Comprehensive chromosome analysis of blastocysts before implantation using array CGH

Background Chromosomal abnormalities are common in embryos produced in vitro and cause implantation failure, miscarriage, and serious medical problems in infants. Because preimplantation genetic screening (PGS) is increasingly being used to detect aneuploidy in embryos with the purpose of improving implantation rates after IVF (in vitro fertilization), we aimed to validate the usefulness of array CGH for the preimplantation genetic screening (PGS) of embryos at the blastocyst stage of development. Results A total of 150 blastocysts were biopsied from couples undergoing IVF and analyzed using array CGH. We found that 54.5% (73/134) of the blastocysts were euploid embryos, whereas 45.5% of the embryos (61/134) had chromosomal abnormalities. Multiple chromosome abnormality was most frequently observed (34.4%), and dual aneuploidy was observed in 26.2% of the embryos. Monosomy (21.3%) appeared more frequently than trisomy (18%). Conclusion Chromosomal microarray analysis provided clinically significant cytogenetic information regarding the frequency and variety of chromosomal abnormalities observed in embryos at the blastocyst stage, suggesting that this is a useful tool for comprehensive aneuploidy screening in IVF.


Background
During IVF procedures, a preimplantation genetic diagnosis (PGD) is used to eliminate embryos carrying genetic diseases prior to implantation. The first application of PGD was successfully performed for couples at risk for transmitting recessive X-linked diseases to male offspring [1], whereby polymerase chain reaction (PCR) was used to determine the sex of the embryos.
Unlike PCR methods, preimplantation genetic screening (PGS) aims to provide a means for identifying potentially viable euploid embryos i.e., screening that may improve pregnancy rates. PGS was first described by Verlinsky et al. [2] and Munne et al. [3]. Although previous methods for embryo screening used fluorescence in situ hybridization (FISH) to analyze chromosomes [4,5], the FISH approach is limited because the technique is unable to screen all chromosomes simultaneously. Conventional comparative genomic hybridization (CGH) has been used to comprehensively screen for aneuploidy in oocytes and embryos [6,7]. However, although useful for selecting euploid embryos, the CGH protocol is not generally used because it is time consuming and complicated. At present, both array CGH (aCGH) and single nucleotide polymorphism (SNP) arrays have been validated as accurate methods for producing comprehensive analyses of chromosome in embryos that are compatible with day-3 biopsies and day-5 replacements in a fresh cycle [8][9][10][11][12]. The difference in mosaicism between embryos at days 3 and 5 has led to a preference for biopsies at the blastocyst stage [13,14].
Here, we describe the results of an embryo analysis and the details of the chromosomal abnormalities found.
The details of the array CGH results derived from aneuploid embryos (n=61) are summarized in Table 2. The type of chromosomal abnormality that was most frequently observed was multiple chromosomal abnormality (34.4%), and the second most frequent was dual chromosomal abnormality (26.2%). Monosomy (21.3%) appeared more frequently than trisomy (18%). Examples of array CGH profiles are shown in Figure 1.
The chromosomes that were most frequently detected to have aneuploidy were, in order, 15, 22, 21, 16, and 18. Chromosomes 4 and 12 were the least frequently found to have aneuploidy.

Discussion
Preimplantation genetic screening for aneuploidy is increasingly used to examine the chromosomes of embryos from couples undergoing IVF [8][9][10]15,16]. The purpose of PGS is to identify embryos that are free from chromosomal abnormalities. The main indications for PGS are maternal age, repeated implantation failure, and repeated miscarriage. We examined 150 embryos from 49 couples with these indications.
As shown in Table 1, we successfully analyzed 89.3% (134/150) of the embryos and found that 45.5% (61/134) of the embryos contained abnormal chromosomes. Although the array CGH method is robust and specific, we observed some failure in amplification and a noisy profile. Some cells containing degraded DNA or samples of low quality resulting from apoptosis can be obtained during the biopsy procedure, causing experimental error.
The results showed excessive single chromosome loss versus single chromosome gain (Table 2); frequent abnormalities in chromosomes 15, 22, 21, 16, and 18; and rare aneuploidy in chromosomes 4 and 12, which are similar to the results of previous reports [17][18][19]. Although the frequency of chromosomal abnormalities varied, aneuploidy occurred in all of the 24 chromosomes (data not shown), suggesting that PGS is necessary for selecting healthy embryos during IVF procedures. Previous studies have shown the importance of screening embryos with improved pregnancy success as a result [19].
In conclusion, array CGH is a useful technique for the detection of chromosomal abnormalities during IVF procedures, as previously described [18]. However, embryo cultures up to days 5 or 6 should be established before performing array CGH experiments on blastocysts, and further evidence is required to determine whether PGS results in enhanced delivery rates [14].

Patient materials
A total of 150 blastocysts were collected from 49 couples who visited the clinic center to undergo IVF between September 2011 and December 2012. All patient materials were obtained and evaluated with informed patient consent and under approval from the Ethics Committees of MGMED clinic center and Seoul Rachel Fertility Center. All patients were provided with counseling regarding PGS using array CGH and signed an informed consent prior to entering the study.

Experimental procedures
The biopsied cells were washed in PBS and collected into PCR tubes. Whole-genome amplification was performed using a kit and following the manufacturer's instructions (Sigma-Aldrich, Saint Louis, MO).
Approximately 3 μg of amplified DNA was used in the array CGH experiments, as described, with slight modifications [20]. Briefly, the amplified DNA was labeled with Cy-3 and Cy-5 dCTP for 3 h using a random priming method. The labeled DNA was purified, dissolved in hybridization buffer, and hybridized overnight. The slides were washed several times and dried as described [21]. Images of the slides were acquired with a GenePix4000B dual-laser scanner (Axon Instruments, Union City, CA) and analyzed with MacViewer software [21].