Preparation of embryos for the study
To establish the qPCT method, 30 reference samples, which are WGA products with normal PGT-A results and no parental contamination were collected, and to perform the simulated contamination test, six discarded aneuploidy embryos were collected. We conducted a prospective clinical study with patients recruited from January 2020 to April 2021. A total of 34 couples with indications for PGT-A due to advanced maternal age, repeated implantation failures or recurrent abortion who had surplus blastocysts from prior conventional IVF available for biopsy were recruited regardless of their prognosis, age, or diagnosis. The couples had no karyotype abnormalities. Approval for this study was obtained from the Ethics Committee of Guangdong Women and Children Hospital (Research Ethics Committee No. 202101109). All these samples were from Reproductive Medical Center of Guangdong Women and Children Hospital and all of the patients signed the informed consent form before any study-specific procedures were performed. The whole protocol described below is summarized in Fig. 3A. The clinical details of 34 couples are summarized in Table 2.
Artificial addition of sperm and cumulus cells to TE cells
To establish an artificial model for detecting parental cells contamination in biopsied TE cells, frozen-thawed sperm and cumulus cell was isolated, aspirated and put into the pipette with biopsy cells. To simulate the freezing process of sperm on the zona pellucida, the method involving the zona pellucida combined with sperm was used (Supplementary, Fig. S1).
Frozen stage 5 or 6 blastocysts were thawed in advance and cultured 2-4 h prior to biopsy, whereas frozen day 3 embryos were thawed 2–3 days in advance. Every blastocyst that reached the blastocyst stage and fulfilled the biopsy criteria was biopsied. Biopsy was performed in 10-μl drops of G-MOPS-PLUS, the blastocyst was fixed and positioned with a clear view of the inner cell mass (ICM) at 9-12 o’clock, and the hatched TE cells were aspirated into the biopsy pipette, followed by three laser pulses of 2.0 ms (ZYLOS-tk®, Hamilton Thorne, MA, USA) to loosen cell connections and the application of the mechanical ‘flicking’ method to cut away the TE cells using the biopsy pipette and holding pipette. When a blastocyst was not hatched, the zona pellucida was perforated by a laser for 2.0 ms, after which the collapse of the blastocyst was induced before biopsy. TE cells were washed and placed in 0.2-ml PCR tubes with 1–2.0 μl PBS and stored at −20°C until further processing. Approximately 10 biopsy cells from one discarded aneuploidy blastocyst were artificially mixed with sperm or cumulus cells and 4–6 TE cells were biopsied for clinical application of qPCT-PGT. All embryos were cryopreserved by vitrification (Life Global®, USA) within 1 h after biopsy and stored at -196°C.
Whole-genome amplification of sperm, cumulus and TE cells
Single-cell WGA were performed in 22 samples of sperm cells (20 cells per sample) and three samples of cumulus cells (one cell per sample) under different conditions used for TE biopsy samples (Fig. 1A and 1B). The multiple annealing and loop-based amplification cycles (MALBAC)-based single-cell WGA kit (ChromSwiftTM, Cat. No. KT110700324, Yikon Genomics Ltd, Suzhou, China), the PicoPLEX single-cell WGA kit (Rubicon Genomics, Ann Arbor, USA) and the multiple displacement amplification (MDA)-based single-cell WGA Kit (Cat. No. 150343, Qiagen, USA) were utilized to amplify DNA from different types of cells according to the manufacturer’s instructions.
Determination of blastocyst ploidy status by NGS
To analyze the ploidy status of blastocysts, the amplified DNA of TE samples was sequenced using a NextSeq 550 sequencer (Cat No. SY-415-1002, Illumina, Inc., USA) with a single-ended read length of 55 bp. Approximately 2 million raw reads were generated for each TE sample. Genome-wide CNVs were analyzed to determine the euploidy or aneuploidy status of each embryo. Aneuploid embryos were diagnosed when the size of CNVs was greater than 4 Mb and the extent of mosaicism was above 30%.
Genotyping assay of blastocysts and parental genomes
To determine the genotype of each blastocyst and parental genome, the Infinium Asian Screening Array (ASA) bead chip (Cat No. 20016317, Illumina, Inc., USA) and the iScan system (Cat No. SY-101-1001, Illumina, Inc., USA) were utilized. Parental genomic DNA was extracted from the peripheral blood of the parents of each blastocyst. Genomic DNA, along with the amplified DNA of TE samples, was linearly amplified, fragmented, precipitated and hybridized according to the manufacturer’s instructions. Signal scanning was performed with the iScan system. Iaap-cli gencall algorithm (Version 1.1.0, Illumina, Inc., USA) was applied to analyze the genotype of each sample, and the BAF and log R ratio (LRR) values of all SNPs were generated simultaneously.
Determination of parental contamination by BAF analysis
As shown in (Fig. 1F), theoretically, the BAFs of genotypes AA, AB and BB should be approximately 0, 0.5 and 1, respectively. For a SNP with paternal AA and maternal BB genotypes or paternal BB and maternal AA genotypes, the genotype of the embryo is genetically expected to be heterozygous AB, with a BAF of approximately 0.5. Deviation from a BAF of 0.5 for the embryo may be caused by parental contamination, DNA copy number aberrations or allelic amplification bias and errors (e.g. allele dropout or preferential amplification of one allele) resulting from single-cell WGA technology. Selecting SNP j with homozygous genotypes but different alleles between the father and mother, maternally and paternally biased SNPs in an embryo were defined according to a BAF ≥0.6 (SM, SNP site for maternal bias) and BAF ≤0.4 (SP, SNP site for paternal bias) given maternal and paternal genotypes of BB and AA, respectively; for maternal and paternal genotypes of AA and BB, maternally and paternally biased SNPs were determined according to a BAF ≤0.4 (SM) and BAF ≥0.6 (SP) (Fig. 1F). Relative parental origin bias (POB) statistics were thus constructed and formulated for each chromosome, i, as follows:
Based on reference samples from euploid embryos without parental contamination, a positively skewed normal distribution of with a mean value of 1.14 was obtained (Fig. S1B). Here, a tested sample with an value significantly greater than 1.14 is indicated to show maternal origin bias, implying maternal contamination (most chromosomes with numbers ≥15 share the same pattern), or chromosome-level maternal-origin DNA replication, or uniparental disomy, or paternal-origin DNA deletion with a P-value < 0.05 (Mean+3*SD); on the contrary, a tested sample with an value significantly less than 1.14 is indicated to show paternal-origin bias, with the possibility of paternal contamination (most chromosomes with numbers ≥15 share the same pattern), or chromosome-level paternal-origin DNA replication, or uniparental disomy, or maternal-origin DNA deletion with a P-value <0.05 (Mean-2*SD, standard deviation). statistics can to some degree eliminate allelic amplification bias from WGA.
Preparation and analyzing of artificially contaminated gDNA
To address the issue of quantification of parental contamination, we establish a standard curve, which represents the correlation between the POB value and the ratio of parental contamination. Parents genomic DNA was extracted from peripheral blood samples, while fetal genomic DNA was extracted from tissue of spontaneous abortion. A commercial genomic DNA extraction kit (DNeasy Blood & Tissue Kit, Cat. No. 69504, Qiagen, USA) was used according to the manufacturer’s instructions. Paternal/fet al and maternal/fet al DNA was mixed in different proportion (Fig. 1G). The artificially contaminated DNA was then diluted to 50pg/μL, and amplified by using a ChromInst Single cell WGA kit (Cat. No. Yikon Genomics Ltd, Suzhou, China) according to the manufacturer’s instructions. Finally, the amplified DNA was analyzed by using the Infinium Asian Screening Array (ASA) bead chip (Cat No. 20016317. Illumina, Inc. USA) and the iScan system (Cat No. SY-101-1001. Illumina, Inc. USA) to obtain the BAF value of each SNP loci. The RPOB was calculated to establish the standard curve of the ratio of parental contamination.
Measurement of clinical outcomes
Biochemical was defined as β-HCG > 5 U/L detected 12 days after blastocyst transfer, but no gestational sac was found by transvaginal ultrasound. Negative was defined as β-HCG <5 U/L detected 12 days after blastocyst transfer. Clinical pregnancy was defined by fetal cardiac activity. Non-invasive prenatal testing (NIPT) was performed at 12-18 weeks of gestation.