Preimplantation genetic diagnosis and selection

Transcription

1 Preimplantation genetic screening 120 Preimplantation genetic diagnosis and selection Serdar Coskun, DVM, PhD 1, Wafa Qubbaj, PhD 1 1 Section of Assisted Reproductive Technology, Department of pathology and Laboratory Medicine, King Faisal Specialist Hospital and Research Center, P.O. Box 3354 MBC 10, Riyadh, 11211, Saudi Arabia Abstract The availability of assisted reproductive technologies and advances in molecular biology gave rise to preimplantation genetic diagnosis (PGD). PGD is an early form of prenatal diagnosis and determines the genotype of an embryo before implantation takes place to avoid the implantation of diseased embryos. This requires couples to adhere to a strict family planning and effective contraceptive strategy, and undergo in vitro fertilization (IVF) treatment. During IVF, sampling of the cells can be performed at different developmental stages from polar body biopsy to trophectoderm cells from the blastocysts. It is indicated in couples with a family history of monogenic autosomal or X-linked recessive or dominant disorders and in detecting chromosomal aberrations of the embryos. The genetic diagnosis is performed using appropriate molecular testing which might include polymerase chain reaction, fluorescent in situ hybridization, comparative genomic hybridization or microarrays. PGD is now a wellestablished procedure and offers an alternative reproductive choice for couples who are at risk of an affected child. In this review, the past, present and future aspect of PGD will be covered. J. Reprod Stem Cell Biotechnol 1(1): , 2010 The authors have nothing to declare. Correspondence: Serdar Coskun, DVM, PhD, Section of Assisted Reproductive Technology, Department of pathology and Laboratory Medicine, King Faisal Specialist Hospital and Research Center, P.O. Box 3354 MBC 10, Riyadh, 11211, Saudi Arabia. [email protected], [email protected] T: , F: Introduction The availability of assisted reproductive technologies following the birth of first in vitro fertilization (IVF) baby in 1978 (Steptoe and Edwards) and the introduction of polymerase chain reaction (PCR, Saiki et al., 1985) gave rise to preimplantation genetic diagnosis (PGD). The procedure is particularly beneficial to families who are at risk of transmitting genetic diseases to their offspring. The first attempt of PGD goes back to 1968 for sexing of rabbit embryos by identifying a sex specific chromatin pattern in biopsied rabbit blastocysts (Gardner and Edwards). Although sex selection of embryos following these developments was applied in animal production, human PGD only became available after gaining ability of perform PCR on a single cell DNA (Li et al., 1988). In the human, the first PGD was achieved by sexing in families with X-linked genetic diseases, namely adrenoleukodystrophy and X-linked mental retardation, by amplifying Y-chromosome specific sequences from biopsied blastomeres (Handyside et al., 1990) using polymerase chain reaction (PCR) procedure. The first successful PGD with live birth for disease specific mutation testing was performed in 1992 in a cystic fibrosis carrier family (Handyside et al., 1992). Following the first misdiagnosis for embryo sexing, using PCR technique, due to the amplification failure of the Y specific sequence (Kontagianni et al., 1991), the use of fluorescent in situ hybridization (FISH) was evolved and adapted to be applied on single cells. Later FISH was the method of choice for sex determination with high efficiency (Harper et al., 1994). FISH can determines the chromosomal constitution of an embryo on interphase stage by using probes, labeled with flurochrome, and targeted specific parts of the chromosomes. Therefore, it was applicable on polar bodies (PBs), blastomeres and on trophectoderm cells (ET) from blastocysts. Interphase FISH has been widely used for aneuploidy screening, embryo sexing and chromosomal abnormalities. It requires cells fixation on glass microscope

2 Preimplantation genetic screening 121 slides, hybridization with DNA labeled probes and later signal visualization using florescent microscope. PGD is an early form of prenatal diagnosis and determines the genotype of an embryo before implantation takes place to avoid the implantation of diseased embryos. The controversial decision of pregnancy termination after having an affected fetus following classical prenatal diagnosis is not an issue in PGD. However, couples have to be exposed to an invasive and expensive infertility treatment which requires adherence to a strict family planning and effective contraceptive strategy. PGD consists of three parts: (i) generation of embryos, (ii) removal of the cell, and (iii) genetic diagnosis. Embryos are generated through in vitro fertilization treatments. In vitro fertilization Although the majority of the patients undergoing PGD are fertile, they have to start an IVF cycle to increase the number of available embryos for testing. Before treatment starts, all couples should undergo proper genetic testing and counseling which should include its indications, techniques, risks, and outcomes. They need to sign an informed consent stating their understanding of all these points. Once the couple is found to be fit for IVF and PGD, the wife starts with controlled ovarian hyperstimulation on day 3 of the menstrual cycle with daily gonadotrophin injections along with pituitary down-regulation. The follicular development in response to the medication is monitored with ultrasound scanning to determine the length of the stimulation, which may last at least 7 days. When there is enough number of mature follicles, human chorionic gonadotrophin (hcg) is given to induce final follicular maturation. Oocytes are collected through ultrasound guided transvaginal aspiration 36 hours after hcg injection (Pauli et al, 2009). The preferred method of fertilization is intracytoplasmic sperm injection (ICSI) which is the injection of a single spermatozoon into each oocyte to avoid any genetic contamination that might arise from excess spermatozoa. Embryo development is monitored daily until the diagnosis is made and the normal embryos are selected for transfer. Embryo transfers are usually performed on days 4-6 post retrieval at morulae/blastocyst stages. Biopsy The biopsy can be performed at three different developmental stages according to the protocols set up by different clinics. (i) at pre-fertilization stage by removing the first polar body from the oocytes and the second polar body following fertilization; (ii) blastomeres from preimplantation stage cleaving embryos; or (iii) trophectoderm cells from the blastocysts. Biopsy is performed under an inverted microscope with micromanipulators attached. The embryo/oocyte is held with a holding pipette and cells are removed with an aspiration needle following opening a hole in the zona pellucida (Figure 1). The opening of this hole can be achieved by several methods including acid treatment, mechanical slitting or laser (De Vos and Van Steirteghem, 2001). Figure 1. Blastomere biopsy. (a) an embryo was held with a holding pipette and a hole was made with the laser source, (b and c) the exposed blastomere was aspirated with a blastomere biopsy pipette, and (d) the blastomere was removed from the embryo by applying gentle suction. Polar bodies are the expelled genetic materials during the course of meiosis and only provide information about the maternal genotype. It was first performed in women who were heterozygous for alpha-1-antitrypsin deficiency (Verlinsky et al., 1990). It was successfully applied to several other diseases and aneuploidy screening by the same group (Verlinsky and Kuliev, 1996). This is usually performed in countries where embryo biopsy is not an acceptable alternative (Intrum et al., 2004; Montag et al., 2009).

3 Preimplantation genetic screening 122 The most common biopsy strategy is the removal of one or two blastomeres from 6-10 cell stage embryos. The blastomere stage biopsy has the advantage of providing more time for diagnosis since the embryos have to be transferred by day 6 and it will reveal the genetic contributions from both parents. The use of calcium and magnesium free media facilitates the blastomere removal since many embryos have a certain degree of compaction on day 3 (Dumoulin et al., 1998). There is an ongoing debate over whether to remove one or two blastomeres from the developing embryos. In a prospective randomized study, two-cell removal has been found to be associated with increased diagnostic efficiency with PCR; however, it reduced the chance of blastocyst formation without affecting the delivery rates. (Goossens et al., 2008). The diagnostic efficiency of FISH was not affected with the number of cell removed for testing. However, in a recent prospective cohort study, two-cell removal has been shown to negatively impact clinical outcome (De Vos et al., 2009). In our hands, removing two cells has been shown to increase the amplification efficiency of whole genome amplification (WGA) and reduce allele dropout (ADO) rate without affecting the pregnancy rates (Coskun et al., 2008). Another alternative is to perform the biopsy at the blastocyst stage on day 5 following fertilization. The blastocyst-stage biopsy provides more cells however little time is available to reach the genetic diagnosis. Cryopreserving blastocysts following biopsy and the transfer of normal embryos in a replacement cycle have been suggested (Magli et al., 2006). However, the center should have a successful blastocyst culture and cryopreservation protocol if blastocyst stage biopsy is used. Vitrification in the cryopreservation of biopsied blastocysts has promising implantation rates (Schoolcraft et al., 2009). Indications Candidates for PGD are couples with a family history of monogenic autosomal or X-linked recessive or dominant disorders. Moreover, PGD can be used in detecting chromosomal aberrations of the embryos either to improve the reproductive outcome of patients undergoing IVF or prevent of aneuploidies in patients with certain chromosomal rearrangements (Kearns et al., 2005). Based on these alterations, special primers or chromosome-specific probes can be developed to ensure the establishment of a pregnancy with disease-free embryos. PGD for monogenic disorders Single gene disorders are triggered by mutations in specific genes transmitted according to a predictable inheritance model which can be dominant, recessive or X-linked. Carriers of recessive diseases are at 25% risk of an affected offspring whereas carriers of dominant diseases are at 50% risk of affected offspring. There are over 6,000 known single gene disorders and they could be observed in 1/300 births ( In principle, PGD could be utilized in any of these disorders as long as disease causing mutations are identified. It is currently estimated that PGD is utilized in the prevention of over 100 different diseases (Preimplantation Genetic Diagnosis International Society-PGDIS, 2008). PGD for late onset diseases and cancer susceptibility genes PGD is already extended to diseases which have late onset or mutations that predispose to familial cancers. In case of late onset diseases, the penetrance is high and the presence of mutation is an almost certain indication that the disease will present later in life. PGD for Huntington s and Alzheimer diseases have been successfully performed (Sermon et al., 1998a, Verlinsky et al., 2002a). For genes linked to predisposition for cancer, the presence of a mutation does not always result in the occurrence of the malignancy. However, there is an increasing demand to utilize PGD for such conditions. PGD is performed for adenomatous polyposis of the colon (Ao et al., 1998), tumor-suppressor gene p53, Von Hippel-Lindau syndrome, retinoblastoma, rhabdoid tumor (Verlinsky et al., 2001b; Rechitsky et al., 2002), neurofibromatosis type 2 (Abou-Sleiman et al., 2002) and BRCA1 (Spits et al., 2007). PGD for stem cell transplantation ("designer babies") Embryos are selected based on their HLA types for this indication of PGD. Once the pregnancy is established and reaches to the term, the cord blood stem cells obtained during the delivery

4 Preimplantation genetic screening 123 can be used to be transplanted to the effected sibling. The PGD baby is a matched stem cell donor for an affected sibling in addition to being free from the familial disease. These embryos are popularly known as "designer babies". Families requiring this procedure have a genetic disease which affects the hematopoietic cells. Therefore, the affected members are usually in need of stem cell transplantation. The chance of being a normal embryo in an autosomal recessive disease is 3 in 4 and the likelihood of being HLA compatible is 1 in 4 which gives an overall likelihood of 3 in 16 embryos being suitable for transfer. The first PGD for this indication was applied to a family with Fanconi anemia (Verlinsky et al., 2001a) and later extended to Wiscott-Aldrich syndrome, Thalassemia, X-linked adrenoleukodystrophy, hyperimmunoglobulin M syndrome (Rechitsky et al., 2004) X-linked chronic granulomatous disease (Reichenbach et al., 2008) and Diamond-Blackfan anemia (Bellavia et al., 2010). Preimplantation genetic screening (PGS) PGS refers to techniques where embryos are screened for aneuploidies. It is usually performed by using fluorescence in-situ hybridization (FISH) technique. FISH involves in single cell biopsy, cell spreading and fixation of the nucleus on a slide, followed by probe hybridization for a limited number of chromosomes labeled with different fluorescent dyes. Afterwards the signals are visualized under a fluorescent microscope and the chromosome copy number is scored. Consequently, only euploid embryos for the tested chromosomes will be considered for transfer while the aneuploid embryos will be discarded. PGS was first reported on polar bodies by Verlinsky et al. (1995) and Munne et al. (1995a), and on cleavage stage embryos by Gianaroli et al. (1997). Since then, PGS has been commonly performed in many IVF centers worldwide. PGS has focused on chromosomes frequently identified as abnormal in prenatal samples or material derived from first-trimester spontaneous abortions, such as chromosomes X, Y, 13, 18 or 21 (5-probe FISH assay). However, the aneuploidy frequencies for specific chromosomes change between the cleavage stage and the first trimester (Munné et al., 2004a), therefore, chromosomes 15, 16, 17 and 22 were later included in the PGD screening (9- probe FISH assay, Munné et al., 1998a, 2004b). The 10-probe assay which consists of probes for chromosomes (X, Y, 8, 9, 13, 15, 16, 18, 21 and 22) and the 12-probe assay which consists of probes for chromosomes (X, Y, 8, 13, 14, 15, 16, 17, 18, 20, 21 and 22) were also introduced (Colls et al., 2009; Munné et al., 2010). Data from spontaneous abortions suggests that the 9-12 probe FISH assay detects only 57 67% of aneuploid embryos (Jobanputra et al., 2002; Munné et al., 2004b; Lathi et al., 2008). Due to limited availability of fluorochromes, testing 9-12 probes requires a second or subsequent round of hybridization, which might cause DNA degeneration and therefore, reduce the hybridization efficiency (Harrison et al., 2000). Some laboratories have reported high efficiency rates of 95-97% for FISH following a first round (Staessen et al., 1999; Harper et al., 1994; Munné et al., 2003) and an efficiency rate of 95% is also reported for single blastomeres when reprobing for a second round (Munné et al., 2003). The no-result rescue strategy (NRR) was introduced recently (Colls et al., 2007, 2009; Uher et al., 2009) and it requires a third round of hybridization using telomeric probes for the chromosome(s) in question. The NRR strategy is recommended for dubious results in order to increase the efficiency of PGD and was recommended in the published guidelines for good PGD laboratory practice (PGDIS, 2008). PGS scoring criteria were applied according to Munné et al. (1998b) and Magli et al. (2001a). The embryos are considered normal if two signals for each tested autosomal chromosome are present, or haploid, triploid or polyploid if one, three or more copies of tested chromosomes are present, respectively. A chaotic pattern is defined as the presence of two or more chromosomal abnormalities in the same nucleus. False-positive results refer to embryos diagnosed as abnormal but found to be normal after reanalysis which consequently reduces the number of transferable embryos. False-negative results occur when embryos diagnosed as being normal are found to be abnormal after reanalysis which might result in misdiagnosis (Gianaroli et al., 1999). In general, FISH error rates are

5 Preimplantation genetic screening 124 centre and technique-related, and depend on the chromosomes and probes being used. PGS error rates using FISH were found to be as low as 5 7% (Colls et al., 2007; Magli et al., 2007) or as high as 50% (Baart et al., 2004). Indications for PGS The indications for PGS are advanced maternal age (AMA), repeated implantation failure (RIF), and repeated miscarriage (RM), severe male factor (SMF) infertility, a previous sporadic genetically abnormal pregnancy, poor embryo quality, previous radiotherapy and single embryo transfer (Harper et al., 2010). AMA is usually defined as maternal age over 37 or 38 years (Harper et al., 2008a). Oocytes from women with AMA are more likely to have chromosome non-disjunction and premature chromatid separation resulting in an aneuploid embryo. High rates of aneuploidies, 40-70%, were reported in oocytes from women with AMA (Battaglia et al., 1996; Pellestor et al., 2003; Staessen et al., 2004). PGS has been reported to improve pregnancy and delivery rates in non-randomized studies for AMA (Munné et al., 1995b, 1999; M?rquez et al., 2000; Gianaroli et al., 1999, 2005), in women with a history of RM (Vidal et al., 1998; Pellicer et al., 1999; Rubio et al., 2003; Werlin et al., 2003; Munné et al., 2005; Platteau et al., 2005; Garrisi et al., 2009) and RIF (Gianaroli et al., 1999, 2001, 2003; Kahraman et al., 2000; Pehlivan et al., 2003; Wilton et al., 2003). Beneficial effects of PGS also reported for women with previous aneuploid conception (Gianaroli, 1999, 2005; Munné, 2004b; Kahraman et al., 2000; Kuliev and Verlinsky, 2004). Although above mentioned studies showed advantages of performing PGS, several randomized controlled studies (RCTs) performed recently in couples with AMA stated that PGS does not improve the clinical pregnancy rate and it is not recommend (Staessen et al., 2004, 2008; Mastenbroek et al., 2007; Hardarson et al., 2008). However, these studies came under several criticisms from PGS advocates (Cohen et al., 2007a; Munne et al., 2007). The criticisms extended from the number of cells removed to suboptimal laboratory conditions. Another limiting factor could be the incomplete chromosomal coverage by FISH. To further investigate and come to a better conclusion, ESHRE launched a multicenter RCT to investigate the usefulness of PGS by using array CGH on biopsied polar bodies (Harper et al., 2010). Carriers of chromosomal aberration Carriers of structural abnormalities such as reciprocal or Robertsonian translocations and pericentric or paracentric inversions, have a reduced fertility rate and these couples are at increased risk of recurrent miscarriages, RIF and chromosomally unbalanced offspring (Chandley, 1998). PGD for translocation carriers has been reported to significantly reduce spontaneous abortions and increase the pregnancy rates (Munné et al., 1998c, 2000, 2006; Verlinsky et al., 2002b, 2005; Gianaroli, at al, 2002; Otani, et al., 2006; Fischer et al., 2009). PGD for reciprocal translocations is usually performed either by using two probes, labeled in different colors, that are located on either side of the breakpoint on one chromosome and one probe anywhere on the other chromosome involved in the translocation (Munné et al., 2000) or by using one sub-telomeric probe for each of the chromosomes involved in the translocation and a control centromeric probe (Scriven et al., 1998). While, in PGD for Robertsonian translocations, two probes labeled in different colors are used, one for each chromosome involved in the translocation (Munné et al., 2000). Blastomeres showing two signals for each probe are classified as being normal or balanced, while any other combination is classified as unbalanced and will not be considered for transfer. Limitations of PGS Causes of misdiagnosis associated with FISH have been stated clearly by Wilton et al. (2009). Among them, a thorough removal of cumulus cells from oocytes or zygotes is crucial in order to minimize the chance of contamination (Thornhill et al., 2005). If the cytoplasm is covering the nucleus, it could prevent probe penetration resulting in hybridization failure. To prevent this, different blastomere spreading techniques have been introduced such as the Tween/ HCl method (Coonen et al. 1994) and the methanol/ acetic acid method (Tarkowski, 1966). In addition, poor-quality embryos might have degenerated interphase chromatin which could affect FISH signals (Uher et al., 2009).

6 Preimplantation genetic screening 125 Mosaicism, which is characterized by the embryo having two or more cell lines, could cause misinterpretation, since the analyzed cell might not be a true representative of the whole embryo (Harper et al., 1995; Munne et al., 1997; Baart et al., 2006). Analyzing two cells per embryo may improve the accuracy but it may have a negative effect on the developmental capacity of an embryo (Tarin et al., 1992; Goossens et al., 2008). For testing chromosomal imbalances in embryos from translocation carriers, the probes should account for all possible segregation patterns for the particular translocation (Thornhill et al., 2005). The probes need to be tested for any cross-hybridization to other chromosomes. If present this must be taken into account during signal scoring and interpretation. An overlap signal results when the two copies of chromosomes lie on the top of each other during nucleus fixation or spreading. Signals from different chromosomes with different fluorochromes can also overlap. If a red and a green signal overlap, it will result in a yellow spot, which can be misinterpreted if a yellow fluorochrome is also used. Interpretation of closely adjacent or split signals can be difficult. A decision has to be made on whether it represents one copy of that chromosome or should be considered as two separate signals representing two chromosomes which are closely adjacent to each other. To be considered as one signal, the distance between signals should be less than the half diameter of the signal. If the nucleus is thick signals might appear at different depths and all signals on different focal planes should be scored. FISH error rates are laboratory and techniquedependent and therefore each laboratory should have optimized it s protocols to suit it s own conditions. The efficiency of the probes should be tested before the clinical case, and the laboratory must not rely on the quality assurance provided by the manufacturer. When performing more than one round of FISH on each nucleus, great care should be taken to minimize the persistence of signals from the first round of FISH as this could result in incorrect interpretation of signals in the second round. Genetic analysis of single cells Preliminary PGD workup (pre-pgd) Genetic testing should be performed on both parents and any affected child to confirm the mutation causing the disease in question and to test for informative linked and/or unlinked markers to be used during the PGD cycle. The testing of extended family members may be required in doubtful situations. A PCR protocol will be developed to test the mutation(s) site simultaneously with other informative polymorphic markers. The developed protocol should also be optimized and validated on single cell level, high amplification efficiency and minimum ADO should be aimed to ensure efficient reliable diagnosis on a single cell. Polymerase chain reaction (PCR) The genetic diagnosis is usually done using PCR which is a powerful molecular technique to amplify a particular DNA fragment to a level that can be analyzed allowing a specific mutation to be detected (Saiki et al., 1985). However, it is still challenging to obtain a reliable diagnosis on a single cell where amplification failure (AF), ADO and extraneous DNA contamination present major challenges. Amplification failure PGD misdiagnosis for an X-linked disorder was reported (Hardy and Handyside, 1992) due to AF of Y-chromosome specific sequences leading to a false diagnosis of a male embryo as female. The reasons for amplification failure in single cell PCR are still unclear but it occurs frequently. However, many recommendations have been reported to minimize its risk, one of them being to ensure that the biopsied cells do not lyse before they have been placed in the PCR tube, and that they should possess a visible nucleus (Sermon et al., 1995; Kontogianni et al., 1996). Allele Dropout ADO is a phenomenon unique to PCR with low quantity DNA. It is the result of preferential amplification (PA) of one allele, leaving the second allele under the detectable level (Figure 2, Findlay et al, 1995; Ray and Handyside, 1996; Rechitsky et al., 1996, 1998). ADO is a potential cause of misdiagnosis in the analysis of autosomal dominant diseases and recessive disorders in couples carrying compound heterozygotes mutations. ADO of one of the mutant alleles in compound heterozygotes, or

7 Preimplantation genetic screening 126 the mutant allele in autosomal dominant diseases, results in a misdiagnosis, subsequently leading to the transfer of an affected embryo. Contamination Peculiar DNA contaminations from sperm, maternal cumulus cells (Figure 3). If both parents carry the same mutation, then ADO of the mutant allele could result in an embryo diagnosis of being normal, when in fact Figure 2. A chromatogram showing ADO. The upper panel is showing two peaks from the genomic DNA and the lower panel shows only a single peak amplified from a single cell genome from the same individuals it is heterozygous, which imposes no serious misdiagnosis in returning back such an embryo (benign misdiagnosis) (Qubbaj et al., 2008). Yet, if ADO occurred to the normal allele, the embryo will be diagnosed as being affected, and this consequently reduces the number of healthy embryos available for transfer. ADO has been reported to occur in ~0.8-36% of single cell PCR amplifications (Ray and Handyside, 1996; Wells and Sherlock 1998; Rechitsky et al., 1998). There are several possible causes of ADO, including DNA degradation, inefficient cell lysis or imperfect PCR conditions. To minimize the risk of any misdiagnosis as a result of ADO, it is advisable to test two cells from each embryo if possible, since the probability of ADO affecting the same allele in both cells is low (Kontogianni et al 1996; Thornhill and Snow, 2002). Optimal lysis conditions and increasing the denaturing temperature during the first cycle of PCR amplification has been recommended to reduce ADO (Ray et al., 1996). Additionally, using an informative linked polymorphism helps to detect any ADO in the gene of interest (Kuliev et al., 1998; Qubbaj et al., 2008). Figure 3. Maternal contamination detected by the inclusion of informative markers. The father has peaks 1 and two, and the mother has 2 and 3. Embryo 1 shows the preferential amplification of peak 2 from the father compared to maternal peak 3. Embryo 2 has 3 peaks; paternal peak 1 and maternal peak 2 and 3. or any carry over DNA might be associated with misdiagnosis. Therefore, all cumulus cells must be carefully removed from the oocyte and the embryo, and excess sperm is avoided by the use of the ICSI method. Biopsied cells should be washed in a medium that has been tested for any contamination before being transferred to the PCR tubes. A separate room with filtered air under positive pressure (pre-pcr room) should be used for single cell PCR preparation and for the cell transfer to the tube. To avoid any PCR template carry-over, equipments and reagents should be dedicated for single cell PCR and they should not be in contact with any previously amplified DNA. All reagents should be tested before a clinical case and preparations should take place in a laminar flow hood. Aliquoting of reagents into several tubes is important to avoid repeated freezing and thawing, and they should be discarded after a few uses. Staff working in the pre-pcr room should wear clean gowns, gloves, and shoe covers. Positive and negative controls should be included with each clinical case and all negative controls should be clear with no amplifications.

8 Preimplantation genetic screening 127 Molecular strategies to overcome problems associated with single cell PCR Different molecular strategies have been incorporated in PGD to help prevent misdiagnosis due to contamination, ADO, AF or PA. These include nested PCR, multiplex PCR (M-PCR), fluorescent PCR (F-PCR), multiple fluorescent PCR (MF-PCR), whole genome amplification (WGA), and the use of microsatellite and other polymorphic markers. Multiplex PCR allows the amplification of multiple loci simultaneously by including several sets of primers specific for different loci within the same PCR mixture. PCR reactions need to be optimized so that different primer sets work at the same annealing temperature and PCR conditions. Primers should not interact with other primers, the PCR fragments of different loci should be distinguishable either by their size or, when using fluorescent labeled primers, by different fluorescent labels in MF-PCR. M-PCR using multiple linked markers confirms the diagnosis and provides the diagnosis where the mutation site has failed to amplify (Findlay et al., 2001). F-PCR improves both the sensitivity and specificity through utilizing fluorescent labeled primers at the 5 end, followed by the detection of the amplified products on an automated fluorescent sequencer. F-PCR has been carried out to detect diseases caused by nucleotide expansions such as CCG repeats in fragile-x syndrome, and for CAG repeats in Huntington disease (Sermon, 1998b, 2001). Fluorescent Gap PCR has also been utilized for alpha thalassaemia (Deng et al., 2006) and for mutations caused by small deletions in betathalassaemia (DeRycke et al., 2001). MF-PCR can be achieved by utilizing more than one set of fluorescently labeled primers (Findlay et al., 1995, 1998). It has been used in many PGD clinical cases including single gene disorders combined with HLA typing (Fiorentino et al., 2004, 2006) and for mutation detection combined with linked informative markers (Kuliev et al., 1998; Rechitsky et al., 1998, 2001; Goossens et al., 2003; Holding et al., 1993). Cell lysis Cell lysis is a very crucial step prior to PCR. Many protocols have been implemented for single cell lysis such as, Proteinase K / sodium dodecyl sulphate (Jeffreys et al., 1988; Holding et al., 1993), Proteinase K, Tween-20 and Triton X-100 (Rechitsky et al., 2001). Alkaline lysis buffer (ALB) has also been used extensively and samples need to be neutralized after it s use before the PCR amplification (Cui, et al., 1989). Detection method Several molecular approaches at a single cell level have been implemented for single gene disorders like single-stranded conformation polymorphism (El Hashemite et al., 1996), denaturing gradient gel electrophoresis (Kanavakis, et al., 1999; Vrettou, et al., 1999) and heteroduplex analysis (Handyside et al., 1992; Ao et al., 1996; Ray et al., 1998). These methods will only predict the presence of mutation(s) but not their nature and will not differentiate between the presence of mutations versus polymorphisms. As a result DNA samples from both parents and an affected (or normal) child should be included as controls while testing the embryos. Direct mutation detection has also been performed using different strategies such as amplification refractory mutation system-pcr (Sherlock, 1998), allele-dependent length polymorphism (Storm et al., 1998), restriction endonuclease analysis (Kuliev et al., 1998; 1999; De Rycke et al., 2001), site specific mutagenesis (Kuliev et al., 1998; 1999; De Rycke et al., 2001), direct sequencing (Hussey, et al., 2002), minisequencing (Fiorentino, et al., 2003, 2004), gap-pcr (Deng et al., 2006) and expanded long template PCR (Sermon et al., 2001). Informative polymorphic linked markers, which are in linkage disequilibrium with a specific disease, can be used as an indirect method for the detection of the mutant chromosome. This approached has been used for fragile-x syndrome (Apessos A., et al., 2001), mytonic dystrophy (Sermon, et al., 1997), and beta thalassaemia (Kuliev et al., 1998; 1999). Primer choice Primer choice is very crucial for PCR efficiency and specificity. There are a number of online tools that can assist in PCR primer design, based on the primer length, GC content, the secondary structure formation and the desired amplicon length. Examples of these tools include free online Primer3 (

9 Preimplantation genetic screening 128 bin/primer3/primer3_www.cgi), Primer Express software from Applied Biosystems. It is always advisable to check the specificity of each primer using the free online BLAST programme ( The genomic DNA sequence information can be retrieved online from Ensemble ( the National Center for Biotechnology Information nucleotide database ( or The Human Gene Mutation Database ( PCR condition PCR should be optimized prior to PGD clinical cases by first using genomic DNA at ng, then downscaled to very low concentration of genomic DNA (100 pg 16 single cells) and followed by single cells (7 pg of DNA) to ensure a high amplification efficiency and low ADO. Optimization of PCR conditions is based on the number of primers used, the annealing temperature, the use of dimethyl sulfoxide (DMSO) and magnesium ion concentration. Selection of polymerase type and PCR buffer type is also important. Ampli Taq polymerase, Faststart Taq DNA polymerase (Roche Applied Science), Expand Long Template Taq DNA Polymerase (Roche), Expand High Fidelity polymerase (Roche) and QIAGEN Multiplex PCR Kit (QIAGEN) have been widely used. Fragment detection method Many methods have been used for the fragment detection from conventional agarose to polyacralamide gel electrophoresis. Fragment analysis following the use of fluorescently labelled primers can also be performed by an automated sequencer. Whole Genome Amplification (WGA) From a single cell genome, only a single reaction can be performed whether using simplex or multiplex PCR. Optimizing various PCR conditions on a single cell level is required for each mutation which is costly and a timeconsuming. Multiplex PCR even requires more extensive optimizations. In addition, preferential amplification and ADO still occur at a significantly higher rate in multiplex-pcr (Findlay et al., 2001). WGA is a technique to increase the DNA quantities from samples with limited DNA contents. It can potentially overcome some of problems associated with single cell PCR and allows genomic DNA PCR protocols to be used in WGA products. There are different PCRbased WGA methods like primer extension preamplification-pcr (PEP-PCR, Zhang et al., 1992), and degenerate oligonucleotide primed PCR (DOP-PCR, Telenius et al., 1992). PEP- PCR was first introduced in 1992 in the analysis of single sperm typing to study genetic recombination. It utilizes total degenerate 15- mer PCR primers and generates a smear of DNA fragments that are visible on an agarose gel. The genomic coverage was at least 78% (Zhang et al., 1992). PEP-PCR has been used in PGD cases for beta thalassaemia (Jiao et al., 2003). DOP-PCR uses partially degenerate sequence oligonucleotides (5 - CCGACTCGAGNNNNNNATGTGG-3 ) which randomly anneal to the genome (Telenius et al., 1992). DOP-PCR amplified single cell genome allowed the first CGH to be performed on a single cell and provided reliable detection of trisomies and sex in a blind study (Wells et al., 1999). However, the amplification products from these techniques are short, characterized by strong biases, have artifacts, have incomplete genomic coverage and suffer inefficient microsatellite amplification (Cheung and Nelsonm 1996; Paunio et al., 1996; Wells et al., 1999). Recently, multiple displacement amplification (MDA) has been introduced which utilizes bacteriophage phi29 DNA polymerase and random hexamer primers under isothermal conditions (Blanco et al., 1989; Dean et al., 2002). MDA is found to overcome certain obstacles, including bias amplification, irreproducibility and misdiagnosis (Hosono et al., 2003) and has been widely applied in PGD clinical cases (Hellani et al., 2005; Burlet et al., 2006; Coskun and Alsmadi, 2007; Renwick et al., 2006; Al-Sayed et al., 2007; Qubbaj et al., 2008). It has been particularly helpful in the introduction of preimplantation genetic haplotyping which utilizes the amplification of several informative markers along with (or without) a specific mutation (Renwick et al., 2006). MDA is still facing some challenges such as template independent DNA synthesis (TIDA) and ADO. TIDA has been addressed by

10 Preimplantation genetic screening 129 technical modifications to the MDA protocol by reducing the reaction volume which resulted in suppression of TIDA (Kumar et al., 2008). ADO is still a challenge for MDA to be commonly used in PGD. A recent study indicated satisfactory genome coverage can be obtained from single cell MDA and showed a reasonable accurate representation of MDA from a minute amount of DNA template by using the SNP mapping array (Ling et al., 2009). The Genomeplex WGA method has also been recently introduced into the single cell field. It is a whole genome amplification method utilizing a proprietary amplification technology based upon random fragmentation of genomic DNA and conversion of the resulting small fragments to PCR amplifiable OmniPlex Library molecules flanked by universal priming sites (Barker et al., 2004). The OmniPlex library is then PCR amplified using universal oligonucleotide primers in a limited number of cycles. It has been successfully used in PGD for β-thalassaemia and HLA typing (Chen et al., 2008). A single cell genome amplified by genomeplex WGA has been accurately used in microarray analysis to differentiate self from the sibling (Treff et al., 2009). Again, ADO rates assessed by the call rates during microarray analysis are high (Treff et al., 2009). Moreover, it is highly labor intensive and time consuming compared to MDA. Preimplantation genetic haplotyping (PGH) Testing of multiple markers to increase the accuracy and to confirm the genetic diagnosis in single cells has earlier been implemented (Kuliev et al., 1998; Rechitsky et al., 1998, 2001; Goossens et al., 2003). The diagnosis by using many linked markers has been recently utilized following WGA and termed as PGH (Renwick et al., 2006). It is a novel approach in PGD to apply a rapid and simple application for any mapped single gene disorders even in the absence of a known pathogenic mutation. Many PGH cycles have been carried out for cystic fibrosis (CF), Duchenne and Becker muscular dystrophy, resulting in birth of babies in the PGD cycles for CF (Renwick et al., 2006, 2010). PGH requires careful study of the pedigree with at least one affected family member and identification of several informative telemetric and centromeric linked markers for the mutated gene. This information can be used to reveal the haplotypes associated with both normal and mutant alleles for the studied family (Dreesen et al., 2000; Renwick et al., 2006). Applying PGH to extended family members can help to identify the incidence of crossover via the presence of recombinant haplotypes (Renwick et al., 2007). Utilizing PGH alongside mutation analysis helps to exclude the risk of misdiagnosis due to ADO. Even with high ADO, testing multiple linked makers located at the 3 and 5 side of the gene of interest will allow haplotypes associated with the mutant alleles to be identified (Renwick et al., 2006; Ogilvie et al., 2009). PGH allows identification ofthe parental origin of each inherited chromosome (paternal or maternal) which also assists in the detection of any DNA contamination. PGH can also identify haploid embryos or monosomies by showing a single allele throughout the tested region. This will be particularly helpful in preventing the transfer of such embryos when the mutation detection shows the normal allele. Comparative genomic hybridization (CGH) Human embryos may have chromosomal abnormalities which cannot be detected by FISH analysis since only up to 12 chromosomes can be tested by FISH under the best conditions (Munne et al., 2010). Consequently, abnormalities that are not tested by FISH will be missed. This led the introduction of CGH into the single cell diagnostics (Wells et al., 1999). CGH was initially developed to analyze aneuploidy in small numbers of cells obtained from solid tumors (Kallionemi et al., 1992). It compares the sample DNA to known normal DNA by labeling them with different fluorochromes and then simultaneously hybridizing them to normal human male metaphase chromosomes fixed on a microscope slide. The differences in fluorescence ratios for each chromosome are analyzed to detect chromosomal unbalances. CGH has been successfully used in single blastomeres obtained from embryos and the cycle resulted in a birth of a healthy baby (Wilton et al., 2001). In spite of the clear advantages, CGH has not found a broad clinical application because the procedure is complicated and takes several days to finish and requires the cryopreservation of the embryos to be used in a replacement cycle (Wilton, 2005). A blastocyst-cgh strategy which included blastocyst vitrification has been recently adopted and resulted in high implantation rates (Schoolcraft et al., 2009). To

11 Preimplantation genetic screening 130 overcome the difficulties associated with CGH, array CGH has been utilized and the birth of several children reported without requiring a cryopreservation step (Sher et al., 2007; Hellani et al., 2008, Fishel et al., 2010). The real question to answer is whether chromosomal screening has any advantage to patients undergoing IVF. For this reason, ESHRE initiated a randomized control trial for advanced maternal age indication by using polar body biopsy and array CGH (Harper et al., 2010). Accuracy and misdiagnosis Misdiagnosis can be considered to be either adverse or benign. Adverse misdiagnoses are recognized following the birth of an affected child or termination of an affected pregnancy following the transfer of embryos diagnosed as unaffected. Benign misdiagnoses result from the birth of an unaffected child carrying a mutation when the transferred embryo was thought to be homozygous normal. Misdiagnosis rates have been estimated at <1% for polymerase chain reaction (PCR) cases (Wilton et al., 2009). This percentage is usually estimated from the published misdiagnosis cases amongst the embryos having been transferred or the number of pregnancies (Wilton et al., 2009). The true level of misdiagnosis in PGD may however remain unknown for several reasons: not all embryos transferred result in pregnancy or a child birth, a proportion of misdiagnoses remained undetected, benign misdiagnoses were not considered, and some centers may not publish all such incidences (Wilton et al., 2009). During patient counseling it is recommended that both published and in-house estimates (individual misdiagnosis rates) should be available to allow informed consent for PGD patients (Wilton et al., 2009). New technologies in preimplantation genetic diagnosis Microarray technology With the initial announcement of the human genome project in 2000, techniques to study whole human genome are becoming widely available and the development of microarrays facilitated the identification of the molecular basis of many genetic diseases (Shinawi and Cheung, 2008). With the availability of WGA, microarrays were able to enter into the PGD field (Hellani et al., 2004). The arrays used in PGD can be considered in two group; (i) array CGH and (ii) Single Nucleotide Polymorphism (SNP) microarrays. Array CGH evolved from CGH and it enables high-resolution, genomewide screening of segmental genomic DNA sequences (Pinkel et al., 1998). Similar to conventional CGH, it involves the competitive hybridization of differentially labeled test and reference DNA samples. There are two alternative array CGH platforms available for chromosomal screening: Bacterial Artificial Chromosome (BAC) arrays and Oligo-Arrays (Maciejewski and Mufti, 2008). BAC arrays contain a few thousand spots comprising large DNA fragments. Oligo-arrays utilize oligonucleotides of short sequences (<100) and are directly synthesized on the surface of the solid support. Although array CGH has been utilized in PGD (Sher et al., 2007; Wells et al., 2008; Hellani et al., 2008, Fishel et al., 2010), it cannot be used in the detection of balanced chromosome rearrangements, mutations, changes in regions of the genome not covered by the array and parental origin of aneuploidies. These disadvantages led to the introduction of SNP microarrays in PGD. SNPs are normally occurring genetic variations that are randomly present throughout the genome. They are the main causes of human genomic variation and are estimated to be present in every 100 to 300 bases (Ward, 2006). SNPs are used in the identification of sets of genetic markers on a chromosome that tend to be inherited together and utilized in molecular karyotyping (Rauch et al., 2004). The use of SNP arrays has been applied to single cell diagnostics and the information obtained from SNP analysis used in DNA fingerprinting of embryos to distinguish sibling embryos (Treff et al., 2009). The same group also successfully used SNP arrays for the detection of 24 chromosome aneuploidy screening (Treff et al., 2010). Recently, two new algorithms of SNP microarrays data analyses were introduced by utilizing parental information to detect aneuploidies (Johnson et al., 2010) and the linkage pattern of Mendelian traits in addition to identifying aneuploidies (Karyomapping, Handyside et al., 2009). These technologies will allow the detection of the parental origin of aneuploidies, DNA fingerprinting of each embryo tested and the linkage-based detection of disease causing

12 Preimplantation genetic screening 131 mutation by the genotyping of SNPs located in close proximity to the mutation site. This potentially opens up the possibility of using SNP-microarray platforms for the concomitant detection of chromosome anomalies and single gene disorders and probably will change the ways in which IVF and PGD are performed in future. Outcome of preimplantation genetic diagnosis and screening The European Society of Human Reproduction and Embryology (ESHRE) PGD Consortium was established in 1997, and collects data from participating centers. The results are published annually in Human Reproduction. Of ESHRE PGD consortium data collected since 1999, nine PGD datasets were published (Goossens et al., 2009). Data were collected from 57 collaborating centers and divided into three main groups, PGD, PGS and PGD-social sexing (SS). A total of 21,743 oocyte retrievals were documented of which 72% reached ET and 26% of ETs resulted in clinical pregnancies (Table 1). technical limitations) and the risks of abuse for non-medical reasons (mainly social sexing). The destruction of these embryos created a lot of discussion among different social and religious groups. Other indications for PGD that involve ethical concerns include HLA typing, cancer predisposition mutations and late onset diseases which may require non-disclosure testing (Stern, et al., 2002). In summary, PGD is now a well-established procedure and offers an alternative reproductive choice for couples who are at risk of an affected child. The new technologies are promising. Whole genome amplification will allow genomic DNA PCR protocols to be used in PGD and will eventually remove the need for PCR protocols to be optimized for single cell diagnosis. This will allow PGD to become easily available wherever molecular diagnostic laboratories are present. Microarrays can also be used following WGA of a single cell genome and this opens up new diagnostic opportunities like combining single gene disorder testing with aneuploidy analysis. PGD data collected from 8,111 cycles included cycles for chromosomal abnormalities, testing for specific X-linked diseases, and monogenic disorders. Monogenic disorders including specific X-linked diseases contributed 3530 PGD cycles to oocyte retrieval (OR). Of these, 2775 cycles reached to embryo transfer (ET) and resulted in 769 clinical pregnancies. Ethical consideration The European Society of Human Reproduction and Embryology (ESHRE) published a report addressing ethical concerns related to PGD (ESHRE Task Force, 2001) which included the destruction of affected pre-implantation embryos (and sometimes normal embryos due to References Abou-Sleiman PM, Apessos A, Harper JC, Serhal P, Winston RM, Delhanty JD. First application of preimplantation genetic diagnosis to neurofibromatosis type 2 (NF2). Prenat Diagn. 2002; 22: Al-Sayed M, Al-Hassan S, Rashed M, Qeba M, Coskun S. Preimplantation genetic diagnosis for Zellweger syndrome. Fertil Steril. 2007; 87:1468.e1-3. Ao A, Ray P, Harper J, Lesko J, Paraschos T, Atkinson G, Soussis I, Taylor D, Handyside A, Hughes M, Winston RM. Clinical experience with preimplantation genetic diagnosis of cystic fibrosis (delta F508). Prenat Diagn 1996; 16:

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