.2

21

22.2 22.1 22.3

**der(14;21)(q10;q10)**

12 13 11.1 11.2 11.1 11.2

q10

22.2 22.1 22.3

14, 21 2xTel14q, 2xLSI21 Normal Der14/21 2xTel14q, 2xLSI21 Balanced 14, der14/21 3xTel14q, 2xLSI21 Trisomy 14 21 1xTel14q, 2xLSI21 Monosomy 14 14 2xTel14q,1xLSI21 Monosomy 21 21, der14/21 3xTel14q, 2xLSI21 Trisomy 21

21

**14**

24.1 24.2 24.3

32.3 32.2 32.1 31

q10

and 21.

(Scriven et al., 2001).

**the carrier parent**

**Segregation of the translocation chromosomes present in the gamete of**

chromosome sets in a sample.

translocation between chromosomes 14 and 21.

can also be used for the detection of other structural abnormalities, provided that the smallest translocated segment is detectable by the resolution of the array platform used. Alternatively when the segments are small FISH can be used. In the case of other structural aberrations, such as inversions, insertions, duplications and deletions, FISH involves the use probes that are included in the segment that is inverted, inserted, duplicated or deleted and one or two other probes on either side (proximally and distally) of that segment in order to be able to detect all different meiotic outcome combinations. Figure 6 below shows examples of strategies used in PGD for an inversion and an intrachromosomal insertion.

Fig. 6. i) Probe strategy for PGD for an intrachromosomal inversion, 46, XX, inv(8)(p21;q24.1) and FISH on the lymphocytes of the carrier parent, ii) probe stratege for PGD for an intrachromosomal insertion, 46, XY, dir ins (7)(p22q32q31.1) FISH on the lymphocytes of the carrier parent. An extra probe was used in this protocol for chromosome 15 to check for ploidy.

Meiotic Behaviour of Chromosomes Involved in Structural

Fig. 7i. Normal euploid aCGH profile of a male embryo

Gain of chromosome 14

Fig. 7ii. aCGH profiles from cleavage stage embryos from 45, XX, der(13;14)(q10;q10)

Loss of chromosome 8

production of chaotic embryos.

Chromosomal Abnormalities Determined by Preimplantation Genetic Diagnosis 251

well as other forms of structural abnormalities such as intrachromosomal insertions (Xanthopoulou et al., 2010). As far as the embryos from translocation carriers are concerned, Iwarsson et al. (2000) reported that the chromosomes involved in the translocation show an even higher degree of mosaicism when compared to control chromosomes. This suggests that the translocation chromosomes not only have a high risk of meiotic malsegregation leading to embryos with unbalanced genomes, but also have a predisposition to segregate unfavourably during the following postzygotic divisions and produce highly mosaic, chaotic embryos (Simopoulou et al., 2003). As a result errors in chromosome segregation during subsequent mitotic divisions can complicate the situation further (Conn et al., 1999). The chaotic nature of the chromosomes in the biopsied samples therefore might therefore produce signals that are not characteristic of a particular segregation pattern. Furthermore Delhanty et al. (1997) observed that there is a patient-related predisposition towards the

In addition to meiotic malsegregation and postzygotic errors resulting in mosaicism, recently testing embryos at PGD using aCGH has revealed a high level of abnormalities affecting other chromosomes apart from those involved in the structural aberration. More specifically Alfarawati et al. (2011) report that 28.9% of the embryos that were balanced for the aberration chromosomes had an aneuploidy for other chromosomes. Follow up analysis on the untransferred embryos can reveal whether there aneuploidies for other chromosomes are meiotic in origin. Figure 7 below shows results from aCGH PGD cases for reciprocal and Robertsonian translocations. In each case the karyotype of the carrier parent is shown together with the abnormalities detected at PGD. Figure 7i shows a normal, euploid profile, whereas the rest of the images show different abnormalities present, which are highlighted.

Gain of 1q

Loss of chromosome 22

#### **5. Evidence obtained from PGD treatment cycles relevant to the meiotic segregation of chromosomes involved in structural abnormalities**

What is unique about studying preimplantation embryos is that all the products from all different modes of segregation from a structural aberration can be seen, which are not viable at later stages of development. Moreover in cases where the structural aberration involves a small segment, unbalanced forms are more likely to be tolerated until later stages of development.

For carriers of reciprocal translocations overall alternate segregation is reported as being the most common segregation pattern (Scriven et al., 2000), followed by adjacent-1 (25%), 3:1 (15%), adjacent-2 (10%) and 4:0 (2%), where most 3:1 segregations came from female carriers (Ogilvie and Scriven, 2002).

However each translocation is unique in terms of the position of the breakpoints and in terms of the length of the translocated segments. As a result different translocations will form different quadrivalents at meiosis I and in each case the configuration of the quadrivalent cross will be different. The quadrivalent configuration partly influences which spindle fibre gets attached to which centromere thus determining the way in which the translocation chromosomes will segregate (Gardner and Sutherland, 2004). The number and the position of chiasmata is also important (Scriven et al., 1998). Each translocation therefore behaves differently at meiosis (Conn et al., 1999) and hence for each translocation the frequency of each mode of segregation will be different and a particular segregation mode might occur at a higher frequency that others (Jalbert et al., 1980). In this way particular translocations will have a prediscposition towards a specific mode of segregation. In order to confirm the meiotic segregation patterns seen on PGD, full follow up analysis is required on the untransferred embryos, which allows us to study the chromosomal ploidy status of those embryos.

For Robertsonian translocations sperm studies have shown that the most common segregation pattern in carriers of Robertsonian translocations is alternate segregation that results in normal or balanced gametes (Ogur et al., 2006), whereas an interchromosomal effect was also seen, as aneuploidy for other chromosomes that were not involved in the translocation were seen. This interchromosomal effect, referring to the translocation chromosomes affecting the recombination and segregation of other chromosomes was also suggested for reciprocal translocations by Estop et al. (2000), but it was not detected in later studies (Oliver-Bonet et al., 2004).

Follow up analysis on untransferred embryos has revealed that cleavage stage embryos show a high level of mosaicism, a situation whereby more than one different cell lines is present in the embryo (Munne et al., 1993, Harper et al., 1995, Delhanty and Handyside, 1995). An extreme form of mosaicism is chaoticism, whereby almost every nucleus present in the embryo will have a different chromosome constitution. Mosaicism has been reported not only in arrested or fragmented embryos but also in embryos of good quality (Delhanty et al., 1997) and has been observed in the embryos of both young and older women (Munne et al., 1995). Different factors are thought to be involved in the formation of mosaic embryos, such as the ovarian stimulation protocol used or the embryo culture conditions, as well as the fact that cell-cycle checkpoints are not fully functional at those early stages of preimplantation embryo development (Delhanty and Handyside, 1995).

A high level of mosaicism has been reported in embryos from reciprocal translocation carriers (Simopoulou et al., 2003), Robertsonian translocation carriers (Conn et al., 1998) as

What is unique about studying preimplantation embryos is that all the products from all different modes of segregation from a structural aberration can be seen, which are not viable at later stages of development. Moreover in cases where the structural aberration involves a small segment, unbalanced forms are more likely to be tolerated until later stages of

For carriers of reciprocal translocations overall alternate segregation is reported as being the most common segregation pattern (Scriven et al., 2000), followed by adjacent-1 (25%), 3:1 (15%), adjacent-2 (10%) and 4:0 (2%), where most 3:1 segregations came from female carriers

However each translocation is unique in terms of the position of the breakpoints and in terms of the length of the translocated segments. As a result different translocations will form different quadrivalents at meiosis I and in each case the configuration of the quadrivalent cross will be different. The quadrivalent configuration partly influences which spindle fibre gets attached to which centromere thus determining the way in which the translocation chromosomes will segregate (Gardner and Sutherland, 2004). The number and the position of chiasmata is also important (Scriven et al., 1998). Each translocation therefore behaves differently at meiosis (Conn et al., 1999) and hence for each translocation the frequency of each mode of segregation will be different and a particular segregation mode might occur at a higher frequency that others (Jalbert et al., 1980). In this way particular translocations will have a prediscposition towards a specific mode of segregation. In order to confirm the meiotic segregation patterns seen on PGD, full follow up analysis is required on the untransferred

embryos, which allows us to study the chromosomal ploidy status of those embryos.

For Robertsonian translocations sperm studies have shown that the most common segregation pattern in carriers of Robertsonian translocations is alternate segregation that results in normal or balanced gametes (Ogur et al., 2006), whereas an interchromosomal effect was also seen, as aneuploidy for other chromosomes that were not involved in the translocation were seen. This interchromosomal effect, referring to the translocation chromosomes affecting the recombination and segregation of other chromosomes was also suggested for reciprocal translocations by Estop et al. (2000), but it was not detected in later

Follow up analysis on untransferred embryos has revealed that cleavage stage embryos show a high level of mosaicism, a situation whereby more than one different cell lines is present in the embryo (Munne et al., 1993, Harper et al., 1995, Delhanty and Handyside, 1995). An extreme form of mosaicism is chaoticism, whereby almost every nucleus present in the embryo will have a different chromosome constitution. Mosaicism has been reported not only in arrested or fragmented embryos but also in embryos of good quality (Delhanty et al., 1997) and has been observed in the embryos of both young and older women (Munne et al., 1995). Different factors are thought to be involved in the formation of mosaic embryos, such as the ovarian stimulation protocol used or the embryo culture conditions, as well as the fact that cell-cycle checkpoints are not fully functional at those early stages of

A high level of mosaicism has been reported in embryos from reciprocal translocation carriers (Simopoulou et al., 2003), Robertsonian translocation carriers (Conn et al., 1998) as

preimplantation embryo development (Delhanty and Handyside, 1995).

**5. Evidence obtained from PGD treatment cycles relevant to the meiotic segregation of chromosomes involved in structural abnormalities**

development.

(Ogilvie and Scriven, 2002).

studies (Oliver-Bonet et al., 2004).

well as other forms of structural abnormalities such as intrachromosomal insertions (Xanthopoulou et al., 2010). As far as the embryos from translocation carriers are concerned, Iwarsson et al. (2000) reported that the chromosomes involved in the translocation show an even higher degree of mosaicism when compared to control chromosomes. This suggests that the translocation chromosomes not only have a high risk of meiotic malsegregation leading to embryos with unbalanced genomes, but also have a predisposition to segregate unfavourably during the following postzygotic divisions and produce highly mosaic, chaotic embryos (Simopoulou et al., 2003). As a result errors in chromosome segregation during subsequent mitotic divisions can complicate the situation further (Conn et al., 1999). The chaotic nature of the chromosomes in the biopsied samples therefore might therefore produce signals that are not characteristic of a particular segregation pattern. Furthermore Delhanty et al. (1997) observed that there is a patient-related predisposition towards the production of chaotic embryos.

In addition to meiotic malsegregation and postzygotic errors resulting in mosaicism, recently testing embryos at PGD using aCGH has revealed a high level of abnormalities affecting other chromosomes apart from those involved in the structural aberration. More specifically Alfarawati et al. (2011) report that 28.9% of the embryos that were balanced for the aberration chromosomes had an aneuploidy for other chromosomes. Follow up analysis on the untransferred embryos can reveal whether there aneuploidies for other chromosomes are meiotic in origin. Figure 7 below shows results from aCGH PGD cases for reciprocal and Robertsonian translocations. In each case the karyotype of the carrier parent is shown together with the abnormalities detected at PGD. Figure 7i shows a normal, euploid profile, whereas the rest of the images show different abnormalities present, which are highlighted.

Fig. 7i. Normal euploid aCGH profile of a male embryo

Fig. 7ii. aCGH profiles from cleavage stage embryos from 45, XX, der(13;14)(q10;q10)

Meiotic Behaviour of Chromosomes Involved in Structural

the mode of segregation (Xanthopoulou et al., 2010).

can segregate at meiosis is shown in figure 8.

**6.2 Maternal age**

Chromosomal Abnormalities Determined by Preimplantation Genetic Diagnosis 253

carriers of Robertsonian translocations were different from those described in male carriers, with females showing a higher level of unbalanced gametes, however this observation was not confirmed by later studies (Munne, 2005). However for one family with an intrachromosomal insertion there seemed to be an effect of the sex of the carrier parent on

Chromosomal insertions are rare forms of structural chromosomal abnormalities, that involve breakpoints on the same chromosome and they can be interchromosomal or intrachromosomal depending on whether the material that is broken off from one chromosome is inserted at another site within the same chromosome or is inserted on another chromosome. Moreover insertions can be direct or inverted depending on whether the orientation of the inserted segment is the same or changes with relation to the centromere. An example of an intrachromosomal insertion and the way that chromosomes

Fig. 8. Chromosome segregation in a carrier of an intrachromosomal insertion at meiosis. Key: N: normal, dupl: duplication of the inserted segment, del: deletion of the inserted

Balanced carriers of direct intrachromosomal insertions are phenotypically normal but are at risk of unbalanced meiotic segregation due to crossing over. At PGD it is possible to study all possible segregation modes, which might not be viable in later stages of development.

Full follow up analysis on 22 untransferred embryos from a female carrier and 19 embryos from a male carrier of the same intrachromosomal insertion, indicated that the female carrier

Advanced maternal age has long been associated with an increase in the rate of aneuploidy, for example trisomy 21, whereas preimplantation embryos from women of advanced maternal age referred for Preimplantation Genetic Screening (PGS) also seem to have an

Ogilvie and Scriven (2002) reported a higher percentage of 3:1 segregation modes in female carriers of reciprocal translocations, resembrling aneuploidy non-disjunction, but no maternal age associations were made. Advanced maternal age is not considered to increase levels of aneuploidy for the chromosomes involved in a structural abnormality in embryos.

produced far more balanced embryos (45% versus 16%, Xanthopoulou et al., 2010).

segment, bal ins: balanced chromosome that carries the insertion

increased level of meiotic errors (Mantzouratou et al., 2007).

The image on the left shows a female embryo with a gain of chromosome 14 as a result of the translocation, but also a loss of chromosome 8. The image on the right shows a female embryo that is balanced for the translocation chromosomes but has a gain of 1q and a loss of chromosome 22.

Fig. 7iii. aCGH profiles from trophectoderm after blastocyst stage biopsy for the reciprocal translocation 46, XX, t(8;17) (q21.1;p11.2).

The image on the top left shows a female embryo with a loss of 8p and a gain of chromosome 17 as a result of the translocation, whereas the image on the top right shows a female embryo that was balanced for the translocation chromosomes but had a loss of 2q. The image in the middle shows a male embryo that was balanced for the translocation chromosomes but had loss of chromosome 15.

As a result, apart from unfavourable meiotic segregation, mosaicism and postzygotic errors contribute to the reproductive challenges faces by couples carrying structural abnormalities. Array CGH therefore might be a more appropriate method for PGD for those patients as it allows screening of all chromosomes and therefore aids in choosing those embryos that are viable.

#### **6. Other factors affecting chromosome segregation at meiosis in carriers of reciprocal translocations**

#### **6.1 Sex of the carrier parent and segregation mode at meiosis**

For couples treated with PGD for reciprocal translocations the overall number of balanced embryos does not seem to be different between male and female carriers (Xanthopoulou et al., 2011). Munne et al. (2000) reported that the meiotic segregation patterns found at female carriers of Robertsonian translocations were different from those described in male carriers, with females showing a higher level of unbalanced gametes, however this observation was not confirmed by later studies (Munne, 2005). However for one family with an intrachromosomal insertion there seemed to be an effect of the sex of the carrier parent on the mode of segregation (Xanthopoulou et al., 2010).

Chromosomal insertions are rare forms of structural chromosomal abnormalities, that involve breakpoints on the same chromosome and they can be interchromosomal or intrachromosomal depending on whether the material that is broken off from one chromosome is inserted at another site within the same chromosome or is inserted on another chromosome. Moreover insertions can be direct or inverted depending on whether the orientation of the inserted segment is the same or changes with relation to the centromere. An example of an intrachromosomal insertion and the way that chromosomes can segregate at meiosis is shown in figure 8.

Fig. 8. Chromosome segregation in a carrier of an intrachromosomal insertion at meiosis. Key: N: normal, dupl: duplication of the inserted segment, del: deletion of the inserted segment, bal ins: balanced chromosome that carries the insertion

Balanced carriers of direct intrachromosomal insertions are phenotypically normal but are at risk of unbalanced meiotic segregation due to crossing over. At PGD it is possible to study all possible segregation modes, which might not be viable in later stages of development.

Full follow up analysis on 22 untransferred embryos from a female carrier and 19 embryos from a male carrier of the same intrachromosomal insertion, indicated that the female carrier produced far more balanced embryos (45% versus 16%, Xanthopoulou et al., 2010).

#### **6.2 Maternal age**

252 Meiosis - Molecular Mechanisms and Cytogenetic Diversity

The image on the left shows a female embryo with a gain of chromosome 14 as a result of the translocation, but also a loss of chromosome 8. The image on the right shows a female embryo that is balanced for the translocation chromosomes but has a gain of 1q and a loss of

Loss of chromosome 15

Fig. 7iii. aCGH profiles from trophectoderm after blastocyst stage biopsy for the reciprocal

The image on the top left shows a female embryo with a loss of 8p and a gain of chromosome 17 as a result of the translocation, whereas the image on the top right shows a female embryo that was balanced for the translocation chromosomes but had a loss of 2q. The image in the middle shows a male embryo that was balanced for the translocation

As a result, apart from unfavourable meiotic segregation, mosaicism and postzygotic errors contribute to the reproductive challenges faces by couples carrying structural abnormalities. Array CGH therefore might be a more appropriate method for PGD for those patients as it allows screening of all chromosomes and therefore aids in choosing those embryos that are

**6. Other factors affecting chromosome segregation at meiosis in carriers of**

For couples treated with PGD for reciprocal translocations the overall number of balanced embryos does not seem to be different between male and female carriers (Xanthopoulou et al., 2011). Munne et al. (2000) reported that the meiotic segregation patterns found at female

**6.1 Sex of the carrier parent and segregation mode at meiosis**

Loss of chromosome 2q

Gain of chromosome 17

chromosome 22.

Loss of 8p

translocation 46, XX, t(8;17) (q21.1;p11.2).

chromosomes but had loss of chromosome 15.

**reciprocal translocations**

viable.

Advanced maternal age has long been associated with an increase in the rate of aneuploidy, for example trisomy 21, whereas preimplantation embryos from women of advanced maternal age referred for Preimplantation Genetic Screening (PGS) also seem to have an increased level of meiotic errors (Mantzouratou et al., 2007).

Ogilvie and Scriven (2002) reported a higher percentage of 3:1 segregation modes in female carriers of reciprocal translocations, resembrling aneuploidy non-disjunction, but no maternal age associations were made. Advanced maternal age is not considered to increase levels of aneuploidy for the chromosomes involved in a structural abnormality in embryos.

Meiotic Behaviour of Chromosomes Involved in Structural

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#### **7. Conclusion**

PGD provides a unique opportunity to study the behaviour of chromosomes involved in structural abnormalities during meiosis. At those early stages of human preimplantation development all the different modes of meiotic segregation which might not be viable at later stages of development can be seen. As described above carriers of structural chromosomal abnormalities face a high risk of malsegregation at meiosis.

#### **8. References**

However women of advanced maternal age referred for PGD for reciprocal translocations have reduced chances of achieving a pregnancy (Xanthopoulou et al., 2010) due to the high level of meiotic aneuploidy for chromosomes other than the ones involved in the aberration

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**7. Conclusion**

**8. References**

**14**

*1,2Italy, 3USA*

**OMICS for the Identification of Biomarkers for**

**Oocyte Competence, with Special Reference**

Maria Elena Dell'Aquila1, Yoon Sung Cho2, Nicola Antonio Martino1,

A crucial component of Assisted Reproductive Technologies (ART) is the assessment of oocyte developmental potential, to allow selection of those oocytes most likely to result in fertilization and pregnancy. Currently, oocyte quality assessment is largely based on the morphological appearance of the cumulus-oocyte complex, however the accuracy of morphological methods, as predictive of oocyte competence, is still suboptimal. Therefore, the development of objective, accurate, fast and reliable tests for assessing oocyte developmental potential remains an important aim of human and veterinary reproductive medicine. The process of oocyte meiotic maturation, which is central to the developmental competence of the oocyte, is regulated by numerous genes (Matzuk & Lamb, 2008; Fauser et al., 2011) and protein pathways (Kubiak, 2011) and is accompanied by significant changes within the oocyte at many levels. Better understanding of oocyte meiotic maturation would allow better support of this process to increase the success of reproductive biotechnologies, and thus overcome some forms of infertility. Recently, global assessment strategies, namely OMICS, investigating genomic, transcriptomic, proteomic, lipidomic, and glycomic profiles of oocytes, cumulus or granulosa cells have become increasingly applied to the study of oocyte physiology and pathology. Also being investigated is the oocyte-cumulus metabolome, via measurements of metabolites in biological fluids, such as follicular or tubal fluid, or in culture media. The establishment of these technologies, which are in their initial stages of application to reproductive biology, can require large sample numbers; only animal models can meet this requirement. Because of their wide availability and the body of existing knowledge regarding their biology, oocytes of large animals provide useful models for investigating the relationship between oocyte developmental competence and OMICS biomarkers. This review summarizes recent literature on the application of OMICS strategies to evaluating developmental competence of human oocytes and oocytes of large

**1. Introduction**

Manuel Filioli Uranio1, Lucia Rutigliano1 and Katrin Hinrichs3

*2Assisted Procreation Unit - Ospedale Accreditato Clinica Santa Maria, Bari, 3Dept. Physiology and Pharmacology, Texas A&M University, College Station,*

*1Dept. Animal Production, University of Bari "Aldo Moro", Bari,*

**to the Mare as a Prospective Model for**

**Human Reproductive Medicine**

### **OMICS for the Identification of Biomarkers for Oocyte Competence, with Special Reference to the Mare as a Prospective Model for Human Reproductive Medicine**

Maria Elena Dell'Aquila1, Yoon Sung Cho2, Nicola Antonio Martino1, Manuel Filioli Uranio1, Lucia Rutigliano1 and Katrin Hinrichs3 *1Dept. Animal Production, University of Bari "Aldo Moro", Bari, 2Assisted Procreation Unit - Ospedale Accreditato Clinica Santa Maria, Bari, 3Dept. Physiology and Pharmacology, Texas A&M University, College Station, 1,2Italy, 3USA*

#### **1. Introduction**

256 Meiosis - Molecular Mechanisms and Cytogenetic Diversity

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2010, Male and female meiotic behaviour of an intrachromosomal insertion

2011, When is old too old for preimplantation genetic diagnosis for reciprocal translocations? *Prenat Diagn.* Jun 28. doi: 10.1002/pd.2813. [Epub ahead of print]

A crucial component of Assisted Reproductive Technologies (ART) is the assessment of oocyte developmental potential, to allow selection of those oocytes most likely to result in fertilization and pregnancy. Currently, oocyte quality assessment is largely based on the morphological appearance of the cumulus-oocyte complex, however the accuracy of morphological methods, as predictive of oocyte competence, is still suboptimal. Therefore, the development of objective, accurate, fast and reliable tests for assessing oocyte developmental potential remains an important aim of human and veterinary reproductive medicine. The process of oocyte meiotic maturation, which is central to the developmental competence of the oocyte, is regulated by numerous genes (Matzuk & Lamb, 2008; Fauser et al., 2011) and protein pathways (Kubiak, 2011) and is accompanied by significant changes within the oocyte at many levels. Better understanding of oocyte meiotic maturation would allow better support of this process to increase the success of reproductive biotechnologies, and thus overcome some forms of infertility. Recently, global assessment strategies, namely OMICS, investigating genomic, transcriptomic, proteomic, lipidomic, and glycomic profiles of oocytes, cumulus or granulosa cells have become increasingly applied to the study of oocyte physiology and pathology. Also being investigated is the oocyte-cumulus metabolome, via measurements of metabolites in biological fluids, such as follicular or tubal fluid, or in culture media. The establishment of these technologies, which are in their initial stages of application to reproductive biology, can require large sample numbers; only animal models can meet this requirement. Because of their wide availability and the body of existing knowledge regarding their biology, oocytes of large animals provide useful models for investigating the relationship between oocyte developmental competence and OMICS biomarkers. This review summarizes recent literature on the application of OMICS strategies to evaluating developmental competence of human oocytes and oocytes of large

OMICS for the Identification of Biomarkers for Oocyte Competence,

**2.1 The mare as a model for human oocyte biology**

interest in the equine species.

with Special Reference to the Mare as a Prospective Model for Human Reproductive Medicine 259

separate: one set of sister chromatids is discarded as the polar body; the other set, in response to a recrudescence of MPF, lines up on a spindle, forming the second metaphase plate (MII). Fertilization causes inactivation of MPF and ERK 2, and the second meiotic division occurs, separating the sister chromatids. One set of chromatids is discarded, as the second polar body; the other set becomes the female pronucleus. For a comprehensive description of major pathways involved in oocyte M-phase entry, see Kubiak et al., 2011 and Tosti & Boni, 2011. Detailed descriptions of OMICS techniques go beyond the intent of this article; corresponding references are provided in the text. We instead focus on the most significant results obtained using these techniques, the role of large animal models in experimental designs that cannot be performed in humans, and on actual and potential contributions of different animal models to understanding of oocyte biology, with particular

Large animal models allow the establishment of a wide variety of experimental designs that can not be applied in humans for obvious ethical reasons, or due to the limited and highly regulated availability of human biological samples. Among large animals, the mare has many attributes that make her a good model for reproduction in women (Carnevale, 2008). These include a long follicular phase, a long interovulatory interval (22 days), presence of a single dominant follicle, formation a large diameter follicle (~40 mm) with a large volume of follicular fluid -- the same volume:body weight ratio as in women; a relatively long time from LH stimulation to ovulation (36 h for both human and horse) and, like the human oocyte, formation of a markedly dense chromatin mass within the germinal vesicle as the oocyte gains meiotic competence or undergoes atresia (Parfenov et al, 1989; Hinrichs et al., 1993). Although seasonality does not occur in women, the equine characteristic of seasonal reproductive activity provides the potential to examine the influence of applied factors when cyclic hormonal patterns are not occurring (Carnevale, 2008). Horses are the best animal model for studies on age-related infertility. Because mares can be of great value, many mares continue to be bred until they experience subfertility, thus animals with naturally-occuring age-related subfertility are available for study. Horses have a long lifespan, thus age-related subfertility occurs at an age (~20-25 years) much closer to that observed in women than is seen in other animal models. In addition, horses, unlike other large domestic species, are selected for attributes other than fertility, such as conformation, athletic prowess, or behavior. Individuals showing subfertility may be worked with intensively to try to obtain foals, thus, they provide an excellent naturally-occurring model for many intrinsic causes of subfertility. Horses have similar metabolic responses to nutrient intake to that in humans, and are used for a wide variety of athletic purposes, thus they can serve to model important physiological or pathological situations affecting reproduction in humans (such as stress, life-style, sports activity, obesity or metabolic syndrome) as well as to examine the effects of external factors such as acute or long-term exposure to drugs or environmental toxicants. In addition to mimicking the situation in humans, the development of particularly large follicles allows the possibility of collecting large amounts of mural granulosa cells (GC) issuing from the follicular wall as well as large amounts of follicular fluid (30 to 50 ml/follicle) that may be used for OMICS studies in a 1:1 comparison with the developmental status of the enclosed oocyte. The cumulus-oocyte complex (COC)

animals. Among the available animal models, the mare is uniquely applicable to investigation of oocyte developmental competence. Horses represent the most economically valuable domestic animal, with progeny from specific mares worth hundreds of thousands of euros. Thus, there is obvious practical interest in the use of assisted reproduction in this species. In addition, similarities between equine and human follicle growth and oocyte maturation make the mare a particularly valuable model for topics at the interface between animal breeding and biomedical research, such as age-related and obesity-related oocyte dysfunction and the effects of exposure to environmental toxicants, as well as for fundamental research on factors involved in meiotic maturation. For these reasons, particular attention will be dedicated in this review to recent OMICS results obtained in the equine species and to discussion of the potential application of this animal model in future investigations.

#### **2. OMICS as innovative strategies for evaluating oocyte quality**

Systems biology is a new and rapidly developing research area in which, by quantitatively describing the interactions among the components of a cell, a systems-level understanding of a biological response can be achieved. Therefore, it requires high-throughput measurement technologies, that is, technologies that can investigate a large number of biological molecules at once. OMICS technologies -- in which aspects of cellular structure or function, such as proteins or RNA transcripts, are studied in their totality (global assessment strategies) -- are opening wider and wider doors into the understanding of all branches of the biology, physiology, and pathology of living organisms. It is likely that information obtained using OMICS will change our concept of "normal" and "pathological," and will enable the efficient evaluation of the effects of extrinsic factors on the status of living systems. Initial studies on the application of OMICS strategies to the oocyte have appeared in the past decade, starting with genomics and transcriptomics, and progressing to the newer fields of glycomics and metabolomics. As noted above, a major concern in the production of viable and competent embryos in vitro is the evaluation of initial oocyte quality and the support of optimal nuclear and cytoplasmic maturation. OMICS approaches to the oocyte will significantly contribute not only to accurate assessment of oocyte quality, but also to the clarification of the mechanisms involved in cell cycle regulation and cell differentiation, thus contributing to the effective utilization of recovered oocytes. Because meiotic maturation and early embryo development involve regulation of the cell cycle and evolution from differentiated, to pluripotent, back to differentiated cells, data generated from study of these processes may also relate to the establishment of innovative targeted cancer treatments and stem cell-based therapies. To introduce the sequential phases of the meiotic process, changes occurring within the oocyte and some of their fundamental regulating factors are briefly described. Upon the luteinizing hormone (LH) surge, M-phase promoting factor (MPF) and the mitogen-activated protein (MAP) kinase ERK 2 (extracellularly-regulated kinase 2) are activated within the oocyte. The oocyte, which at this time is in prophase of meiosis, has replicated chomosomes contained within a nucleus (termed the germinal vesicle, GV). Activation of MPF and ERK 2 trigger nuclear envelope breakdown and chromatin condensation. The condensed chromosomes are subsequently aligned on the spindle of the first meiotic division, forming the metaphase I plate. At this time MPF levels decrease, while ERK 2 levels remain high. The homologous chromosomes separate: one set of sister chromatids is discarded as the polar body; the other set, in response to a recrudescence of MPF, lines up on a spindle, forming the second metaphase plate (MII). Fertilization causes inactivation of MPF and ERK 2, and the second meiotic division occurs, separating the sister chromatids. One set of chromatids is discarded, as the second polar body; the other set becomes the female pronucleus. For a comprehensive description of major pathways involved in oocyte M-phase entry, see Kubiak et al., 2011 and Tosti & Boni, 2011. Detailed descriptions of OMICS techniques go beyond the intent of this article; corresponding references are provided in the text. We instead focus on the most significant results obtained using these techniques, the role of large animal models in experimental designs that cannot be performed in humans, and on actual and potential contributions of different animal models to understanding of oocyte biology, with particular interest in the equine species.

#### **2.1 The mare as a model for human oocyte biology**

258 Meiosis - Molecular Mechanisms and Cytogenetic Diversity

animals. Among the available animal models, the mare is uniquely applicable to investigation of oocyte developmental competence. Horses represent the most economically valuable domestic animal, with progeny from specific mares worth hundreds of thousands of euros. Thus, there is obvious practical interest in the use of assisted reproduction in this species. In addition, similarities between equine and human follicle growth and oocyte maturation make the mare a particularly valuable model for topics at the interface between animal breeding and biomedical research, such as age-related and obesity-related oocyte dysfunction and the effects of exposure to environmental toxicants, as well as for fundamental research on factors involved in meiotic maturation. For these reasons, particular attention will be dedicated in this review to recent OMICS results obtained in the equine species and to discussion of the potential application of this animal model in future

Systems biology is a new and rapidly developing research area in which, by quantitatively describing the interactions among the components of a cell, a systems-level understanding of a biological response can be achieved. Therefore, it requires high-throughput measurement technologies, that is, technologies that can investigate a large number of biological molecules at once. OMICS technologies -- in which aspects of cellular structure or function, such as proteins or RNA transcripts, are studied in their totality (global assessment strategies) -- are opening wider and wider doors into the understanding of all branches of the biology, physiology, and pathology of living organisms. It is likely that information obtained using OMICS will change our concept of "normal" and "pathological," and will enable the efficient evaluation of the effects of extrinsic factors on the status of living systems. Initial studies on the application of OMICS strategies to the oocyte have appeared in the past decade, starting with genomics and transcriptomics, and progressing to the newer fields of glycomics and metabolomics. As noted above, a major concern in the production of viable and competent embryos in vitro is the evaluation of initial oocyte quality and the support of optimal nuclear and cytoplasmic maturation. OMICS approaches to the oocyte will significantly contribute not only to accurate assessment of oocyte quality, but also to the clarification of the mechanisms involved in cell cycle regulation and cell differentiation, thus contributing to the effective utilization of recovered oocytes. Because meiotic maturation and early embryo development involve regulation of the cell cycle and evolution from differentiated, to pluripotent, back to differentiated cells, data generated from study of these processes may also relate to the establishment of innovative targeted cancer treatments and stem cell-based therapies. To introduce the sequential phases of the meiotic process, changes occurring within the oocyte and some of their fundamental regulating factors are briefly described. Upon the luteinizing hormone (LH) surge, M-phase promoting factor (MPF) and the mitogen-activated protein (MAP) kinase ERK 2 (extracellularly-regulated kinase 2) are activated within the oocyte. The oocyte, which at this time is in prophase of meiosis, has replicated chomosomes contained within a nucleus (termed the germinal vesicle, GV). Activation of MPF and ERK 2 trigger nuclear envelope breakdown and chromatin condensation. The condensed chromosomes are subsequently aligned on the spindle of the first meiotic division, forming the metaphase I plate. At this time MPF levels decrease, while ERK 2 levels remain high. The homologous chromosomes

**2. OMICS as innovative strategies for evaluating oocyte quality**

investigations.

Large animal models allow the establishment of a wide variety of experimental designs that can not be applied in humans for obvious ethical reasons, or due to the limited and highly regulated availability of human biological samples. Among large animals, the mare has many attributes that make her a good model for reproduction in women (Carnevale, 2008). These include a long follicular phase, a long interovulatory interval (22 days), presence of a single dominant follicle, formation a large diameter follicle (~40 mm) with a large volume of follicular fluid -- the same volume:body weight ratio as in women; a relatively long time from LH stimulation to ovulation (36 h for both human and horse) and, like the human oocyte, formation of a markedly dense chromatin mass within the germinal vesicle as the oocyte gains meiotic competence or undergoes atresia (Parfenov et al, 1989; Hinrichs et al., 1993). Although seasonality does not occur in women, the equine characteristic of seasonal reproductive activity provides the potential to examine the influence of applied factors when cyclic hormonal patterns are not occurring (Carnevale, 2008). Horses are the best animal model for studies on age-related infertility. Because mares can be of great value, many mares continue to be bred until they experience subfertility, thus animals with naturally-occuring age-related subfertility are available for study. Horses have a long lifespan, thus age-related subfertility occurs at an age (~20-25 years) much closer to that observed in women than is seen in other animal models. In addition, horses, unlike other large domestic species, are selected for attributes other than fertility, such as conformation, athletic prowess, or behavior. Individuals showing subfertility may be worked with intensively to try to obtain foals, thus, they provide an excellent naturally-occurring model for many intrinsic causes of subfertility. Horses have similar metabolic responses to nutrient intake to that in humans, and are used for a wide variety of athletic purposes, thus they can serve to model important physiological or pathological situations affecting reproduction in humans (such as stress, life-style, sports activity, obesity or metabolic syndrome) as well as to examine the effects of external factors such as acute or long-term exposure to drugs or environmental toxicants. In addition to mimicking the situation in humans, the development of particularly large follicles allows the possibility of collecting large amounts of mural granulosa cells (GC) issuing from the follicular wall as well as large amounts of follicular fluid (30 to 50 ml/follicle) that may be used for OMICS studies in a 1:1 comparison with the developmental status of the enclosed oocyte. The cumulus-oocyte complex (COC)

OMICS for the Identification of Biomarkers for Oocyte Competence,

reviews by Seli et al., 2010; Geraedts et al., 2010).

(Antonello et al., 2011).

analysis of 370.000 human SNPs.

with Special Reference to the Mare as a Prospective Model for Human Reproductive Medicine 261

chromosome complement of the two PBs by Comparative Genomic Hybridization (CGH;

Conventional CGH, initially developed by cancer biologists, was applied to human early embryos around 10 years ago (Voullaire et al., 2000 reviewed by Geraedts et al., 2010). These authors performed CGH in association with whole genome amplification by degenerate oligonucleotide-primed polymerase chain reaction. CGH is a competitive hybridization of two fragmented genomes (test and reference genomes) to the chromosomes of a metaphase plate of a normal subject. The tested and subject genomes are labeled with different (red and green) fluorescent dyes so that an increase of red staining will indicate the presence of duplicated regions, an increase of green staining will indicate the presence of deleted regions, whereas the lack of predominance of one of the two colors will indicate normal chromosome structure. This technique allows examination of the whole chromosomal complement, but requires extensive time to get the results. In recent years, CGH-microarray tools have been developed in which the labeled DNAs are affixed to DNA on a microscope slide rather than to metaphase chromosomes. A variety of microarray-CGH platforms are available. As an example, the Cambridge-based company BlueGnome offers an array-based CGH protocol which allows analysis of biopsied PBs within 11 hrs (SurePlex amplification protocol; 24sure analysis, BlueGnome; "http://www.bluegnome.co.uk/"; Geraedts et al., 2010). As regard, Geraedts et al., (2011) and Magli et al., (2011) reported clinical results and technical aspects of a proof-of-principle study performed in associated ART centers in which all mature metaphase II oocytes from patients who consented to the study, fertilized by ICSI, were analyzed. The first and second PBs were biopsied and analysed separately for chromosome copy number by array CGH. If either or both of the PBs were found to be aneuploid, the corresponding zygote was then also processed by array CGH for concordance analysis. It was concluded that the ploidy of a zygote can be predicted with acceptable accuracy by array CGH analysis on both PBs. Interestingly, on the male side, the application of CGH arrays to single human sperm cells has been recently reported

In the aim to move from chromosomal structure to single mutation analysis, SNP (Single Nucleotide Polymorphism) arrays have been developed. A single-nucleotide polymorphism (SNP, pronounced snip) is a DNA sequence variation occurring when a single nucleotide — Adenine (A), Thymine (T), Cytosine (C), or Guanine (G) — differs in a sequence between members of a species or paired chromosomes in an individual. Many common SNPs have only two alleles. Within a population, SNPs can be assigned a minor allele frequency — the lesser of the two allele frequencies for a population. There are variations among human populations, so a SNP allele that is common in one geographical or ethnic group may be rare in another. Unlike the CGH-microarray platforms which involve simultaneous hybridization of differentially labeled DNAs to the same microarray, SNP-microarrays assess test and reference samples, separately, in parallel. From 10.000 to 500.000 SNPs may be evaluated simultaneously. For example, using the Affymetrix platform, analysis of 250.000 SNPs in first PB biopsies (Treff et al., 2010a) and in Day-3 embryos (Treff et al., 2010b) have been reported (review by Seli et al., 2010). The Illumina platform allows the

Because of their utility in recognizing variations associated with disease, recent genetic epidemiology studies have been dominated by genome-wide association studies using

of the mare is particularly large, thus allowing a 1:1 evaluation of biological parameters of cumulus cells (CCs) predictive of oocyte meiotic and developmental competence. Moreover, follicular COCs in the mare can be recovered from immature follicles, with initial COC morphological features indicating viability (compact cumulus) or atresia (apoptosis, expanded cumulus) of their surrounding follicle, thus supporting study of the effects of follicle immaturity and early or late atresia on oocyte competence (Hinrichs and Williams, 1997; Dell'Aquila et al., 2003). The equine oocyte is approximately 200 microns in diameter, with good visibility of the perivitelline space and the first polar body (PB), so that mature oocytes are easily identified on morphological examination. The horse oocyte possesses a unique distribution of cytoplasmic lipid droplets, which assume polar aggregation in metaphase II (MII) oocytes, and whose biological meaning is under investigation (Ambruosi et al., 2009). Horses make a valuable model for oocyte assessments associated with penetration of the zona pellucida (e.g. PB biopsy) because, in contrast to species such as cattle and sheep, methods for fertilization via intracytoplasmic sperm injection (ICSI) are well established in the horse (Hinrichs et al., 2005; Choi et al., 2006). Sperm injection is necessary to achieve fertilization after penetration of the zona pellucida for investigative purposes, as the defect in the zona would lead to polyspermy if standard in vitro fertilization (IVF) were to be performed. These features make the equine oocyte a particularly useful model for the establishment of OMICS strategies that could be not only applied to better understanding of human assisted reproductive medicine, but also directly applicable to the horse industry.

#### **2.2 Oocyte genomics – The polar body biopsy and genomic analysis for predicting half of the DNA constitution of an embryo: from FISH to CGH/CNV/SNP-based arrays**

The genomic DNA constitution of the oocyte determines the sequence of produced transcripts and proteins, and constitutes half of the early embryo phenotype. The most widely used diagnostic tool for oocyte and embryo genomic investigation to date has been Fluorescent In Situ Hybridization (FISH), a cytogenetic technique which identifies specific DNA sequences on chromosomes by means of fluorescent probes that bind to those parts of the chromosome with which they show a high degree of sequence similarity.

Since the first report in humans (Griffin et al., 1992), several studies have been published reporting the evaluation of human day 3 embryos (4-8 cell stage) for up to 8 pairs of chromosomes (chromosomes 13, 15, 16, 17, 18, 21, 22, and X/Y; review by Seli et al., 2010). However, in recent meta-analysis studies, reported from 2008 to 2010, it has become clear that preimplantation genetic screening by using FISH is not justified. This is because it causes damage to the embryo, it requires embryo cryopreservation and transfer in a subsequent cycle, and it does not significantly contribute to the identification and exclusion of aneuploid embryos. Use of FISH has been reported to be associated with lower implantation rates and it shows errors such as false positives due to mosaicism or false negatives due to the limited number of chromosomes analyzed and the limited targeted regions. Therefore, authoritative scientific committees, including the European Society of Human Reproduction and Embryology (ESHRE) and the American Society of Reproductive Medicine (ASRM), decided to conduct a study to determine whether biopsy of the first and second polar bodies (PBs) of the oocyte would enable the timely identification of the chromosomal status of an oocyte. This aim could be reached by analyzing the complete

of the mare is particularly large, thus allowing a 1:1 evaluation of biological parameters of cumulus cells (CCs) predictive of oocyte meiotic and developmental competence. Moreover, follicular COCs in the mare can be recovered from immature follicles, with initial COC morphological features indicating viability (compact cumulus) or atresia (apoptosis, expanded cumulus) of their surrounding follicle, thus supporting study of the effects of follicle immaturity and early or late atresia on oocyte competence (Hinrichs and Williams, 1997; Dell'Aquila et al., 2003). The equine oocyte is approximately 200 microns in diameter, with good visibility of the perivitelline space and the first polar body (PB), so that mature oocytes are easily identified on morphological examination. The horse oocyte possesses a unique distribution of cytoplasmic lipid droplets, which assume polar aggregation in metaphase II (MII) oocytes, and whose biological meaning is under investigation (Ambruosi et al., 2009). Horses make a valuable model for oocyte assessments associated with penetration of the zona pellucida (e.g. PB biopsy) because, in contrast to species such as cattle and sheep, methods for fertilization via intracytoplasmic sperm injection (ICSI) are well established in the horse (Hinrichs et al., 2005; Choi et al., 2006). Sperm injection is necessary to achieve fertilization after penetration of the zona pellucida for investigative purposes, as the defect in the zona would lead to polyspermy if standard in vitro fertilization (IVF) were to be performed. These features make the equine oocyte a particularly useful model for the establishment of OMICS strategies that could be not only applied to better understanding of human assisted reproductive medicine, but also directly

**2.2 Oocyte genomics – The polar body biopsy and genomic analysis for predicting half of the DNA constitution of an embryo: from FISH to CGH/CNV/SNP-based arrays**  The genomic DNA constitution of the oocyte determines the sequence of produced transcripts and proteins, and constitutes half of the early embryo phenotype. The most widely used diagnostic tool for oocyte and embryo genomic investigation to date has been Fluorescent In Situ Hybridization (FISH), a cytogenetic technique which identifies specific DNA sequences on chromosomes by means of fluorescent probes that bind to those parts of

Since the first report in humans (Griffin et al., 1992), several studies have been published reporting the evaluation of human day 3 embryos (4-8 cell stage) for up to 8 pairs of chromosomes (chromosomes 13, 15, 16, 17, 18, 21, 22, and X/Y; review by Seli et al., 2010). However, in recent meta-analysis studies, reported from 2008 to 2010, it has become clear that preimplantation genetic screening by using FISH is not justified. This is because it causes damage to the embryo, it requires embryo cryopreservation and transfer in a subsequent cycle, and it does not significantly contribute to the identification and exclusion of aneuploid embryos. Use of FISH has been reported to be associated with lower implantation rates and it shows errors such as false positives due to mosaicism or false negatives due to the limited number of chromosomes analyzed and the limited targeted regions. Therefore, authoritative scientific committees, including the European Society of Human Reproduction and Embryology (ESHRE) and the American Society of Reproductive Medicine (ASRM), decided to conduct a study to determine whether biopsy of the first and second polar bodies (PBs) of the oocyte would enable the timely identification of the chromosomal status of an oocyte. This aim could be reached by analyzing the complete

the chromosome with which they show a high degree of sequence similarity.

applicable to the horse industry.

chromosome complement of the two PBs by Comparative Genomic Hybridization (CGH; reviews by Seli et al., 2010; Geraedts et al., 2010).

Conventional CGH, initially developed by cancer biologists, was applied to human early embryos around 10 years ago (Voullaire et al., 2000 reviewed by Geraedts et al., 2010). These authors performed CGH in association with whole genome amplification by degenerate oligonucleotide-primed polymerase chain reaction. CGH is a competitive hybridization of two fragmented genomes (test and reference genomes) to the chromosomes of a metaphase plate of a normal subject. The tested and subject genomes are labeled with different (red and green) fluorescent dyes so that an increase of red staining will indicate the presence of duplicated regions, an increase of green staining will indicate the presence of deleted regions, whereas the lack of predominance of one of the two colors will indicate normal chromosome structure. This technique allows examination of the whole chromosomal complement, but requires extensive time to get the results. In recent years, CGH-microarray tools have been developed in which the labeled DNAs are affixed to DNA on a microscope slide rather than to metaphase chromosomes. A variety of microarray-CGH platforms are available. As an example, the Cambridge-based company BlueGnome offers an array-based CGH protocol which allows analysis of biopsied PBs within 11 hrs (SurePlex amplification protocol; 24sure analysis, BlueGnome; "http://www.bluegnome.co.uk/"; Geraedts et al., 2010). As regard, Geraedts et al., (2011) and Magli et al., (2011) reported clinical results and technical aspects of a proof-of-principle study performed in associated ART centers in which all mature metaphase II oocytes from patients who consented to the study, fertilized by ICSI, were analyzed. The first and second PBs were biopsied and analysed separately for chromosome copy number by array CGH. If either or both of the PBs were found to be aneuploid, the corresponding zygote was then also processed by array CGH for concordance analysis. It was concluded that the ploidy of a zygote can be predicted with acceptable accuracy by array CGH analysis on both PBs. Interestingly, on the male side, the application of CGH arrays to single human sperm cells has been recently reported (Antonello et al., 2011).

In the aim to move from chromosomal structure to single mutation analysis, SNP (Single Nucleotide Polymorphism) arrays have been developed. A single-nucleotide polymorphism (SNP, pronounced snip) is a DNA sequence variation occurring when a single nucleotide — Adenine (A), Thymine (T), Cytosine (C), or Guanine (G) — differs in a sequence between members of a species or paired chromosomes in an individual. Many common SNPs have only two alleles. Within a population, SNPs can be assigned a minor allele frequency — the lesser of the two allele frequencies for a population. There are variations among human populations, so a SNP allele that is common in one geographical or ethnic group may be rare in another. Unlike the CGH-microarray platforms which involve simultaneous hybridization of differentially labeled DNAs to the same microarray, SNP-microarrays assess test and reference samples, separately, in parallel. From 10.000 to 500.000 SNPs may be evaluated simultaneously. For example, using the Affymetrix platform, analysis of 250.000 SNPs in first PB biopsies (Treff et al., 2010a) and in Day-3 embryos (Treff et al., 2010b) have been reported (review by Seli et al., 2010). The Illumina platform allows the analysis of 370.000 human SNPs.

Because of their utility in recognizing variations associated with disease, recent genetic epidemiology studies have been dominated by genome-wide association studies using

OMICS for the Identification of Biomarkers for Oocyte Competence,

oocyte and embryo competence.

tissues.

with Special Reference to the Mare as a Prospective Model for Human Reproductive Medicine 263

Another promising genomic investigation area is Epigenetics, the study of changes in gene expression and thus cellular phenotype caused by mechanisms other than changes in the underlying DNA sequence – hence the name *epi-* (Greek: *επί*- over, above, outer) *genetics*. Examples of such changes are DNA methylation and deacetylation of the histones, the proteins around which DNA are wrapped. Both of these changes serve to suppress gene expression without altering the sequence of the silenced genes. These changes may remain through cell divisions for the remainder of the cell's life, and some epigenetic changes in germ cells may potentially last for multiple generations. Epigenetic changes in eukaryotic biology are the basis of the process of cell differentiation. During embryonic morphogenesis, the totipotent cells of the zygote become the various pluripotent cell lines of the embryo, which in turn become fully differentiated cells. This is accomplished by activating some genes while inhibiting others. Current epigenetic research focuses on chromatin modifications occurring during sequential phases of fertilization (sperm chromatin decondensation, pronuclear formation with DNA duplication and syngamy) and early development (chromosome condensation and assembly in the first metaphase plate of the first mitotic division and the subsequent series of mitotic divisions to the blastocyst stage; Burton & Torres Padilla, 2011). These events may be studied by comparing embryos produced in vivo with those obtained using different technologies, such as IVF, ICSI, parthenogenesis or somatic cell nuclear transfer (Cremer & Zakhartchenko, 2011). Soon, the emergence of quantitative high-throughput microarray technology should allow the development of epigenomic arrays for the evaluation of embryo whole-genome epigenetic status, thus opening the new field of epigenomics (Callinan & Feinberg, 2006) to the study of

The methylation pattern of DNA in oocytes may be a key factor for the improvement of efficiency of in vitro embryo production, because it is related to oocyte competence. A recent study (Simarro Fagundes et al., 2011) reported on a differentially-methylated region located in exon 10 of the imprinted gene *IGF2* This study evaluated immature vs *in vitro*-matured bovine oocytes from small (1–3 mm in diameter) and large follicles (≥ 8.1 mm in diameter). It was observed that after IVM, oocytes from ≥ 8.1 mm follicles were less methylated (18.51%) than were those from 1- to 3-mm follicles (49.62%). As oocytes from the larger follicles are more developmentally competent, the less methylated pattern appears to be associated with higher oocyte quality. It was concluded that the methylation pattern of specific genes could be used as a molecular marker for epigenetic reprogramming status in oocytes, helping the development of new in vitro embryo production protocols. A broader study on this wave (Smallwood et al., 2011) reported the first integrated epigenomic analysis of mammalian oocytes (GV vs MII oocytes) and preimplantation embryos (blastocyst stage) identifying over a thousand CG islands methylated in matured oocytes. The authors observed that CG islands were preferentially located within active transcription units, supporting a general transcription-dependent mechanism of methylation, and that very few CG islands were protected from post-fertilization reprogramming, the majority showing incomplete demethylation in Day-3 blastocysts. This study revealed the extent and dynamics of CG island methylation in oocytes, which is a prerequisite for defining the full repertoire of imprinted genes and the mechanistic basis of parent-of-origin expression effects in somatic

Epigenomic studies have not been reported to date in equine oocytes; however, in the promising field of genomic investigations, the equine oocyte would serve as an excellent

SNPs. However, another form of structural genomic variation, termed copy number variation (CNV), is also widespread throughout the genome. These genomic structural variations range from 1 to 5 Mb and can be highly polymorphic between individuals, and thus can be used for epidemiological study. CNVs in the form of large-scale insertions and deletions, as well as inversions and translocations, may have important roles in meiotic recombination, human genome evolution and gene expression. Many genetic diseases are based on CNVs. However, because they consist of quantitative rather than qualitative changes, show variability in copy numbers and are confounded by the diploidy of the human genome, the detailed genetic structure of CNVs cannot be readily studied by available techniques. Thus, the establishment CNV-microarrays is currently under investigation. New microarray-based technologies will enable more accurate mapping of CNVs, and CNV maps of the human genome are being refined with increasing resolution. The study of CNVs and their effects on human health and disease therefore present a dynamic and exciting challenge for researchers in the field of genetic epidemiology (Wain and Tobin, 2011). The importance of CNVs in human preimplantation genetic screening, or to animal oocyte and embryo testing, has not been reported to date.