Preimplantation Genetic Screening: Comparative Genomic Hybridization (CGH)

Abstract: Comparative Genomic Hybridization (CGH) as a valuable alternative to Fluorescence In Situ Hybridization (FISH) for embryo Preimplantation Genetic Screening (PGS). 

Nowadays, after some studies have recently questioned the validity of Preimplantation Genetic Screening (PGS) using Fluorescence In Situ Hybridization (FISH), there is an unsolved essential dilemma what method is the most accurate, transparent and inclusive for comprehensive chromosome screening: Fluorescence In Situ Hybridization (FISH) or Comparative Genomic Hybridization (CGH)? This essential dilemma outlines the controversy between the experts’ opinions: Fluorescence In Situ Hybridization (FISH) versus Comparative Genomic Hybridization (CGH)? Which method should be used for comprehensive chromosome screening? What are the basic indicators for every method? What consequences entail embryo development in the future the comprehensive chromosome screening using Fluorescence In Situ Hybridization (FISH)? What consequences entail embryo development in the future the comprehensive chromosome screening using Comparative Genomic Hybridization (CGH)? Consequently, these both methods should be investigated with further outlining of their accuracy and efficiency. This dilemma is continuously being discussed among embryologists. Consequently, this dilemma presupposes: 

1. Substantiating the status of Fluorescence In Situ Hybridization (FISH) and Comparative Genomic Hybridization (CGH); 
2. Analyzing the functional peculiarities of both methods;
3. Comparison of accuracy and efficiency of both methods for establishing the positive and negative aspects of both methods.

Introduction

In Vitro Fertilization (IVF) Treatment cycles usually involve the production of multiple embryos. One of the most important and unsolved problems in In–Vitro Fertilization is to decide which embryos are more suitable to implant and therefore should be transferred and which embryos should be cryopreserved for further usage. Synchronically (in the time–aspect) with top–quality embryo selection process, the use of sequential, stage–specific media combined with low–oxygen tension culture systems and the introduction of vitrification strategy in IVF have permitted blastocyst culture and Cryopreservation to be accomplished with high efficiency [McArthur et al., 2005; Schoolcraft et al., 2010]. However, the viability of individual embryos is highly variable. Even among a cohort of sibling embryos competence can vary greatly. The great challenge for IVF clinics is to correctly identify the most viable embryos and prioritize them for further transfer to the uterus, especially in cases when it is vital to exclude the embryos with chromosome abnormalities. Currently, the decision of which embryo(s) to transfer is made on the basis of morphologic assessments conducted in the IVF laboratory. Blastomere number, size and shape, and the presence or absence of extracellular fragments are the relevant characteristics that form the current basis for non–invasive evaluations of developmental competence [Giorgetto et al., 1995]. It has already been demonstrated that the majority of pregnancies result from the transfer of good morphology embryos with the expected number of blastomeres; however, only a few of those embryos implant and develop successfully.

The weakness of correlation between conventional methods of embryo evaluation and chromosomal complement has lead the introduction of Preimplantation Genetic Screening (PGS) as a complementary Treatment during In Vitro Fertilization (IVF) cycles to avoid the transfer of aneuploid embryos and improve the delivery rate per transfer cycle [Munné et al., 1993]. It is of paramount importance that the method of Preimplantation Genetic Screening (PGS) is accurate and that the technique itself does not diminish the developmental potential of the individual embryo tested [Northrop et al., 2010; Treff et al., 2010a; Fragouli and Wells, 2012].

Preimplantation Genetic Screening (PGS) is currently applied to evaluate the presence of aneuploidies in embryos of couples at risk of occurrence the chromosome abnormalities, i.e. Advanced Maternal Age (AMA), Recurrent Miscarriage (RM), recurrent IVF failure or severe male factor [Donoso et al., 2007]. The technique routinely used in IVF laboratories for PGS is Fluorescence In Situ hybridization (FISH), which is a fast and easy method to perform. Until recently, Fluorescent In Situ Hybridization (FISH) of blastomeres, biopsied at the cleavage stage, was thought to be the best method for screening preimplantation embryos for numerical chromosome abnormalities, a major presumed factor causing low pregnancy rates in medically Assisted Reproduction. Nowadays, after some studies have recently questioned the validity of Preimplantation Genetic Screening (PGS) using Fluorescence In Situ Hybridization (FISH), there is an unsolved essential dilemma what method is the most accurate, transparent and inclusive for comprehensive chromosome screening: Fluorescence In Situ Hybridization (FISH) or Comparative Genomic Hybridization (CGH)? This essential dilemma outlines the controversy between the experts’ opinions: Fluorescence In Situ Hybridization (FISH) versus Comparative Genomic Hybridization (CGH)? Which method should be used for comprehensive chromosome screening? What are the basic indicators for every method? What consequences entail embryo development in the future the comprehensive chromosome screening using Fluorescence In Situ Hybridization (FISH)? What consequences entail embryo development in the future the comprehensive chromosome screening using Comparative Genomic Hybridization (CGH)? Consequently, these both methods should be investigated with further outlining of their accuracy and efficiency. This dilemma is continuously being discussed among embryologists. Consequently, this dilemma presupposes: 

1. Substantiating the status of Fluorescence In Situ Hybridization (FISH) and Comparative Genomic Hybridization (CGH); 
2. Analyzing the functional peculiarities of both methods; 
3. Comparison of accuracy and efficiency of both methods for establishing the positive and negative aspects of both methods.

Embryos are prone to chromosomal abnormalities, mainly due to age–dependent chromosome segregation errors during meiosis I [Battaglia et al., 1996]. The consequences of chromosomal imbalance or, in other words, chromosomal abnormalities could cause miscarriage (early pregnancy loss) or severe chromosomal diseases [Munné 2006]. High embryonic losses occur during cleavage with, at best, only 50% in vitro fertilized oocytes competent to reach the blastocyst stage [Hardy et al., 2002]. Aneuploidy is the leading cause of implantation failure and early spontaneous abortion of the embryo [Macklon et al., 2002; Spandorfer et al., 2004; Menasha et al., 2005], but does not represent a strong negative selection barrier for embryo preimplantation development to the blastocyst stage [Alfarawati et al., 2011].

Chromosomal abnormalities can be prevented in In Vitro Fertilization (IVF) therapy by performing Preimplantation Genetic Screening (PGS) of all 24 chromosomes. Preimplantation Genetic Screening (PGS) is widely used to select in vitro–fertilized embryos free of chromosomal abnormalities and to improve the clinical outcome of In Vitro Fertilization (IVF) Treatment cycle. Chromosome analysis and the interpretation of the results can be challenging and the terminology confusing. There are now a number of fundamentally different techniques in routine use, including karyotyping, Fluorescence In Situ Hybridization (FISH), Comparative Genomic Hybridization, microarray (BAC and oligonucleotide aCGH, SNP microarray) and multiple ligation–dependent probe amplification. Specific systems of international nomenclature have been developed to cope with them and to improve and maintain international collaboration [Shaffer et al., 2009]. Multiple clinical trials have confirmed the clinical efficacy of Preimplantation Genetic Screening (PGS), including increasing implantation and clinical pregnancy rates, as well as decreasing miscarriage rates [Yang et al., 2012; Forman et al., 2013; Keltz et al., 2013; Scott et al., 2013].

With the development of Comprehensive Chromosome Screening (CCS) platforms, the promise of aneuploidy screening is becoming a reality in Assisted Reproduction Technologies [Voullaire et al., 2000; Wells and Delhanty, 2000; Fishel et al., 2010; Harper and Harton, 2010; Schoolcraft et al., 2010; Treff et al., 2010b; Gutiérrez–Mateo et al., 2011]. The main disadvantage of Preimplantation Genetic Screening (PGS) is that it requires biopsy
of the preimplantation embryo, which can limit the clinical applicability of Preimplantation Genetic Screening (PGS) due to the invasiveness and complexity of the process.

Comparing the above–mentioned Preimplantation Genetic Screening (PGS) methods for transparent and Comprehensive Chromosome Screening (CCS) currently in clinical use, what is essential to mention is although mosaicism has been reported to exist in preimplantational embryos, affecting the accuracy of Preimplantation Genetic Screening (PGS) [Munne et al., 1994; Bielanska et al., 2002b; Li et al., 2005], another reason why the implantation rate is not improved by Preimplantation Genetic Screening (PGS) using FISH is probably the fact that the whole set of chromosomes is not analyzed and aneuploidies in chromosomes that are not screened could be hampering the implantation or development of transferred embryos. Comparative Genomic Hybridization (CGH) is a well–established procedure which allows for the study of all chromosomes simultaneously. The main advantage of Comparative Genomic Hybridization (CGH) over FISH is that the whole chromosomal complement can be ascertained and this also allows for the detection of not only chromosomal imbalances generated by aberrant segregation but also structural imbalances of fragments larger than 10–20 Mb [Griffin et al., 1998; Malmgren et al., 2002]. However, Comparative Genomic Hybridization (CGH) is a rather labour–intensive technique and it takes ∼4 days to obtain results. For this reason, two strategies have been developed to enable its application in PGD. The first strategy is the use of the first polar body (1PB) [Wells et al., 2002; Sher et al., 2007; Obradors et al., 2008, 2009] or the first and second polar bodies (1PB and 2PB) [Fragouli et al., 2010] as indirect indicators of the oocyte’s chromosomal complement, in which case, only maternal contribution of first or first and second meiotic division, respectively, is determined and paternal meiotic and post–zygotic errors cannot be detected. In order to obtain a more complete analysis, a second strategy has been developed, based on the Comparative Genomic Hybridization (CGH) analysis of one blastomere from Day–3 embryos [Wilton et al., 2001; Voullaire et al., 2002; Sher et al., 2009] or of several cells removed from the trophectoderm at the blastocyst stage [Fragouli et al., 2010; Schoolcraft et al., 2010]. Nevertheless, owing to the duration of Comparative Genomic Hybridization (CGH), these two approaches require the cryopreservation of the biopsied embryos and the transfer of those diagnosed as euploid in a subsequent cycle.

Therefore what is vital to mention is that the scientists Rius M., Obradors A., Daina G., Cuzzi J., Marquès L., Calderón G., Velilla E., Martínez–Passarell O., Oliver–Bonet M. and Benet J. in their investigation attempted to develop and validate a Comparative Genomic Hybridization (CGH) variant conserving all advantages of the conventional Comparative Genomic Hybridization (CGH), including its cost, but avoiding the cryopreservation of the embryos, making the Comparative Genomic Hybridization (CGH) methodology suitable for Preimplantation Genetic Screening (PGS) of Day–3 embryos, without the need of Cryopreservation [Rius M. et al., 2010].

1. The essence of the Comparative Genomic Hybridization (CGH) methodology represented in the study “Reliability of short Comparative Genomic Hybridization in fibroblasts and blastomeres for a comprehensive aneuploidy screening: first clinical application” written by Rius M., Obradors A., Daina G., Cuzzi J., Marquès L., Calderón G., Velilla E., Martínez–Passarell O., Oliver–Bonet M., Benet J. 

Isolation and lysis of fibroblasts and blastomeres

A drop of a highly diluted cell suspension from a confluent culture was placed on a small plate under a stereoscopic microscope and a single fibroblast was isolated using a 170–μm denuding pipette. The fibroblast was washed in four Phosphate–Buffered Saline (PBS)/0.1% Polyvinyl Alcohol (PVA) droplets and transferred to a Polymerase Chain Reaction (PCR) tube. For the embryos, the zona pellucida was removed using Tyrode’s acid or pronase (3 mg/ml) and blastomeres were isolated, washed and stored in the same way as fibroblasts. A minimum of two blastomeres were obtained from eight of the embryos and a single blastomere was obtained from two of the embryos. Every Polymerase Chain Reaction (PCR) tube containing either one fibroblast or one blastomere was properly coded with a different number from the original, so that the Comparative Genomic Hybridization (CGH) analysis was conducted blindly [Rius M. et al., 2010].

Although single fibroblasts from a female were used as reference in the fibroblast lines analyses, single lymphocytes from a male were used in the blastomeres experiments. Both fibroblasts and lymphocytes were obtained from confluent cultures and stored individually in Polymerase Chain Reaction (PCR) tubes [Rius M. et al., 2010].

Lysis of fibroblasts, blastomeres or lymphocytes were performed adding 1 µl of sodium dodecyl sulphate (17 µM) and 2 µl of proteinase K (125 µg/ml) to each sample, this was overlaid with light mineral oil, and the tubes were incubated at 37°C for 1 hour, followed by 10 minutes at 95°C to inactivate proteinase K [Rius M. et al., 2010].

Whole genome amplification 

Single cell DNA was amplified using degenerate oligonucleotide primed Polymerase Chain Reaction (PCR) (DOP–PCR) as previously described [Gutierrez–Mateo et al., 2004a] but in this case a shortened reaction, with fewer Polymerase Chain Reaction (PCR) cycles, was tested to reduce the processing time from 5 hours to 3 hours 40 minutes: the sample was heated to 95°C for 5 minutes; 10 cycles of 95°C for 1 minutes, 30°C for 1.5 minutes and 68°C for 3 minutes; 20 cycles of 95°C for 1 minute, 62°C for 1 minute and 68°C for 2.5 minutes with a final extension step of 68°C for 7 minutes. The Polymerase Chain Reaction (PCR) programme was carried out in a Gradient thermocycler (Biometra, Goettingen, Germany) and electrophoresis on a 1.5% agarose gel was used to evaluate the correct DNA amplification (smear between 200 and 4000 bp) of each sample [Rius M. et al., 2010].

Labelling and precipitation of probes

Whole genome amplification products were fluorescently labelled by nick translation, with a step of 15°C for 1 hour 30 minutes and another step of 70°C for 10 minutes. Test DNA (fibroblasts or blastomeres) was labelled with Spectrum Red–dUTP, whereas reference DNA (fibroblasts or lymphocytes) was labelled with Spectrum Green–dUTP. In order to obtain a homogeneous control DNA probe, different reference DNAs were mixed after their individual labelling. After nick translation, reference and test DNA were mixed in equimolar proportions and ethanol precipitated with 10 µg of Cot–1–DNA. The precipitation process took 1 hour at −80°C and 30 minutes of centrifugation. The pellet was dried and dissolved in 8 µl of hybridization mixture (50% formamide, 2× standard saline citrate, 10% dextran sulphate, pH 7) [Rius M. et al., 2010].

Comparative Genomic Hybridization (CGH)

Hybridization was performed on normal male (46, XY) metaphase spreads with some modifications of a previous method [Gutierrez–Mateo et al., 2004a], including a microwave step and agitation of the slides during hybridization. Immediately after a 30–minutes exposure in a 75 W microwave in humid conditions, a reduction in the hybridization step, from 72 hours to 12 hours, was achieved when the slides were kept in a moist chamber at 37°C with rotary agitation (65 rpm/min) for the whole incubation period, using a 1309–1CE rotator [Rius M. et al., 2010].

Image capture and Comparative Genomic Hybridization (CGH) analysis

The capture of metaphases was performed with a Nikon eclipse 90i epifluorescence microscope. An average of 12 metaphases per cell was captured and evaluated using Isis CGH software developed by MetaSystems. The ratio between red and green fluorescence is 1:1 when there is the same proportion of reference and test DNA. The thresholds used to diagnose losses and gains were 0.8 and 1.2, respectively. Deviations of the ratio within the threshold cut-off of 0.8 or 1.2 were also taken into account to evaluate the sensitivity of the technique [Rius M. et al., 2010].

Criterion of Сomparative Genomic Hybridization (CGH) analysis

It is important to mention that chromosomes that are potentially gained or lost artifactually, i.e. chromosomes 17, 19 and 22 [Moore et al., 1997; Voullaire et al., 2002; Gutierrez–Mateo et al., 2004a] were excluded from analysis when all three chromosomes were simultaneously gained or lost in the same cell; otherwise they were considered as being real aneuploidies [Rius M. et al., 2010].

In the present work, there is no distinction between chromosome or chromatid gain or loss has been considered because, in our experience analyzing 1PBs and their corresponding metaphase II (MII) using CGH and FISH, respectively, the CGH loss or gain profiles of the 1PBs were indistinguishably equivalent to losses or gains of either chromosome or chromatid in the MIIs [Gutierrez–Mateo et al., 2004b].

Validation of 12-h hybridization results

After whole genome amplification of single cell DNA, the product can be divided and labelled, obtaining two sets of nick translation products. We used one set to perform the 12 hours hybridization and the other to validate the short–CGH results using the standard, 72 h–CGH [Rius M. et al., 2010].

Modifications in the standard Comparative Genomic Hybridization (CGH) Protocol

In isolated fibroblasts, the application of the shortened whole genome amplification procedure (3 hours 40 minutes DOP–PCR) yielded an amount of DNA product similar to that obtained with the standard one (5 hours DOP–PCR). Only 2 out of 32 fibroblasts failed to give any smear in the 1.5% agarose gel, so the amplification efficiency was 93.75% [Rius M. et al., 2010].

High–quality, homogeneous hybridizations were obtained when the short–CGH variant (12 hours of hybridization) was applied to isolated fibroblasts using DNA probes obtained after 3 hours 40 minutes DOP–PCR. Consequently, that led to reliable CGH profiles with very few deviations, showing the high sensitivity of the modified technique, which was similar to the one obtained using the standard, 72 h–CGH. The short–CGH allowed the detection of all the characteristic aneuploidies in each cell line in 100% of the single fibroblasts (n = 30) analysed. No other aneuploidies or imbalances were observed [Rius M. et al., 2010].

After developing the short procedure for single fibroblasts it was tested for isolated blastomeres. All the methodological modifications were also validated in single blastomeres, except for the shortened whole genome amplification. The 3 hours 40 minutes DOP–PCR was not as successful as in fibroblasts; for this reason, the 5–hour DOP–PCR was kept for isolated blastomeres, as in the standard CGH procedure [Rius M. et al., 2010].

Agreement between short–CGH and FISH

The short–CGH procedure was applied in a total of 48 isolated blastomeres from 10 embryos, which were discarded after PGS by FISH with nine chromosome probes (9–chr–FISH: 13, 15, 16, 17, 18, 21, 22, X and Y). In all 48 blastomeres the standard 72 h–CGH and short–CGH gave the same results, therefore it was considered that total agreement was found between the two approaches. Reanalysis of blastomeres which had discordant results was performed by re–hybridization with telomeric FISH probes for particular chromosomes involved in the disagreement (blastomeres 5.0, 6.0, 7.0, 8.0 and 9.0), and the results were in total agreement with short–CGH results [Rius M. et al., 2010].

Aneuploidy detection by short–CGH (Comparative Genomic Hybridization)

Although the first interpretation of FISH results had led to the conclusion that all discarded embryos were aneuploid, at least for the analyzed blastomere (except for embryo 4, which was rejected for having stopped its development), out of the 48 blastomeres evaluated by short–CGH, 14 blastomeres (29.2%) belonging to three embryos did not present any aneuploidy and 34 blastomeres (70.8%) were aneuploid. The chromosomes most frequently involved in aneuploidy were 22 and 16, but also aneuploidies for chromosomes 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 14, 15, 17, 18, 19, 20, 21, X and Y were detected. Overall, the scientists found 94 aneuploid events, 41 (43.6%) of them corresponding to chromosomes which are not analyzed by 9–chr–FISH. Mosaicism was found in five of the eight embryos that had more than one blastomere analyzed (embryos 1, 2, 3, 5 and 9). Complementary aneuploidy events between two blastomeres of the same embryo were observed (blastomeres 1.5 and 1.6, blastomeres 3.1 and 3.2 and blastomeres 9.2 and 9.10). Moreover, the comprehensive analysis of embryo three showed a chaotic segregation [Rius M. et al., 2010].

Reliability of short-CGH (Comparative Genomic Hybridization) analysis

In 41.6% (20/48 blastomeres) of the analyzed blastomeres concordance was found when comparing short–CGH results with 9–chr–FISH. However, in six of them, the CGH procedure allowed for the detection of aneuploidies in chromosomes which were not analyzed by FISH. Partial agreement was found in 22.9% (11/48) of the studied blastomeres, since either not all of the aneuploidies diagnosed by 9–chr–FISH were corroborated by short–CGH, or aneuploidies that could have been detected by 9–chr–FISH were observed in the CGH analysis. Finally, discordance between short–CGH and 9–chr–FISH was observed in 35.4% (17/48) of the blastomeres, as none of the diagnosed aneuploidies were corroborated by short–CGH. At that point, apart from a possible CGH error, originating from overamplification of some single cell genomic regions (which in fact were not observed when aneuploid fibroblasts were previously analyzed), the following two considerations about those contradictory results could be discussed: mosaicism and/or FISH error. Mosaicism is a phenomenon that has been observed extensively, as five of the eight embryos that had more than one blastomere analyzed were mosaic, and it has been widely reported, affecting about 30% of the cleavage–stage embryos [Munne et al., 1994]. On the other hand, the re–hybridization of discordant blastomeres with telomeric FISH probes revealed that discordance between the short–CGH and 9–chr–FISH results originated from FISH errors. In fact, it has been described that intrinsic FISH factors, such as chromosome overlapping or loss of micronuclei during fixation, may account for the diagnosis of false monosomies [Munne et al., 1996; Bahce et al., 2000] (i.e. blastomeres 5.0, 6.0 and 8.0). Otherwise, false trisomies (i.e. blastomeres 7.0 and 9.0) might be diagnosed as a result of split signals caused by the overlapping of similar fluorochromes [Munne et al., 1998]. The mentioned FISH errors produced an incorrect diagnosis that led to the discarding of embryos 7 and 8 for being aneuploid while, subsequently, by short–CGH they were diagnosed as being euploid. For this reason, it is important to consider the value of short–CGH versus FISH and the need for caution in the diagnosis [Rius M. et al., 2010].

Thus, the main source of error in a diagnosis by short–CGH would be embryo mosaicism, but contrary to what has been stated [Staessen et al., 2008], it does not imply that PGS success using CGH is similar to that using FISH. Although it is true that by analyzing more chromosomes it is possible to detect more aneuploidies that do not necessarily exist in the rest of the embryo, this is not a reason to reject a comprehensive and highly reliable analysis such as CGH. To date, the low implantation rate described after PGS [Mastenbroek et al., 2007; Hardarson et al., 2008; Staessen et al., 2008] has been observed in studies that used FISH as the screening method. Conversely, when PGS has been applied to the whole chromosome complement, even in couples with little chance of success, the implantation rate of transferred embryos has increased [Hellani et al., 2008]. More extensive series of PGS, analyzing the full karyotype in isolated blastomeres, are needed before this short–CGH approach is rejected or supported. But this is independent of the fact that mosaicism is one of the major causes of diagnostic errors and that it will never be entirely eliminated. In this way, this study is a preliminary validation of short–CGH results by 72 h–CGH and FISH reanalysis, which presents the short–CGH approach as a reliable technique to be applied in the aneuploidy analysis of blastomeres [Rius M. et al., 2010].

Conclusion

In conclusion, the scientists Rius M., Obradors A., Daina G., Cuzzi J., Marquès L., Calderón G., Velilla E., Martínez–Passarell O., Oliver–Bonet M., Benet J. postulated that a short–CGH single cell analysis has been successfully developed. Additionally it was noted that using the short–CGH procedure, all specific aneuploidies of the analyzed fibroblasts, and exclusively them, were identified. The short-CGH technique is a valuable alternative to FISH for predetermined chromosomes because it provides a complete aneuploidy screening of all chromosomes. Single fibroblast DNA amplification product obtained when DOP–PCR was shortened to 3 hours 40 minutes was similar to that obtained by the standard 5–hour DOP–PCR Protocol. Nonetheless, the key to success of the short protocol was the fact that the slides were kept in rotary agitation inside the humid chamber for the entire 12 hours incubation, after having treated them in the microwave at 75 W for 30 minutes. It was supposed that probably, rotary movement permits a better diffusion of probe molecules through complementary sequences of the denatured chromatin of lymphocyte metaphases in the slide, allowing for faster and more uniform hybridization. It was revealed that determination of male gender in isolated fibroblasts from male cell lines was considered as an indicator of in situ hybridization quality. That is, 12 metaphases per case were captured and 24 chromosomes were analyzed for each autosome, whereas only half of them were available for chromosomes X and Y. Thus, using fibroblast DNA from a female as reference, the loss of chromosome X and the gain of chromosome Y were detected analyzing only 12 chromosomes for each sex chromosome. Therefore, it meant CGH was performed successfully and high–quality was achieved [Rius M. et al., 2010].

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