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Preimplantation genetic testing for human blastocysts with potential parental contamination using a quantitative parental contamination test (qPCT): an evidence-based study

Open AccessPublished:August 21, 2022DOI:https://doi.org/10.1016/j.rbmo.2022.08.103

      Abstract

      Research question

      Is it possible to develop a quantitative method for detecting parental DNA contamination in conventional IVF using preimplantation genetic testing for aneuploidy (PGT-A)?

      Design

      In this study, a quantification method was established for the parental contamination test (qPCT), which ensured more reliable results, and then verified its effectiveness for vitrified conventional IVF embryos. A total of 120 surplus vitrified blastocysts from patients who underwent prior routine IVF cycles were available for study.

      Results

      The results of the prospective clinical study of qPCT-PGT-A showed that the maternal contamination rate was 0.83% (1/120) and that the risk of paternal contamination was negligible. The 24 frozen embryo transfer cycles resulted in 16 clinical pregnancies, including 13 live births, one late inevitable miscarriage and two ongoing pregnancies.

      Conclusions

      The risk of PGT in embryos with potential parental contamination is relatively low, and PGT-A is applicable for vitrified conventional IVF embryos.

      KEYWORDS

      Introduction

      For preimplantation genetic testing (PGT) to be clinically applicable, intracytoplasmic sperm injection (ICSI) is usually needed, rather than conventional IVF, owing to potential parental contamination from spermatozoa and cumulus cells attached to the zona pellucida during conventional IVF (
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      • Harper J.C.
      ESHRE PGD Consortium/Embryology Special Interest Group–best practice guidelines for polar body and embryo biopsy for preimplantation genetic diagnosis/screening (PGD/PGS).
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      Preimplantation diagnosis for single gene disorders.
      ;
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      • Rubio C.
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      ESHRE PGT Consortium good practice recommendations for the detection of structural and numerical chromosomal aberrations.
      ;
      • Kokkali G.
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      ESHRE PGT Consortium and SIG Embryology good practice recommendations for polar body and embryo biopsy for PGT.
      ). As reported, spermatozoa cannot be amplified by routine whole genome amplification (WGA) protocols (
      • De Munck N.
      • El Khatib I.
      • Abdala A.
      • El-Damen A.
      • Bayram A.
      • Arnanz A.
      • Melado L.
      • Lawrenz B.
      • Fatemi H.M.
      Intracytoplasmic sperm injection is not superior to conventional IVF in couples with non-male factor infertility and preimplantation genetic testing for aneuploidies (PGT-A).
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      • Griffin D.
      Investigation of the risk of paternal cell contamination in PGT and the necessity of intracytoplasmic sperm injection.
      ), because of the tightly packed DNA used for trophectoderm biopsy samples. Because cumulus cells are deliberately removed in IVF and preimplantation genetic testing for aneuploidy (PGT-A), the use of conventional IVF has been preliminarily explored in PGT-A in fresh cycles in recent years (
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      Pre-implantation genetic diagnosis-should we use ICSI for all?.
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      • Arnanz A.
      • Melado L.
      • Lawrenz B.
      • Fatemi H.M.
      Intracytoplasmic sperm injection is not superior to conventional IVF in couples with non-male factor infertility and preimplantation genetic testing for aneuploidies (PGT-A).
      ;
      • Niu X.
      • Long J.
      • Gong F.
      • Wang W.
      Does ICSI for in vitro fertilization cause more aneuploid embryos?.
      ). For patients who have experienced repeated implantation failure or miscarriage caused by chromosomal abnormalities in previous embryo transfer cycles, it is feasible to biopsy existing cryopreserved embryos from IVF. Without the initial intention of carrying out PGT, many spermatozoa and cumulus cells can exist simultaneously in the zona pellucida of some frozen blastocysts from conventional IVF. Although spermatozoa cannot be amplified by conventional methods, they can be amplified by the multiple displacement amplification (MDA)-WGA method and can also be amplified by the SurePlex DNA Amplification System, with minor modifications (Tran et al., 2019). It has not been reported whether spermatozoa can be easily amplified after being frozen in liquid nitrogen. To date, the effect of cumulus cell contamination on PGT-A results has not been reported. In general, the feasibility of PGT-A for cryopreserved IVF embryos has not been reported.
      In this scenario, a parental contamination detection method is required for identifying contaminated embryos to avoid potential false negative results in the application of IVF-PGT-A. So far, several approaches using single nucleotide polymorphism (SNP) genotyping information or short tandem repeat (STR) markers to trace maternal genetic materials from fetal or miscarriage specimens have been reported (
      • Liao G.J.
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      • Lo Y.M.
      Noninvasive prenatal diagnosis of fetal trisomy 21 by allelic ratio analysis using targeted massively parallel sequencing of maternal plasma DNA.
      ;
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      • Sigurjonsson S.
      • Tao R.
      • Demko Z.
      Reliability of 46,XX results on miscarriage specimens: a review of 1,222 first-trimester miscarriage specimens.
      ;
      • Buchovecky C.M.
      • Nahum O.
      • Levy B.
      Assessment of Maternal Cell Contamination in Prenatal Samples by Quantitative Fluorescent PCR (QF-PCR).
      ). Such approaches require sufficient DNA for genetic testing. Esteki et al. (2015) proposed a concurrent whole genome haplotyping approach, referred to as haplarithmisis, to resolve the parental origin of copy number variations (CNV) in single cells, but the method is easily affected by low-quality amplification effects, such as high allele dropout (ADO) (
      • Patassini C.
      • Garolla A.
      • Bottacin A.
      • Menegazzo M.
      • Speltra E.
      • Foresta C.
      • Ferlin A.
      Molecular karyotyping of human single sperm by array- comparative genomic hybridization.
      ;
      • Zamani Esteki M.
      • Dimitriadou E.
      • Mateiu L.
      • Melotte C.
      • Van der Aa N.
      • Kumar P.
      • Das R.
      • Theunis K.
      • Cheng J.
      • Legius E.
      • Moreau Y.
      • Debrock S.
      • D'Hooghe T.
      • Verdyck P.
      • De Rycke M.
      • Sermon K.
      • Vermeesch J.R.
      • Voet T.
      Concurrent whole-genome haplotyping and copy-number profiling of single cells.
      ).
      In the present study, a new quantification method for parental contamination testing (referred to as qPCT) was first developed, based on allelic ratios obtained from SNP genotypes, which fully considers amplification bias, including preferential amplification or ADO effects. The qPCT assay was validated by comparing results before and after the artificial addition of spermatozoa and cumulus cells to trophectoderm cells from discarded aneuploid blastocysts. A total of 120 vitrified embryos were included from 34 couples of advanced age who experienced repeated implantation failure or miscarriage caused by chromosomal abnormalities and who had surplus vitrified embryos from previous conventional IVF cycles that were available for biopsy. By using next-generation sequencing (NGS) with our qPCT method, euploid embryos were selected from 24 transfers with no parental contamination, resulting in 13 healthy live births, one late miscarriage and two ongoing clinical pregnancies.

      Materials and methods

      Ethics approval

      Approval for this study was obtained from the Ethics Committee of Guangdong Women and Children Hospital on 27 April 2021 (Research Ethics Committee number 202101109). All samples were obtained from the Reproductive Medical Center of Guangdong Women and Children Hospital, and all the patients provided informed consent before any study-specific procedures were carried out.

      Preparation of embryos for study

      To establish the qPCT method, 30 reference samples, which included WGA products with normal PGT-A results and no parental contamination, were collected. To conduct the simulated contamination test, six discarded aneuploid embryos and the associated sperm and cumulus cells, were collected from three couples who underwent ICSI-PGT-A at our centre. A prospective clinical study was conducted with patients recruited from January 2020 to April 2021. A total of 34 couples with indications for PGT-A owing to advanced maternal age, repeated implantation failures or recurrent miscarriage, and who had surplus blastocysts from previous conventional IVF that were available for biopsy, were recruited regardless of their prognosis, age or diagnosis. The whole protocol described below is presented in Figure 1.
      Figure 1:
      Figure 1Concept validation and establishment of qPCT. A left panel and B left panel: Isolated sperm and cumulus cells. Bar = 10 µm. A middle panel and B middle panel: Electrophoresis for WGA products of sperm and cumulus cells. M: 100 bp DNA marker.The 500bp and 1Kbp DNA marker were labeled with green and blue arrow, respectively; NC: negative control; A-MAL: 20 sperm cells amplified by MALBAC; A-Pico: 20 sperm cells amplified by Picoplex; A-MDA: 20 sperm cells amplified by MDA. B-MAL: single cumulus cell amplified by MALBAC; B-Pico: single cumulus cell amplified by Picoplex; B-MDA: single cumulus cell amplified by MDA. A right panel and B right panel: CNV-seq results of WGA products amplified from sperm and cumulus cells using MALBAC/Picoplex/MDA. Upper: sperm cells; lower: single cumulus cell; NC: negative control. C: Schematic of the assignment of SNP sites for paternal/maternal bias. SM: SNP site for maternal bias; SP: SNP site for paternal bias. D: Standard curve of qPCT established by artificially mixing parental and foetal gDNA. X-axis: ratio of added maternal/paternal gDNA; Y-axis: POB value (log10); Red triangle: maternal contamination samples; Blue rhombus: paternal contamination samples. E: qPCT results for artificially contaminated gDNA samples. X-axis: chromosome ID; Y-axis: POB value; M5: 50% maternal contamination sample (maternal DNA: foetal DNA = 1:1); N0: no contamination sample; P5: 50% paternal contamination sample (paternal DNA: foetal DNA = 1:1) CNV, copy number variation; POB value, parental origin bias; qPCT, quantitative parental contamination test.

      Artificial addition of spermatozoa and cumulus cells to trophectoderm cells

      To establish an artificial model for detecting parental cell contamination in the biopsied trophectoderm cells of the six discarded aneuploid embryos, the matching frozen–thawed sperm and cumulus cells were isolated, aspirated and placed into pipettes with biopsy cells. To simulate the freezing process of spermatozoa on the zona pellucida, the method involving the zona pellucida combined with spermatozoa was used (Supplementary Figure 1).

      Blastocyst biopsy

      For the clinical test embryos, vitrified stage-5 or stage-6 blastocysts were warmed in advance and cultured 2–4 h before biopsy, whereas vitrified day-3 embryos were warmed 2–3 days in advance. Every embryo that reached the blastocyst stage and fulfilled the biopsy criteria was biopsied. The biopsy was placed in 10-µl drops of G-MOPS-PLUS (Vitrolife, Sweden). The blastocyst was fixed with a clear view of the inner cell mass and positioned at 9–12 o'clock, and the hatched trophectoderm cells were aspirated into the biopsy pipette; this was 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 trophectoderm 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. Trophectoderm cells were washed and placed in 0.2-ml polymerase chain reaction tubes with 1–2.0-µl phosphate buffered saline and stored at −20°C until further processing. Approximately 10 biopsy cells from one discarded aneuploid blastocyst were artificially mixed with spermatozoa or cumulus cells, and four to six trophectoderm cells were biopsied for the clinical application of qPCT-PGT. All embryos were cryopreserved by vitrification (Life Global®, Paramus, NJ, USA) within 1 h after biopsy and stored at –196°C.

      Whole-genome amplification of spermatozoa, cumulus and trophectoderm cells

      Single-cell WGA was carried out for 22 samples of frozen sperm cells (20 cells per sample) and three samples of cumulus cells (one cell per sample) under different conditions used for trophectoderm biopsy samples (Figure 1a and Figure 1b). Patients from our centre donated their spermatozoa and granulosa cells for scientific research. The multiple annealing and loop-based amplification cycle (MALBAC)-based single-cell WGA kit (catalogue number KT110700324) (Yikon Genomics Ltd, Suzhou, China), PicoPLEX single-cell WGA kit (Rubicon Genomics, Ann Arbor, USA) and MDA-based single-cell WGA Kit (catalogue number 150343) (Qiagen, Germantown, MD, USA) were used to amplify DNA from different types of cells according to the manufacturer's instructions.

      Determination of blastocyst ploidy status by next generation sequencing

      To analyse the ploidy status of the vitrified blastocysts, the amplified DNA of the trophectoderm samples was sequenced using a NextSeq 550 sequencer (catalogue number SY-415-1002) (Illumina, Inc., San Diego, CA, USA) with a single-ended read length of 55 bp. Approximately 2 million raw reads were generated for each trophectoderm sample. Genome-wide CNV were analysed to determine the euploid or aneuploid status of each embryo. Embryos were diagnosed as aneuploid when the size of the CNV 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 bead chip (catalogue number 20016317) (Illumina, Inc., San Diego, CA, USA) and iScan system (catalogue number SY-101-1001) (Illumina, Inc., San Diego, CA, USA) were used. Parental gDNA was extracted from the peripheral blood of the parents of each blastocyst, and gDNA along with the amplified DNA of the trophectoderm samples was linearly amplified, fragmented, precipitated and hybridized according to the manufacturer's instructions. The iScan system was used for signal scanning. The Iaap-cli gencall algorithm (Version 1.1.0) (Illumina, Inc., San Diego, CA, USA) was applied to analyse the genotype of each sample, and the B allele frequency (BAF) and log R ratio values of all SNPs were generated simultaneously.

      Determination of parental contamination by B allele frequency analysis

      As shown in Figure 1c, theoretically, the BAFs of genotypes AA, AB and BB should be approximately 0, 0.5 and 1, respectively. For an 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 in an embryo may be caused by parental contamination, DNA copy number aberrations or allelic amplification bias and errors. e.g. allele dropout or the preferential amplification of one allele, resulting from single-cell WGA technology. Single nucleotide polymorphism ‘j’ with homozygous genotypes and different alleles between the father and mother were selected, and maternally and paternally biased SNPs in an embryo were defined according to a BAF 0.6 or above (SM, SNP site for maternal bias) and BAF 0.4 or below (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 or below (SM) and BAF 0.6 or above (SP) . Relative parental origin bias (POB) statistics were, therefore, constructed and formulated for each chromosome, i, as follows:
      RPOBi=jSMi,jjSPi,j


      Trophectoderm biopsy specimens with euploidy collected through ICSI were used as the reference samples without parental contamination, and a positively skewed normal distribution of RPOBi with a mean value of 1.14 was obtained (Supplementary Figure 2B). Here, a tested sample with an RPOBi value significantly greater than 1.14 is indicated to show maternal origin bias, implying maternal contamination (most chromosomes with numbers 15 or above share the same pattern), chromosome-level maternal-origin DNA replication, uniparental disomy or paternal-origin DNA deletion with P < 0.05 (mean +3*SD); in contrast, a tested sample with an RPOBi value significantly less than 1.14 is indicated to show paternal-origin bias, with the possibility of paternal contamination (most chromosomes with numbers 15 or above share the same pattern), chromosome-level paternal-origin DNA replication, uniparental disomy, or maternal-origin DNA deletion with P < 0.05 (mean-2*SD). RPOB statistics can, to some degree, eliminate allelic amplification bias from WGA.

      Preparation and analysis of artificially contaminated gDNA

      To address the issue of the quantification of parental contamination, a standard curve was established, which represents the correlation between the POB value and the ratio of parental contamination. Parental genomic DNA was extracted from peripheral blood samples, whereas fetal genomic DNA was extracted from spontaneous miscarriage tissue. A commercial genomic DNA extraction kit (DNeasy Blood & Tissue Kit, catalogue number 69504) (Qiagen, Germantown, MD, USA) was used according to the manufacturer's instructions. Paternal/fetal and maternal/fetal DNA was mixed in different proportions (Figure 1d). The artificially contaminated DNA was then diluted to 50 pg/µl and amplified by using a ChromInst Single cell WGA kit (catalogue number YK001B Yikon Genomics Ltd, Suzhou, China) according to the manufacturer's instructions. Finally, the amplified DNA was analysed by using the Infinium Asian Screening Array bead chip (catalogue number 20016317) (Illumina, Inc. San Diego, CA, USA) and iScan system (catalogue number SY-101-1001) (Illumina, Inc. San Diego, CA, USA) to obtain the BAF value of each SNP locus. The RPOB was calculated to establish the standard curve of the ratio of parental contamination.

      Measurement of clinical outcomes

      A biochemical pregnancy was defined as a beta-HCG level above 5 U/l detected 12 days after blastocyst transfer, but with no gestational sac found by transvaginal ultrasound. No pregnancy was defined as a beta-HCG level below 5 U/l detected 12 days after blastocyst transfer. A clinical pregnancy was defined by fetal cardiac activity. Non-invasive prenatal testing was carried out at 12−18 weeks of gestation.

      Results

      Whole genome amplification of spermatozoa and cumulus cells

      To study the effect of liquid nitrogen freezing on the amplification of spermatozoa and granular cells, WGA and copy number variation sequencing (CNV-seq) were carried out for 22 samples of frozen–thawed sperm cells and three samples of frozen–thawed cumulus cells (Figure 1a and Figure 1b). As shown, no DNA smears were observed in the MALBAC-WGA and Picoplex-WGA products of the sperm samples, whereas positive results were obtained from the MDA-WGA products. Cumulus cell DNA, however, was easily amplified by the three methods. The MALBAC-CNV-seq and Picoplex-CNV-seq of sperm samples also failed to generate qualified results, showing low genome coverage ranging from 0.31% to 1.08% and relatively high coefficients of variation ranging from 23–50%. Three cumulus cell samples, however, presented high-quality CNV-seq results, with the three samples showing normal karyotypes. These results are partially in accordance with previous studies (
      • De Munck N.
      • El Khatib I.
      • Abdala A.
      • El-Damen A.
      • Bayram A.
      • Arnanz A.
      • Melado L.
      • Lawrenz B.
      • Fatemi H.M.
      Intracytoplasmic sperm injection is not superior to conventional IVF in couples with non-male factor infertility and preimplantation genetic testing for aneuploidies (PGT-A).
      ), indicating the difficulty of amplifying sperm DNA.

      Establishment of qPCT

      To establish the qPCT method, 30 reference samples were first collected from euploid embryos without parental contamination, which were defined as embryos with normal ploidy and XY chromosomes. The POB values of these reference samples were calculated based on the BAF of selected SNP loci (Figure 1c and Supplementary Figure 2), which approximately followed a positively skewed normal distribution, with a mean of 1.14 and SD of 0.30. Therefore, the POB range for non-contaminated euploid embryos was defined as 0.54 to 2.04 (mean –2*SD to mean + 3*SD), and a qualitative standard of parental contamination was established.
      To further address the issue of the quantification of parental contamination, the gDNA of tissue samples from spontaneous miscarriages and blood samples from parents were mixed in specific proportions. The mixed gDNA was diluted to an approximate concentration of 50 pg/µl and then amplified, sequenced and analysed in accordance with the PGT-A procedure. Genotyping assays were also performed. As a result, the POB values of the artificially added contaminant fetal gDNA showed a positive correlation with the proportion of mixed parental gDNA (Figure 1d and Figure 1e). The standard curve was used for the relative quantification of parental contamination.

      Detection of contamination in artificially contaminated trophectoderm samples

      To verify the accuracy and repeatability of qPCT in detecting parental contamination in biopsy cells, gDNA was added from the peripheral blood of parents to the WGA products. The detection rate was 100% (10/10) (Supplementary Figure 3). No significant contamination, however, was detected in the artificially contaminated trophectoderm samples containing approximately 10 biopsy cells and one cumulus cell (Table 1).
      TABLE 1VALIDATION RESULTS OF THE QUANTITATIVE PARENTAL CONTAMINATION TEST (qPCT) METHOD IN THE ARTIFICIAL MODEL
      Type of artificial contaminationDiscarded aneuploid embryoKaryotypeNumber of added sperm cell or cumulus cellResults of PGT-AResults of parental contaminationRatio of parental contamination, %
      The cut-off for quantitative parental contamination test (qPCT) is set at 10%.
      Paternal146,XY,+15(× 3),–22(× 1)346,XY,+15,–22No0
      Paternal46,XY,+15(× 3),–22(× 1)546,XY,+15,–22No0
      Paternal46,XY,+15(× 3),–22(× 1)846,XY,+15,–22No0
      Paternal46,XY,+15(× 3),–22(× 1)2046,XY,+15,–22No0
      Paternal245,XY,–20(× 1)345,XY, –20No0
      Paternal45,XY,–20(× 1)545,XY, –20No0
      Paternal45,XY,–20(× 1)8NA
      Failed next-generation sequencing.
      NA
      Failed next-generation sequencing.
      NA
      Failed next-generation sequencing.
      Paternal45,XY,–20(× 1)2045,XY, –20No0
      Paternal347,XX+16(× 3)347,XX,+16No0
      Paternal47,XX+16(× 3)547,XX+16No0
      Paternal47,XX,+16(× 3)847,XX,+16No0
      Paternal47,XX,+16(× 3)2047,XX,+16No0
      Maternal445,XX,–20(× 1)145,XX,–20(× 1)
      Over 10 biopsy trophectoderm cells mixed with single cumulus cell or failed addition of the single cumulus cell.
      No0
      Maternal45,XX,–20(× 1)246,XX,–20(× 1,mos,73%)Yes20
      Maternal45,XX,–20(× 1)346,XX,–20(× 1,mos,71%)Yes32
      Maternal545,XX–22(× 1)145,XX –22(× 1,mos,89%)No0
      Maternal45,XX–22(× 1)246,XX,–22(× 1,mos,70%)Yes22
      Maternal45,XX–22(× 1)346,XX,–22(× 1,mos,68%)Yes34
      Maternal645,XY,–22(× 1)846,XY,+X(× 2,mos,66%),–Y(× 0,mos,52%)Yes59
      Maternal45,XY,–22(× 1)1246,XY,+X(× 2,mos,45%),–Y(× 0,mos,45%),–22(× 1,mos,41%)Yes60
      Maternal45,XY,–22(× 1)1546,XY,+ Xp(p22.33→p22.2, ∼15Mb, × 2), +Xp(p21.1→p11.21, ∼26Mb, × 2), +Xq(× 2,mos,70%), –Y(× 0,mos,63%)Yes>70
      Maternal45,XY,–22(× 1)4046,XY,+X(× 2,mos,63%),–Y(× 0,mos,62%)Yes>70
      a The cut-off for quantitative parental contamination test (qPCT) is set at 10%.
      b Failed next-generation sequencing.
      c Over 10 biopsy trophectoderm cells mixed with single cumulus cell or failed addition of the single cumulus cell.
      To reveal the correlation between the PGT-A results and parental contamination, discarded aneuploid blastocysts were selected, different numbers of sperm cells or cumulus cells were added to the biopsied trophectoderm samples and PGT-A and qPCT were carried out for these samples. The results showed that no paternal contamination was detected, and no change in the PGT-A results was observed, regardless of the number of added sperm cells (Figure 2a and Table 1). On the other hand, the proportion of maternal contamination increased gradually with the increase in the number of added cumulus cells, and a notable correlation was observed (Figure 2b and Table 1).
      Figure 2:
      Figure 2Validation of quantitative parental contamination test (qPCT) in the artificial model. (A) sperm cells (three to eight cells) were artificially added to approximately 10 biopsy trophectoderm cells; (B) cumulus cells (one to three cells) were artificially added to approximately 10 biopsy trophectoderm cells. CNV, copy number variation.

      Prospective clinical study of qPCT-PGT-A in warmed IVF blastocysts

      After the systematic validation of the qPCT assay, qPCT-PGT-A was carried out for a total of 120 vitrified blastocysts from 34 recruited patients as described in the Materials and methods section. The qPCT-combined PGT-A procedure is shown in Figure 3a. The qPCT results revealed a maternal contamination rate of 0.83% (1/120, case 182866), and no paternal contamination was detected, as expected (Table 2 and Figure 3b). Notably, one sample from case 200633 was defined as aneuploid with suspected paternal contamination (Table 2 and Supplementary Figure 4A). To confirm this, the sample was collected and lysed for heteroploidy detection using the whole embryo, and a triploid result of paternal origin of was revealed (Table 2 and Supplementary Figure 4B). The PGT-A results showed that the euploidy rate of these vitrified blastocysts was 47.50% (57/120), and at least one euploid blastocyst was obtained for frozen embryo transfer for 28 of these patients (Table 2). Single blastocyst transfer was carried out in 24 patients, resulting in 13 healthy births, one late miscarriage and two ongoing clinical pregnancies (Table 2). The last ongoing pregnancy still under observation. The detailed clinical indications and outcomes are presented in Table 2.
      Figure 3:
      Figure 3Clinical application of quantitative parental contamination test (qPCT)-combined preimplantation genetic testing for aneuploidy (PGT-A) in human conventional IVF embryos. (A) workflow of qPCT-combined PGT-A; (B) clinical sample with positive qPCT results. Case ID 182866: maternal contamination; (C) three clinical application scenarios of preimplantation genetic testing (PGT). Fresh intracytoplasmic sperm injection (ICSI)-PGT: PGT in fresh blastocysts from ICSI cycles. Fresh IVF-PGT, PGT in fresh blastocysts from conventional IVF cycles. Frozen IVF-PGT, PGT in vitrified–warmed blastocysts from conventional IVF cycles. (D) typical IVF embryos undergoing trophectoderm cell biopsy. Bar = 10 µm. The red arrows indicate the spermatozoa adhered to the zona pellucida. Blue arrows indicate granular cells adhered to the zona pellucida. CNV, copy number variation; ICSI, intracytoplasmic sperm injection; NGS, next-generation sequencing; SNP, single nucleotide polymorphism.
      TABLE 2CLINICAL INFORMATION ON THE 34 PATIENTS UNDERGOING PREIMPLANTATION GENETIC TESTING FOR ANEUPLOIDY WITH QUANTITATIVE PARENTAL CONTAMINATION TEST (qPCT).
      Patient IDFemale age, yearsPGT-A indication

      after previous IVF and embryo transfer
      Total embryos tested, nStage 4, 5 and 6 blastocystEuploid embryos determined by PGT-AqPCT assayFrozen embryos transferredPregnancy outcome after FETNIPT
      17180435Abnormal pregnancy-labour history84,2,25Negative1Late miscarriage
      19085738Abnormal pregnancy-labour history20,1,11Negative1Negative
      19253235Repeated implantation failure21,1,01Negative1Biochemical
      19090929Abnormal pregnancy-labour history50,2,35Negative1Live birthNormal
      19248236Abnormal pregnancy-labour history53,1,13Negative1Live birthNormal
      17068643Repeated implantation failure22,0,01Negative1Live birthNormal
      19174941Repeated implantation failure10,1,01Negative1BiochemicalNormal
      18286638Repeated implantation failure51,4,02Positive
      One out of five IVF embryos, which form case 182866, was detected as maternal contamination.
      1Live birthNormal
      19070538Abnormal pregnancy-labour history30,3,01Negative
      20173639Abnormal pregnancy-labour history21,1,00Negative
      16028733Repeated implantation failure40,2,22Negative
      17320340Abnormal pregnancy-labour history11,0,01Negative1Biochemical
      20117332Abnormal pregnancy-labour history20,2,02Negative1Live birthNormal
      20147841Repeated implantation failure50,5,01Negative1Negative
      20110034Abnormal pregnancy-labour history32,1,03Negative1Negative
      20107540Repeated implantation failure21,1,00Negative
      20147727Repeated implantation failure30,3,00Negative
      20035040Repeated implantation failure11,0,00Negative
      20054042Repeated implantation failure32,0,11Negative
      20008131Abnormal pregnancy-labour history51,4,03Negative1Live birthNormal
      16262043Abnormal pregnancy-labor history10,0,11Negative1Live birthNormal
      19293627Repeated implantation failure50,5,02Negative1Negative
      19298629Repeated implantation failure72,5,05Negative1Ongoing pregnancy
      16254532Abnormal pregnancy-labor history20,2,00Negative
      20210431Abnormal pregnancy-labor history40,4,02Negative1Live birth
      20063331Abnormal pregnancy-labor history51,4,01False-positive
      Paternal origin of triploid. FET, frozen embryo transfer; NIPT, non-invasive prenatal testing; PGT-A, preimplantation genetic testing for aneuploidy; qPCT, quantitative parental contamination test.
      1Live birth
      20217038Abnormal pregnancy-labor history11,0,01Negative1Live birth
      19154524Repeated implantation failure32,1,03Negative1Negative
      20132140Repeated implantation failure50,5,01Negative1Live birth
      19047239Abnormal pregnancy-labor history64,2,02Negative1Live birth
      20011339Abnormal pregnancy-labor history32,1,00Negative
      19316528Abnormal pregnancy-labor history32,0,12Negative1Live birth
      16229635Abnormal pregnancy-labor history30,1,21Negative
      20147942Repeated implantation failure80,8,03Negative1Ongoing pregnancy
      Total12034,72,1457One embryo2416 Clinical pregnancies
      a One out of five IVF embryos, which form case 182866, was detected as maternal contamination.
      b Paternal origin of triploid.FET, frozen embryo transfer; NIPT, non-invasive prenatal testing; PGT-A, preimplantation genetic testing for aneuploidy; qPCT, quantitative parental contamination test.

      Discussion

      Although spermatozoa has always been considered to be the main source of paternal contamination, several studies have shown that sperm DNA is difficult to amplify by using routine protocols for trophectoderm biopsy samples in fresh PGT-A cycles (
      • Patassini C.
      • Garolla A.
      • Bottacin A.
      • Menegazzo M.
      • Speltra E.
      • Foresta C.
      • Ferlin A.
      Molecular karyotyping of human single sperm by array- comparative genomic hybridization.
      ;
      • De Munck N.
      • El Khatib I.
      • Abdala A.
      • El-Damen A.
      • Bayram A.
      • Arnanz A.
      • Melado L.
      • Lawrenz B.
      • Fatemi H.M.
      Intracytoplasmic sperm injection is not superior to conventional IVF in couples with non-male factor infertility and preimplantation genetic testing for aneuploidies (PGT-A).
      ;
      • Lynch C.
      • Armstrong E.
      • Charitou M.
      • Gordon T.
      • Griffin D.
      Investigation of the risk of paternal cell contamination in PGT and the necessity of intracytoplasmic sperm injection.
      ), but little evidence indicating the risk and ratio of paternal contamination in cryopreserved conventional IVF PGT-A cycles has been reported. In the present study, we carried out a comprehensive investigation of paternal contamination with a three-step study design. First, MALBAC-WGA of isolated frozen-thawed sperm cells was conducted, and the results of both agarose gel electrophoresis and CNV-seq showed amplification failure, even if 20 sperm cells were tubed (Figure 1a). Although the immobilized motile spermatozoa were subjected to freeze–thawing, the spermatozoa remained intact and did not show signs of degeneration. Additionally, similar negative results were found in recent studies using PicoPlex and SurePlex WGA (
      • De Munck N.
      • El Khatib I.
      • Abdala A.
      • El-Damen A.
      • Bayram A.
      • Arnanz A.
      • Melado L.
      • Lawrenz B.
      • Fatemi H.M.
      Intracytoplasmic sperm injection is not superior to conventional IVF in couples with non-male factor infertility and preimplantation genetic testing for aneuploidies (PGT-A).
      ;
      • Lynch C.
      • Armstrong E.
      • Charitou M.
      • Gordon T.
      • Griffin D.
      Investigation of the risk of paternal cell contamination in PGT and the necessity of intracytoplasmic sperm injection.
      ). In contrast, sperm DNA could be amplified by using MDA (Figure 1a), indicating a higher risk of paternal contamination in the application of MDA-based PGT in IVF embryos. Second, we carried out PGT-A and qPCT with artificially mixed trophectoderm and sperm cells, resulting in no change in the PGT-A results and no paternal contamination. Finally, we conducted a prospective clinical study with 120 vitrified conventional IVF blastocysts; unsurprisingly, no paternal contamination was detected (Table 2). This evidence indicates that the risk of paternal contamination is negligible and that PGT can be applied for cryopreserved conventional IVF embryos with the MALBAC system.
      Cumulus cells have always been the main source of maternal genetic contamination in trophectoderm samples. Unlike sperm DNA, cumulus cell DNA can be easily amplified during the PGT procedure. A notable correlation between the number of cumulus cells and proportion of maternal contamination was observed by using the qPCT method (Table 1). The results suggested that severe maternal contamination might lead to false-negative results, and partial maternal contamination would cause mosaicism. Therefore, this qPCT method can be selected in cases of high suspicion of parental DNA contamination.
      In the PGT scenario, biopsy specimens from blastocysts usually require single-cell whole genome amplification to provide sufficient DNA for genetic testing. However, ADO (PCR failure for one allele), preferential amplification (hyperamplification of one allele) resulting from WGA technology, or both, cause SNP genotyping to be unreliable (
      • Zamani Esteki M.
      • Dimitriadou E.
      • Mateiu L.
      • Melotte C.
      • Van der Aa N.
      • Kumar P.
      • Das R.
      • Theunis K.
      • Cheng J.
      • Legius E.
      • Moreau Y.
      • Debrock S.
      • D'Hooghe T.
      • Verdyck P.
      • De Rycke M.
      • Sermon K.
      • Vermeesch J.R.
      • Voet T.
      Concurrent whole-genome haplotyping and copy-number profiling of single cells.
      ). Therefore, many proposed parental cell contamination testing methods involving gDNA samples from tissue without WGA cannot be used in PGT applications (
      • Liao G.J.
      • Chan K.C.
      • Jiang P.
      • Sun H.
      • Leung T.Y.
      • Chiu R.W.
      • Lo Y.M.
      Noninvasive prenatal diagnosis of fetal trisomy 21 by allelic ratio analysis using targeted massively parallel sequencing of maternal plasma DNA.
      ;
      • Lathi R.B.
      • Gustin S.L.
      • Keller J.
      • Maisenbacher M.K.
      • Sigurjonsson S.
      • Tao R.
      • Demko Z.
      Reliability of 46,XX results on miscarriage specimens: a review of 1,222 first-trimester miscarriage specimens.
      ;
      • Buchovecky C.M.
      • Nahum O.
      • Levy B.
      Assessment of Maternal Cell Contamination in Prenatal Samples by Quantitative Fluorescent PCR (QF-PCR).
      ). The haplotype algorithm and karyomapping technology were used to address the contamination issue in WGA products, but the performance was easily affected by low-quality amplification effects, such as ADO or preferential amplification bias (
      • Zamani Esteki M.
      • Dimitriadou E.
      • Mateiu L.
      • Melotte C.
      • Van der Aa N.
      • Kumar P.
      • Das R.
      • Theunis K.
      • Cheng J.
      • Legius E.
      • Moreau Y.
      • Debrock S.
      • D'Hooghe T.
      • Verdyck P.
      • De Rycke M.
      • Sermon K.
      • Vermeesch J.R.
      • Voet T.
      Concurrent whole-genome haplotyping and copy-number profiling of single cells.
      ). Moreover, these approaches failed to evaluate the degree of parental contamination, which is important to avoid the potential false negative result of embryos with a high degree of contamination in the application of PGT-A without embryo waste. For this reason, a parental orientation test was conducted based on the allelic ratio determined via BAF analysis to address the parental contamination issue in IVF-PGT. The features of our method are as follows: BAF analysis, considering the effect of contamination on the relative allele frequency; relative POB statistics are used, with SNP site selection of certain paternal and maternal genotypes, eliminating the confounding factor of random preferential amplification bias or even ADO from the WGA product; reference samples are applied to give the normal range of genetic bias from parents; and a mixed assay of the parents and child is conducted to measure parental contamination. Taken together, our method is sensitive for detecting parental contamination and is also available for the quantification of parental contamination. Moreover, combined with WGA-specific BAF pattern resolution, parental contamination or heteroploidy may be further discriminated (Supplementary Figure 4). According to the principle of qPCT, we believe that this method could be combined with other WGA protocols. Additionally, preimplantation genetic testing for monogenic disorders as well as preimplantation genetic testing for chromosomal structural rearrangements are theoretically feasible for conventional IVF insemination methods using qPCT. The method does not require specialized equipment or complex experimental procedures; therefore, it can be fully adapted for routine use in a molecular diagnostic laboratory. This approach, however, requires parental genetic information for combined analysis, thus limiting its large-scale clinical application to some degree. Moreover, a method of parental contamination removal is urgently needed to provide accurate PGT results for biopsy samples, even samples contaminated by parental genetic material. Epigenetic signal discrimination between embryonic and parental cell DNA may be a promising way to trace the cellular origin of DNA samples, which might filter out the effect of parental contamination in the future (
      • Chen Y.
      • Gao Y.
      • Jia J.
      • Chang L.
      • Liu P.
      • Qiao J.
      • Tang F.
      • Wen L.
      • Huang J.
      DNA methylome reveals cellular origin of cell-free DNA in spent medium of human preimplantation embryos.
      ).
      The biopsy of existing frozen IVF blastocysts to meet patients’ clinical needs increases embryo utilization and reduces physical and economic burdens. Therefore, IVF physicians and embryologists must understand and select the programmes that are available to them. Unlike in ICSI-PGT-A or fresh IVF-PGT-A (
      • Liao G.J.
      • Chan K.C.
      • Jiang P.
      • Sun H.
      • Leung T.Y.
      • Chiu R.W.
      • Lo Y.M.
      Noninvasive prenatal diagnosis of fetal trisomy 21 by allelic ratio analysis using targeted massively parallel sequencing of maternal plasma DNA.
      ;
      • Lathi R.B.
      • Gustin S.L.
      • Keller J.
      • Maisenbacher M.K.
      • Sigurjonsson S.
      • Tao R.
      • Demko Z.
      Reliability of 46,XX results on miscarriage specimens: a review of 1,222 first-trimester miscarriage specimens.
      ;
      • Buchovecky C.M.
      • Nahum O.
      • Levy B.
      Assessment of Maternal Cell Contamination in Prenatal Samples by Quantitative Fluorescent PCR (QF-PCR).
      ), cumulus cells are not required to be completely removed in conventional IVF when there is no PGT expectation (Figure 3c). As a result, it is difficult to avoid sperm cells, cumulus cells, or both, attached to the spherical zona pellucida during trophectoderm cell biopsy (Figure 3d). Moreover, the DNA of spermatozoa or granulosa cells disrupted by a laser might be dispersed into the biopsy fluid during biopsy. Therefore, it is necessary to detect parental contamination in biopsy cells under such conditions. Frozen IVF blastocyst biopsy samples can be resuscitated in advance, so the blastocyst trophectoderm cells can hatch from the holes generated by freezing and draining water or artificial openings made after warming. In the present study, 71.67% of the blastocysts were stage-5 and stage-6 blastocysts with trophectoderm cells located far away from the zona pellucida (Table 2), which could partly explain the reason for the low contamination detection rate in this study. Admittedly, the major disadvantages of the applied clinical strategy are that it requires invasive embryo biopsy, and that repeated cryopreservation of embryos is usually unavoidable. There are still safety concerns with these procedures (
      • Koch J.
      • Costello M.F.
      • Chapman M.G.
      • Kilani S.
      Twice-frozen embryos are no detriment to pregnancy success: a retrospective comparative study.
      ;
      • Zeng Y.
      • Lv Z.
      • Gu L.
      • Wang L.
      • Zhou Z.
      • Zhu H.
      • Zhou Q.
      • Sha J.
      Preimplantation genetic diagnosis (PGD) influences adrenal development and response to cold stress in resulting mice.
      ; 2017;
      • Zhao H.C.
      • Zhao Y.
      • Li M.
      • Yan J.
      • Li L.
      • Li R.
      • Liu P.
      • Yu Y.
      • Qiao J.
      Aberrant epigenetic modification in murine brain tissues of offspring from preimplantation genetic diagnosis blastomere biopsies.
      ;
      • Wu Y.
      • Lv Z.
      • Yang Y.
      • Dong G.
      • Yu Y.
      • Cui Y.
      • Tong M.
      • Wang L.
      • Zhou Z.
      • Zhu H.
      • Zhou Q.
      • Sha J.
      Blastomere biopsy influences epigenetic reprogramming during early embryo development, which impacts neural development and function in resulting mice.
      ;
      • Cimadomo D.
      • Capalbo A.
      • Ubaldi F.M.
      • Scarica C.
      • Palagiano A.
      • Canipari R.
      • Rienzi L.
      The Impact of Biopsy on Human Embryo Developmental Potential during Preimplantation Genetic Diagnosis.
      ). In future, rapid non-invasive chromosome screening or mutation identification combined with qPCT will be a promising research area (
      • Xu J.
      • Fang R.
      • Chen L.
      • Chen D.
      • Xiao J.P.
      • Yang W.
      • Wang H.
      • Song X.
      • Ma T.
      • Bo S.
      • Shi C.
      • Ren J.
      • Huang L.
      • Cai L.Y.
      • Yao B.
      • Xie X.S.
      • Lu S.
      Noninvasive chromosome screening of human embryos by genome sequencing of embryo culture medium for in vitro fertilization.
      ;
      • Liu W.
      • Liu J.
      • Du H.
      • Ling J.
      • Sun X.
      • Chen D.
      Non-invasive pre-implantation aneuploidy screening and diagnosis of beta thalassemia IVSII654 mutation using spent embryo culture medium.
      ;
      • Fang R.
      • Yang W.
      • Zhao X.
      • Xiong F.
      • Guo C.
      • Xiao J.
      • Chen L.
      • Song X.
      • Wang H.
      • Chen J.
      • Xiao X.
      • Yao B.
      • Cai L.Y.
      Chromosome screening using culture medium of embryos fertilised in vitro: a pilot clinical study.
      ;
      • Huang L.
      • Bogale B.
      • Tang Y.
      • Lu S.
      • Xie X.S.
      • Racowsky C.
      Noninvasive preimplantation genetic testing for aneuploidy in spent medium may be more reliable than trophectoderm biopsy.
      ;
      • Jiao J.
      • Shi B.
      • Sagnelli M.
      • Yang D.
      • Yao Y.
      • Li W.
      • Shao L.
      • Lu S.
      • Li D.
      • Wang X.
      Minimally invasive preimplantation genetic testing using blastocyst culture medium.
      ). As the clinical strategy yielded a clinical pregnancy rate of 66.67% without adverse outcomes, however, patients with existing cryopreserved conventional IVF embryos could be counselled regarding this new option.
      In conclusion, the risk of PGT in embryos with potential parental contamination is relatively low, indicating the feasibility of PGT-A for vitrified conventional IVF embryos. The method we established in this study is a practical and easy-to-implement clinical approach for detecting potential parental contamination. This method can be selected in cases of high suspicion of parental DNA contamination.

      Acknowledgements

      This study was funded by grants from the National Key Research and Development Program of China (2018YFC1002604) and the National Natural Science Foundation of China (81800184). The data used, analysed, or both, during the present study are available from the corresponding author upon reasonable request.

      Appendix. Supplementary materials

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      Biography

      Doctor Fenghua Liu is Deputy Dean of Guangdong Women and Children Hospital, a panelist of the National Assisted Reproductive Technology of China, and Executive Director of Guangdong Medical Association. Her main areas of interest are reproductive medicine, gynaecological endocrine diseases, treatment of infertility, PGT-A and PGT-M.
      Key message
      During PGT-A for human cryopreserved conventional IVF embryos, paternal source pollution is negligible, whereas maternal pollution is relatively low. The method established in this study is a practical and easy-to-implement clinical approach for detecting potential parental contamination. It can be selected in cases of high suspicion of maternal DNA contamination.