Preclinical Validation of a Microarray Method for Full Molecular Karyotyping of Blastomeres in a 24-hour Protocol

BACKGROUND: Pre-implantation genetic screening (PGS) has been used in an attempt to determine embryonic aneuploidy. Techniques that use new molecular methods to determine the karyotype of an embryo are expanding the scope of PGS. METHODS: We introduce a new method for PGS, termed “Parental Support” (PS), which leverages microarray measurements from parental DNA to “clean” single cell microarray measurements on embryonic cells and explicitly computes confidence in each copy number call. The method distinguishes mitotic and meiotic copy errors, and determines parental source of aneuploidy. RESULTS: Validation with 459 single cells of known karyotype indicated that per-cell false positive and false negative rates are roughly equivalent to the “gold standard” metaphase karyotype. The majority of the cells were run in parallel with a clinical commercial PGS service. Computed confidences were conservative and roughly concordant with accuracy. To examine ploidy in human embryos, the method was then applied to 26 disaggregated cryopreserved cleavage stage embryos, for a total of 134 single blastomeres. Only 23.1% of the embryos were euploid, though 46.2% of embryos were mosaic euploid. Mosaicism affected 57.7% of the embryos. Counts of mitotic and meiotic errors were roughly equivalent. Maternal meiotic trisomy predominated over paternal trisomy, and maternal meiotic trisomies were negatively predictive of mosaic euploid embryos. CONCLUSIONS: We have performed a major preclinical validation of a new method for PGS and found that the technology performs approximately as well as a metaphase karyotype. We also directly measured the mechanism of aneuploidy in cleavage stage human embryos and found high rates and distinct patterns of mitotic and meiotic aneuploidy. Single cells were isolated from buccal swabs, semen samples, adult blood, immortalized cell lines, and Day 3 cryopreserved embryos. Single tissue culture (lymphocytes) and buccal cells were isolated using a sterile stripper tip (Midatlantic Diagnostic, Mt. Laurel, NJ, USA) affixed to a pipette (Drummond Scientific, Broomall, PA, USA) and a stereoscope (Leica, Wetzlar, Germany). Embryos were thawed, and then individual blastomeres were separated using a micromanipulator (Transferman NK2-Eppendorf, Westbury, NY, USA) after zona pellucida drilling using acidified Tyrode’s solution. Sperm cells were manually isolated using a micromanipulator (Transferman NK2-Eppendorf). Aside from sperm, single cells were washed sequentially four times with wash buffer (5.6mg/ml KCl, 6mg/ml bovine serum albumin). Two different lysis/amplification protocols were used in the analysis: (i) Rubicon Whole Genome Amplification (Ann Arbor, MI, USA), and (ii) multiple displacement amplification (MDA) with Proteinase K Buffer (PKB). Protocol (i) was performed according to the manufacturer’s instructions. For protocol (ii), cells were placed in 5µl PKB (Arcturus PicoPure Lysis Buffer, 50mM DTT), incubated at 56°C for one hour, and then heat inactivated at 95°C for ten minutes. MDA reactions were incubated at 30°C for 2.5 hours and then 65°C for five minutes. Genomic DNA from bulk tissue (Epicentre MasterAmp Buccal Swabs, Madison, WI, USA) was isolated using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany). No template controls (buffer blanks) were performed for each amplification method. All buffer blanks produced intensities equivalent to the noise floor (an intensity of 1000 in the 95th percentile on the green detection channel). Amplified single cells and bulk parental tissue were genotyped using the Illumina (San Diego, CA, USA) Infinium II genome-wide genotyping single nucleotide polymorphism (SNP) microarrays (HapMap CNV370Quad or CytoSNP-12 chips). For bulk tissue (i.e., parent samples), the standard Infinium II protocol (www.illumina.com) was used and required call rates of >99% using standard BeadStudio allele calling. All single cells were genotyped using a modified Infinium II genotyping protocol, such that the entire protocol, from single cell lysis through array scanning, was completed in fewer than 24 hours. The microarray protocol was performed as per the manufacturer’s instructions, except that the duration of the amplification and hybridization steps were reduced to 7 hours and 6 hours, respectively. Single cell array measurements that fell below the noise floor (an intensity of 1000 in the 95th percentile on the green detection channel) were removed from downstream analysis. For the MDA and Rubicon protocols, this represented approximately 1.6% and 1.3% of all amplifications, respectively.