Sureplex dna amplification system

As detailed above, EVs isolated from EF (n=21) in different menstrual cycle stages (P+2 n=7, P+5 n=7, P+8 n=7) were DNase treated to eliminate extravesical DNA contamination. MVs-derived DNA was amplified using the Sureplex DNA amplification system (Illumina) according to the manufacturer’s instructions. This amplification step increases the DNA yield obtained per sample to ensure a sufficient amount for qPCR analysis without altering the proportion of the different sequences. Samples were purified using AMPure XP beads (Beckman Coulter), and the DNA was quantified using D1000 ScreenTape in a TapeStation 4200 instrument (Agilent). Only DNA samples containing >0.1 ng/µL were used for the qPCR analysis. The mitochondrial ATP8 gene was amplified in a qPCR machine using the following primers: Forward 5'-CTAAAAATATTAAACACAAACTACCACCTACCTC-3' and Reverse 5'-GTTCATTTTGGTTCTCAGGGTTTGTTATAA-3'. Standard curves were created by qPCR amplifying serial dilutions of the ATP8 amplicon and used to calculate the copy number of mtDNA in 250 pg of DNA per reaction. Half of the MVs samples were used to calculate the concentration of MVs by NTA analysis, and the mtDNA copy number was normalized with the number of MVs (mtDNA copy number/MV).

Embryos were kept in culture in a time-lapse incubator, single-step culture media (LifeGlobal^®, Paramus, NJ, USA), with 5% oxygen concentration, as described previously (17 (link)). Embryos were transferred at the stage of the blastocyst. When performed, the PGT-A procedure was carried out as previously described (23 (link)). Embryos were biopsied and vitrified on days 5 and 6 of development. Genetic testing on the embryo was performed by array comparative genomic hybridization (a-CGH) using accessible kits and software (SurePlex^® DNA Amplification System, 24Sure^® Microarray Pack, BlueFuseMulti^®, Illumina^®) following the manufacturer’s instructions.

Zona of day-3 embryos was initially drilled by laser
beam. Primary criteria for the embryo selection was the
same as described for Cleavage-stage biopsy. The embryo
culture continued and those developing to the blastocyst
stage were biopsied using laser technology as previously
described (22 (link), 24 (link)). Only well-defined inner cell mass
blastocysts with hatching trophectoderm were biopsied.
Three to eight trophectoderm cells were biopsied.
After biopsy, the embryos were washed in 1X
phosphate buffered saline (PBS, Gibco, USA) and they
were transferred to digestion buffer with minimum PBS
for further genetic analysis. The lysis and Whole Genome
Amplification step was performed by SurePlex®DNA
Amplification System (Illumina, USA).

Sample cell preparation, cell lysing procedures, DNA extraction, and whole genome amplification (WGA) were performed using the SurePlex DNA Amplification System (Illumina, San Diego, CA, USA), according to the manufacturer’s recommended conditions. The DNA concentration of the product after amplification was measured using a Qubit 3.0 Fluorometer (ThermoFisher Scientific) with the Qubit dsDNA HS Assay kit (ThermoFisher Scientific).
Following WGA, the samples were treated using a VeriSeq PGS Kit (Illumina, San Diego, CA, USA), according to the manufacturer’s instructions. NGS was performed using a MiSeq testing device (Illumina, San Diego, CA, USA). The obtained data were analyzed using Bluefuse Multi software to obtain the karyotype information of the sample.

A proportion of stimulation cycles were also designed for chromosome screening, either PCR analysis of chromosome 21 content or CCS by CGH. Embryo screening for chromosome 21 content was performed simultaneously with thalassemia screening from 2011 onwards for a small number of cycles. At least three informative STR markers on chromosome 21 were examined to ensure the presence of enough markers to aid the identification of aneuploidy. This was performed as described above for thalassemia testing. In 2014, CCS by CGH was adopted for the majority of PGD patients screened for monogenic disorders regardless of whether HLA matching was required. CGH was performed prior to thalassemia screening, and only embryos with no detectable anomalies were subsequently tested for thalassemia mutations and, if required, HLA typing. CGH was performed on embryo biopsies using the SurePlex DNA Amplification System (whole genome amplification kit) and the BlueGnome 24sure BAC array (Illumina) as per the manufacturer's instructions.