The Genetic Profiling of Embryos Prior to Implantation - Essay Example

Abstract PGD is the genetic profiling of embryos prior to implantation, PGD and PGS have been mostly conducted using PCR and FISH for the analysis. The shortcoming with PCR when it comes to PGD and PGS analysis is that PCR needs adequate quantities of pure and high-quality DNA samples, which at times can be a challenge to obtain from a sole cell. Shortcomings with FISH in PGS and PGD include that FISH is not able to fully access all the chromosomes: a human cell has 23 pairs of chromosomes while FISH analysis is only able to do accurate assessment of just 10-12 chromosomes with every biopsied cell. Next Generation Sequencing (NGS) and comparative genome hybridisation (CGH) are new technologies used for all-inclusive chromosome testing of IVF embryos. NGS technology facilitates unprecedented scalability and hence in PGS and PGD it enables identification and detection of high percentage of genetic mutations and abnormalities within the DNA. NGS is not only less costly but the technology is also able to produce large quantities of nucleic acid sequences within a comparatively short period. Similarly, CGH technique allows examination of 23 chromosomes and also provides a more comprehensive image of the entire length of the chromosome, and this can even identify and detect imbalance of chromosomal segments. Ethical issues associated with CHG and NGS in pre-natal diagnosis include issues of elective abortion; ethical dilemma between respecting maternal autonomy versus acting beneficently to the fetus; use of proxy decision makers for the fetus; generation and interpretation of the generated data; and how to disclose “incidental information”. Efficacy of recent developments in pre-implantation genetic diagnosis (Next Generation Sequencing (NGS) and comparative genome hybridisation (CGH) Introduction Pre-implantation genetic diagnosis (PGD) refers to the genetic profiling of embryos before they are implanted and at times profiling of oocytes before they are fertilized (Munne, 2012, p.463). When used in screening for specific genetic disease, the key advantage of PGD is that the method avoids selective termination of pregnancy because the technique ensures that there is a high likelihood that the baby is free of the disease under consideration (Tan et al, 2014, P.2). In PGD, in vitro fertilization (IVF) is used to obtain oocytes or embryos for examination. PGD is an adjunct to assisted reproductive technology. On the other hand, pre-implantation genetic screening PGS represents procedures that do not screen specific diseases but utilize PGD techniques in determining embryos at risk. The PGD procedure enables DNA of eggs or embryos to be studied in order to choose the embryos that have specific mutations for genetic diseases (Coco, 2014, 272). Next Generation Sequencing (NGS) and comparative genome hybridisation (CGH) are new technologies used for all-inclusive chromosome testing of IVF embryos. In particular, NGS has become cost-effective, accurate and comprehensive genetic analysis method in non-invasive prenatal diagnosis and it is also increasingly being used in both PGD and PGS (Zheng et al, 2015, p.4). According to Tan et al (2014, p.2) NGS and CGH for Pre-implantation genetic diagnosis are likely to replace other technologies as the predominant techniques for pre-implantation genetic screening (PGS) because of their high accuracy and reduced costs. The focus of this essay is to assess the suitability next generation sequencing (NGS) and comparative genome hybridisation (CGH) for PGD/PGS and critically examine the theoretical advantages and evidence base that support the adoption of these technologies. Previous methods used with focus on FISH For a long time, PGD and PGS have been mostly conducted using PCR and FISH for the analysis. PCR is utilized for the diagnosis of single gene defects and this includes dominant and recessive disorders. PCR is a method where specific DNA sequence is copied several times to enable its analysis (Thornhill & Snow, 2002, p.9). As a result, PCR multiplies a single DNA molecule into billion molecules. However, PCR needs adequate quantities of pure and high-quality DNA samples, which at times can be a challenge to obtain from one cell. According to Thornhill & Snow (2002, p.9) for PGD analysis, PCR-based technique is used in analyzing the targeted gene within the DNA sample. Fluorescent in-situ hybridisation (FISH) is another technique that has been used PGD analysis. FISH has been utilized in translocation carriers (White et al, 2006, 354). FISH utilized chromosome-specific DNA probes in metaphase chromosomes or inter-phase nuclei. Further, FISH has been utilized in screening embryos for spontaneous chromosome aneuploidy or PGS with an aim of trying to enhance the efficacy of assisted reproduction. Scriven et al (2011, p.2) supports FISH as the method of choice in PGD and PGS and explains that FISH enumerates the specific chromosomes within inter-phase cells from pre-implantation embryos (Fauzdar et al, 2013, p.35). Because the organization of the DNA is complex within the inter-phase nuclei, FISH is in developing DNA probes for all reciprocal translocation to identify the imbalances. Thornhill & Snow (2002, p.10) further elaborates that FISH is utilized in sex determination for X-associated diseases, chromosomal abnormalities as well as aneuploidy screening. When FISH is used in evaluating the genetic make-up of an embryo, the embryos are grown to the Day 3 stage, and then one cell is obtained from every embryo. Attachment of cells to a glass side then follows, and packaging; the cells are then sent to the fertility clinic’ genetic testing laboratory for examination. The FISH method entails examining the DNA fragments that are specific to every chromosome. Probes are the put on the slide with the cell from the embryo. The cells then attach to the chromosome target (Scriven et al, 2011, p.3). Shortfalls and problems of FISH technique However, usage of FISH for screening embryos before transfer (PGS) has been questioned because of various reasons. First, evidence shows that FISH is not able to fully access all the chromosomes: a human cell has 23 pairs of chromosomes while FISH analysis is only able to do accurate assessment of just 10-12 chromosomes with every biopsied cell (Scriven et al, 2011, p.3). This implies that there is a probability of a number of abnormal embryos that are not capable to form a successful pregnancy, remaining undetected and might be transferred. Chromosomes that can be analyzed using FISH probes consist of X, Y, 1, 13, 16, 18, and 21. Lee et al (2014, p.18) adds that the main challenge with FISH is that the technique cannot allow analysis of more that selected number of chromosomes at a time. Lee et al (2014, p.20) also emphasizes that FISH-based testing only allows a limited number of chromosomes to be evaluated and explains that FISH only allows 5-12 chromosomes whereas there are 24 different chromosomes within a human cell. And because there are only 1 or 2 cells from a single day 3 embryos, it is hypothesized that the chromosomal makeup of 1 or 2 cells represents all the remaining cells within that embryo (Jensen, 2014, p.1350). This is worsened by the fact that there are high levels of chromosome abnormality as well as mosaicism (state where cells in the same individual have varying genetic make-up) during the cleavage stage embryos, and hence analysis of one cell might not represent the genetic status of the entire embryo (Assche et al, 1999, p.685). In addition, studies have shown that human embryos have a remarkable ability to self-correct where some abnormal cells during the cleavage phase embryo have been demonstrated to stop division, which leaves cell division to the normal cells and as a result a normal embryo is formed (Assche et al, 1999, p.685). According to (Scriven et al, 2011, p.3) another problem with using FISH is that day 3 embryo biopsy and PGS lowers the IVF success rates. This is because FISH technique normally analyses 5 chromosomes out of 3 and hence the FISH test misses numerous chromosomal abnormalities. This results to transfer of abnormal embryos along with the normal cells (Assche et al, 1999, p.685). In addition, the biopsies of day 3 removes 1 or 2 cells from a 6-10 cell embryo and this necessitates a comparatively big hole within the embryo shell and as a result a substantial percentage of the “biomass” of the embryo is removed, which may lead to the loss of important genetic makeup. In addition, evidence indicates that carrying out embryo biopsy on day 3 and conducting the genetic analysis using FISH technique does not result to an increased probability of patients having successful IVF cycle (Lee et al, 2014, p.20). Due to the shortcomings with FISH, CGH and Next Generation Sequencing (NGS) represent enormously superior platforms for PGS. NGS is a new technology used in comprehensive assessment of IVF embryos (Yang et al, 2015, p.3). Next generation sequencing (NGS) NGS refers to the DNA sequencing technique that has revolutionized genomic research because using this technique; it is possible to sequence an entire human genome in one day. According to Tan et al (2014, p.2), NGS technology facilitates unprecedented scalability and hence in PGS and PGD it enables identification and detection of high percentage of genetic mutations and abnormalities within the DNA (Goodwin et al, 2016, p.333). Evidence shows that NGS is not only precise and comprehensive, but also cost-effective and hence it is increasingly being used in human medicine, which include non-invasive prenatal diagnosis and well as in PGS and ODS applications (Behjati & Tarpey 2013, p. 238). According to Chiu et al (2008, p.460) there is evidence that shows that NGS can accurately detect aneuploid and unbalanced rearrangements in trophectodermal biopsies. This is supported by Dahdouh et al (2015, p. 452) who shows that NGS has been severally and successfully used in PGD. A case report that used NGS for PGD and PGS there was increased accuracy and there was increased detection rate of chromosomal abnormalities at a reduced cost. In addition, Behjati & Tarpey (2013, p. 238) explains that NGS is cost-effective when it comes to sequencing and also the accuracy has improved because of incessant technical developments, and this includes some bench-top sequencing platforms. NGS has the ability to attain high coverage of mtDNA. In this study, a comparative copy number of mtDNA for blastocysts using NGS testing was examined and a substantial variation between the copy number of mtDNA for the euploid blastocysts and the chromosomally abnormal blastocysts was identified. This demonstrated the ability of embryonic mitochondria analysis using NGS in evaluating the potential for embryonic development. Dahdouh et al (2015, p. 452) also found out that NGS-based PGD/PGS along with vitrification can be successfully applied within clinical practice. In this study, NGS was able to detect and identify some segmental imbalances that can be omitted using other techniques such as SNP, which can avoid probable risks of false signals. Behjati & Tarpey (2013, p. 238) opine that NGS-based PGD/PGS can be appropriate for detecting genetic abnormalities and can even provide services for the populations with genetic risk. Further advantages of next-generation sequencing (NGS) technique include the ability to produce large quantities of nucleic acid sequences within a comparatively short period (Behjati & Tarpey 2013, p. 238). Comparative Genomic Hybridization (CGH) In CGH, the nucleus of the embryo is labeled using florescent dye while labeling of the control cell line is done using another color, for example red or green. Comparison of the two colors is done and in case the chromosomal analysis indicates an excess of color red, this indicates that the embryo nucleus has an additional chromosome (Feichtinger et al, 2015, p.3). In case the chromosomal analysis indicates color green, this means that the embryo nucleus is missing within one of the chromosomes. CGH technique allows examination of 23 chromosomes and also provides a more comprehensive image of the entire length of the chromosome, and this can even identify and detect imbalance of chromosomal segments (Feichtinger et al, 2015, p.3). CGH technique takes about 72 hours and this implies that embryos should be first frozen to give the required time to obtain a diagnosis. CGH is thus successful in testing for chromosomal abnormalities where data points from DNA in the embryo samples is used in screening for additional or missing chromosomes and this is how the abnormal chromosomes are detected (Feichtinger et al, 2015, p.3). Currently, CGH complete karyotype chromosome analysis (23 pairs) of day 5 blastocyst-stage embryos is clinically available using Comparative Genomic Hybridization. The advantages of this technology is that it utilizes whole genomic amplification of the DNA from the embryo biopsy, and the fluorescent green labeling of the sample DNA follows and it is hybridized using normal DNA, which is labeled fluorescently red. Specialized software collects images and then a comparison between red and green for every chromosome, and as a result a molecular karyotype is generated (Russo et al, 2014, p.60). Accordingly, a CGH can detect and identify an “imbalance” within the chromosomal material and can also detect all trisomies and monosomies (aneuploidy) as well as some huge structural translocation imbalances. Further advantages of CGH include the ability of the technique to analyze many cells which leads to vastly accurate results (Aricò et al, 2014, p.8). In addition, in this technique also tests Trophectoderm cells (future placenta) and because not all cells involved within the formation of the fetus are analyzed, this can decrease any likely damage from the embryo biopsy (Munne, 2012, p.470). In addition, this method tests all 23 chromosome pairs and this results to complete chromosome analysis, unlike FISH which can only analyze 5 chromosome pairs. CGH solves the problem of inadequate analysis by assessing all 23 chromosome pairs, and this allows complete identification of the screened embryos and hence allows identification of the normal embryos and transferring (Russo et al, 2014, p.60). This technique is thus superior in that there is no likelihood of abnormal cells being left out with the normal cells during the analysis. In addition, in CGH, there is complete eradication of diagnostic errors allied to mosaicism. In addition, CGH provides a quantitative analysis that is based on comparison of the relative quantity of test DNA, for example a blastomere to the quantity of the known control DNA from a chromosomally normal person (Beijani & Shaffer, 2006, p.530). DNA from the two sources are identified and labeled differently and hybridized to probes. This makes this technique extremely accurate since numerous copies of every probe are placed on the microarray and every chromosome is tested at numerous distinct loci (Munne, 2012, p.470). Comparison between CGH and NGS In recent years, NGS has been used in gene analysis and research filed as well as in clinical diagnosis. Up to date, NGS has been predominantly effective in PGD analysis and also in detecting chromosomal aneuploidy (Russo et al, 2014, p.62). NGS has also been shown to be effective in prenatal diagnosis and also in chromosomal screening. Evidence shows that NGS can accurately detect aneuploid and unbalanced rearrangements in trophectodermal biopsies. NGS has also been severally and successfully used in PGD and has a high ability to detect chromosomal abnormalities (Russo et al, 2014, p.58). Similarly, CGH technique also has shown efficacy in detecting genetic abnormalities in PGD. Evidence shows that CGH method provides a very comprehensive picture of the chromosomes being analyzed and hence through this technique it is possible to identify and detect imbalance of chromosomal segments and abnormal genetic compositions (Russo et al, 2014, p.62). Furthermore, Yang et al (2015, p.6) explains that CGH has been successful in testing for chromosomal abnormalities where data points from DNA in the embryo samples and in missing chromosomes and this is how the abnormal chromosomes are detected. Dao, Agarwal & Nagy (2011, p.840) also conducted a study and found that the efficacy of CGH and NGS is high in PGD/PGS and also for chromosomal aneuploidy. In addition, evidence indicates that both NGS and CGH have been compatible in prenatal diagnosis. However, unlike CGH, in NGS it is possible to obtain clear clinical effects of chromosomal abnormalities regardless of the position of chromosome and the size of the micro-deletion/micro-duplication. In addition, with NGS it is again possible to obtain clear effects of chromosomal abnormities and at the same time sequence analysis of the similar locus on the other allele. This ensures that any potential patho-genetic SNPs are excluded and this is not possible with CGH. Moreover, NGS technologies also deliver huge amounts of genomic information allows many aspects to be analysed (Lin et al, p.502). A study conducted by Aleksandrova et al (2016, p.4) showed that for the first IVF birth that used NGS during PGS, the results showed NGS was efficient and safe and thus can be successfully use for pre-implantation screening. NGS also allows simultaneous screening of other genetic disorders and abnormalities due to the ability of the technique to perform an accurate single cell 24 chromosome aneuploidy screening (Treff et al, 2010, p. 2018). As a result, Aleksandrova et al (2016, p.5) argues that NGS is more effective and has more advantages than CGH because it reduces the cost for DNA sequencing, can detect the entire chromosome aneuploides and also NGS can extensively be used in choosing the best embryo for implantation due to its ability to detect segmental changes in the embryo. As Aleksandrova et al (2016, p.5) suggests, NGS allows IVF couples to extensively use PGS in selecting the most competent embryos for transfer and thus NGS-based PGS provides the best alternative to other available CCS techniques. Ethical/controversial issues with CGH and NGS in pre-natal diagnostics The key reason for prenatal diagnosis is to provide the family with information about the pregnancy to allow the outcome to be improved or the pregnancy to be terminated in case the pregnancy is severely affected. Even though ethical issues involved in CGH and NGS for pre-natal diagnosis are overlapped by ethical issues common in other diagnostic procedures, pre-natal diagnosis is made more complex by the controversy regarding the moral status of the fetus and choice of elective abortion as the type of treatment (Johnson & Elkins, 1988). While generally termination of pregnancies after 2nd trimester is justifiable in case the fetus has a fatal postnatal condition, the disposition of a fetus facing a non-life threatening condition such as Down’s syndrome brings more controversy. Johnson & Elkins (1988) further explains that more controversy comes in when women with positive screening results opt for elective abortion instead of undergoing an ultimate work-up. This brings up the ethical issue of maternal rights versus fetal rights. According to Jong et al (2010, p.275) the ethical dilemma is the conflict of either respecting maternal autonomy versus acting beneficently to the fetus. Another ethical dilemma is the requirement for using proxy decision makers for the fetus. Apart in circumstances where the mother is not in a position to make sound decisions, it is the duty of parents to be proxy decision makers. Johnson & Elkins (1988) further opines that CGH and NGS involves vast amount of information and as a result encompass findings that might be complex to interpret and explain. Unclear findings may lead to further research and extensive testing to an extent of even testing parents to determine the inheritance of abnormal results, and this might confront parents with unanticipated findings regarding themselves as well (Jong et al, 2010, p.275). According to Johnson & Elkins (1988) the ethical dilemma is whether such findings are beneficial or harmful to both the fetus and the parents. However, in this case the key ethical dilemma lies on terminating the pregnancy basing on unclear results which can result to unbearable guilt for the parents and in case the pregnancy is not terminated due to unclear findings there is anxiety if the problem can persist postnatal period (Jong et al, 2010, p.275) In addition, CGH and NGS have been used in clinical diagnostics and this includes pre-natal diagnostics. Normally, single gene analysis is supposed to be limited to situations of low genetic heterogeneity ought to be given preference if there is a highly heterogeneous disease and in differential diagnosis. This shows the large amount of data that is used in these techniques (Precone et al, 2015, p.5). The major controversial issue regarding CGH and NGS techniques is the huge quantity of data that is generated and how it’s interpreted. The higher the genomic region that is analyzed, the higher the number of variants of uncertain implication identified (Precone et al, 2015, p.5). Another controversy and ethical issue with these techniques include the fact that incidental findings (mutations with known pathogenicity even though unrelated to the medical condition whose test was being performed) (Clarke, 2014, p.18). The key ethical issue here is if the patient is supposed to be informed regarding the incidental findings. However, it is noteworthy that a good patient-informed consent process is supposed to be a component of the pre-test genetic counseling in order to ensure patients are prepared for such types of outcomes and enquire for their concerns about the knowledge of the findings (Brownstein et al, 2012, p.4). The issue of incidental findings also brings the controversy of whether the patient can re-contacted in future with new interpretations of the incidental findings. As a result, there is a hot debate on whether a consensus should be established regarding the “incidental information” that should be disclosed to the patient (Clarke, 2014, p.20). Finally, there is controversy of whether IFs or VUSs are supposed to be revealed to patients after the performance of a genome/exome sequence or if the results of any potential relevance ought to be always revealed. Management of this issue has significant connotations for the preliminary explanation of the test to patients and the consent process. The general assumption is that all sequence information is supposed to be stored, even though this might not be sustainable or practical (Clarke, 2014, p.20). Conclusion The essay has tackled different methods used in Pre-implantation genetic diagnosis (PGD) and PGS. For a long time, FISH technique has been used in trans-locating carriers and in screening embryos for spontaneous chromosome aneuploidy or PGS in order to improve the efficiency of assisted reproduction. However, FISH technique has its shortfalls that include the technique is unable to access all chromosomes during analysis because the technique can only analyze about 10-12 chromosomes while a human cell has 23 pairs of chromosomes. This shows that there is a potential of some abnormal cells not being detected. Shortfalls with FISH technique have led to the development of Next Generation Sequencing (NGS) and Comparative Genome Hybridisation (CGH) techniques that are more effective and successful in detecting all abnormalities during the testing IVF embryos. For example, evidence shows that NGS is extremely cost effective, accurate and also comprehensive in prenatal diagnosis. CGH has also been shown to be effective in prenatal diagnosis. Comparison of the two techniques (Next Generation Sequencing (NGS) and Comparative Genome Hybridisation (CGH)) shows that both are effective in detecting abnormalities and in prenatal diagnosis, although NGS is more effective because with NGS it is again possible to obtain clear effects of chromosomal abnormities and at the same time sequence analysis of the similar locus on the other allele. This ensures that any potential patho-genetic SNPs are excluded and this is not possible with CGH. However, these techniques have not been without controversy and ethical issues. The main ethical issues and controversy is about the large data involved and the issue whether “incidental information” should be disclosed to the patient or not. Reference list Aleksandrova N, Shubina E, Ekimov A et al, 2016, Comparison of the results of preimplantation genetic screening obtained by a-CGH and NGS methods from the same embryos, Gynecological Endocrinology , 32,(2). Assche E, Staessen C, Vagetti W, Bonduelle M, Vandervorst M & Inge L, 1999, Preimplantation genetic diagnosis and sperm analysis by fluorescence in-situ hybridization for the most common reciprocal translocation, Mol Hum Reprod, 5 (7), pp: 682-690. Aricò A, Ferraresso S, Bresolin S, Marconato L, Comazzi S, Te Kronnie G, et al., 2014, Array-Based Comparative Genomic Hybridization Analysis Reveals Chromosomal Copy Number Aberrations Associated with Clinical Outcome in Canine Diffuse Large B-Cell Lymphoma, PLoS ONE. 9(11):e111817.  http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0111817 Beijani B & Shaffer L, 2006, Application of Array-Based Comparative Genomic Hybridization to Clinical Diagnostics, J Mol Diagn, 8(5), pp: 528–533. Brownstein Z, Bhonker Y & Avraham K, 2012, High-throughput sequencing to decipher the genetic heterogeneity of deafness, Genome Biology, 13(245). Behjati S & Tarpey P, 2013, What is next generation sequencing? Arch Dis Child Educ Pract Ed, 98(6), pp: 236–238. Chiu RW, Chan KC, Gao Y, et al, 2008, Noninvasive prenatal diagnosis of fetal chromosomal aneuploidy by massively parallel genomic sequencing of DNA in maternal plasma, Proc Natl Acad Sci, 105(120), pp:458–463. Clarke A, 2014,Managing the ethical challenges of next-generation sequencing in genomic medicine, Br Med Bull, 111 (1), pp: 17-30. Coco R, 2014, Reprogenetics: Preimplantational genetics diagnosis, Genet Mol Biol, 37(1), pp: 271–284. Dao K, Agarwal A & Nagy, 2011, Preimplantation genetic screening: does it help or hinder IVF treatment and what is the role of the embryo? J Assist Reprod Genet, 28(9), pp: 833–849. Dahdouh E, Balayla J & Audiber F, 2015, Preimplantation Genetic Diagnosis and Screening, J Obstet Gynaecol Can,37(5):451–463. Fauzdar A, Chowdhry M, Makroo R, Mishra M, et al, 2013, Rapid-prenatal diagnosis through fluorescence in situ hybridization for preventing aneuploidy related birth defects, Indian J Hum Genet, 19(1): 32–42. Feichtinger M, Stopp T, Göbl C, Feichtinger E, Vaccari E, Mädel U, et al., 2015, Increasing Live Birth Rate by Preimplantation Genetic Screening of Pooled Polar Bodies Using Array Comparative Genomic Hybridization, PLoS ONE, 10(5): e0128317. https://doi.org/10.1371/journal.pone.0128317 Goodwin S, McPherson J & McCombie R, 2016, Coming of age: ten years of next-generation sequencing technologies, Nature Reviews Genetics, 1(17), pp: 333–351. Jensen E, 2014, Technical Review: In Situ Hybridization, View issue TOC, 297(8), pp: 1349–1353. Jong A, Wybo D, Frints S & Wert G, 2010, Non-invasive prenatal testing: ethical issues explored, Eur J Hum Genet, 18(3), pp: 272–277. Johnson S & Elkins T, 1988, Ethical issues in prenatal diagnosis, Clin Obstet Gynecol, 31(2), pp: 408-17. Lee V, Chow J, Lau E, Yeung W & Ernest N, 2014, Comparison between fluorescent in-situ hybridisation and array comparative genomic hybridisation in preimplantation genetic diagnosis in translocation carriers, Hong Kong Med J, 21(1), pp:16–22. Lin Z, Farooqui A, Li G, Wong G, Mason A, Banner D, Kelvin A, Kelvin D & Leon A, 2014, Article Next-generation sequencing and bioinformatic approaches to detect and analyze influenza virus in ferrets, J Infect Dev Ctries, 8(4), pp:498-509. < http://www.jidc.org/index.php/journal/article/view/24727517/1048> Munne S, 2012, Preimplantation Genetic Diagnosis for Aneuploidy and Translocations Using Array Comparative Genomic Hybridization, Curr Genomics, 13(6), pp: 463–470. Precone V, Monaco V, Esposito M, Palma F & Argenio V, 2015, Cracking the Code of Human Diseases Using Next-Generation Sequencing: Applications, Challenges, and Perspectives, BioMed Research International, 2015 (2015), pp:1-15. Russo C, Giacomo G, Claudio G, Padula F, Megan M et al, 2014, Comparative study of aCGH and Next Generation Sequencing (NGS) for chromosomal microdeletion and microduplication screening, J Prenat Med, 8(3-4), pp: 57–69. Scriven, P, Kirby T, Ogilvie C, 2011, FISH for Pre-implantation Genetic Diagnosis, J. Vis. Exp, (48), e2570, doi:10.3791/2570. Tan Y, Yin X, Zhang S, Jiang H, Tan K, Li J, Gong F et al, 2014, Clinical outcome of preimplantation genetic diagnosis and screening using next generation sequencing, Gigascience, 3(30). Thornhill A & Snow K, 2002, AMolecular Diagnostics in Preimplantation Genetic Diagnosis, J Mol Diagn, 4(1): 11–29. Treff NR, Su J, Tao X, et al. Accurate single cell 24 chromosome aneuploidy screening using whole genome amplification and single nucleotide polymorphism microarrays. Fertil Steril 2010;94:2017–21. White H, Levkov L, Malmgren H et al, 2006, PGD for dystrophin gene deletions using fluorescence in situ hybridization, Mol Hum Reprod,12 (5), pp: 353-356. Yang Z, Lin J, Fong W, Li P, Zhao R et al, 2015, Randomized comparison of next-generation sequencing and array comparative genomic hybridization for preimplantation genetic screening: a pilot study, BMC Medical Genomics,8(30). Zheng H, Jin H, Liu J & Wang W, 2015, Application of next-generation sequencing for 24-chromosome aneuploidy screening of human preimplantation embryos, Mol Cytogenet, 8(38). Read More

Previous methods used with focus on FISH For a long time, PGD and PGS have been mostly conducted using PCR and FISH for the analysis. PCR is utilized for the diagnosis of single gene defects and this includes dominant and recessive disorders. PCR is a method where specific DNA sequence is copied several times to enable its analysis (Thornhill & Snow, 2002, p.9). As a result, PCR multiplies a single DNA molecule into billion molecules. However, PCR needs adequate quantities of pure and high-quality DNA samples, which at times can be a challenge to obtain from one cell.

According to Thornhill & Snow (2002, p.9) for PGD analysis, PCR-based technique is used in analyzing the targeted gene within the DNA sample. Fluorescent in-situ hybridisation (FISH) is another technique that has been used PGD analysis. FISH has been utilized in translocation carriers (White et al, 2006, 354). FISH utilized chromosome-specific DNA probes in metaphase chromosomes or inter-phase nuclei. Further, FISH has been utilized in screening embryos for spontaneous chromosome aneuploidy or PGS with an aim of trying to enhance the efficacy of assisted reproduction.

Scriven et al (2011, p.2) supports FISH as the method of choice in PGD and PGS and explains that FISH enumerates the specific chromosomes within inter-phase cells from pre-implantation embryos (Fauzdar et al, 2013, p.35). Because the organization of the DNA is complex within the inter-phase nuclei, FISH is in developing DNA probes for all reciprocal translocation to identify the imbalances. Thornhill & Snow (2002, p.10) further elaborates that FISH is utilized in sex determination for X-associated diseases, chromosomal abnormalities as well as aneuploidy screening.

When FISH is used in evaluating the genetic make-up of an embryo, the embryos are grown to the Day 3 stage, and then one cell is obtained from every embryo. Attachment of cells to a glass side then follows, and packaging; the cells are then sent to the fertility clinic’ genetic testing laboratory for examination. The FISH method entails examining the DNA fragments that are specific to every chromosome. Probes are the put on the slide with the cell from the embryo. The cells then attach to the chromosome target (Scriven et al, 2011, p.3). Shortfalls and problems of FISH technique However, usage of FISH for screening embryos before transfer (PGS) has been questioned because of various reasons.

First, evidence shows that FISH is not able to fully access all the chromosomes: a human cell has 23 pairs of chromosomes while FISH analysis is only able to do accurate assessment of just 10-12 chromosomes with every biopsied cell (Scriven et al, 2011, p.3). This implies that there is a probability of a number of abnormal embryos that are not capable to form a successful pregnancy, remaining undetected and might be transferred. Chromosomes that can be analyzed using FISH probes consist of X, Y, 1, 13, 16, 18, and 21.

Lee et al (2014, p.18) adds that the main challenge with FISH is that the technique cannot allow analysis of more that selected number of chromosomes at a time. Lee et al (2014, p.20) also emphasizes that FISH-based testing only allows a limited number of chromosomes to be evaluated and explains that FISH only allows 5-12 chromosomes whereas there are 24 different chromosomes within a human cell. And because there are only 1 or 2 cells from a single day 3 embryos, it is hypothesized that the chromosomal makeup of 1 or 2 cells represents all the remaining cells within that embryo (Jensen, 2014, p.1350). This is worsened by the fact that there are high levels of chromosome abnormality as well as mosaicism (state where cells in the same individual have varying genetic make-up) during the cleavage stage embryos, and hence analysis of one cell might not represent the genetic status of the entire embryo (Assche et al, 1999, p.685). In addition, studies have shown that human embryos have a remarkable ability to self-correct where some abnormal cells during the cleavage phase embryo have been demonstrated to stop division, which leaves cell division to the normal cells and as a result a normal embryo is formed (Assche et al, 1999, p.685).

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