Extent of Chromosomal Mosaicism Influences the Clinical Outcome of in Vitro Fertilization

EXTENT OF CHROMOSOMAL MOSAICISM INFLUENCES THE CLINICAL OUTCOME OF IN VITRO FERTILIZATION TREATMENTS

Francesca Spinella, Ph.Da, Francesco Fiorentino, Ph.Da*, Anil Biricik, Ph.Da, Sara Bono, Ph.Da, Alessandra Ruberti, BScb, Ettore Cotroneo, Ph.Da, Marina Baldi, Ph.Da, Elisabetta Cursio, BSc b, Maria Giulia. Minasi, BScb, and Ermanno Greco, M.Db.

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aGENOMA Group - Molecular Genetics Laboratories, Via di Castel Giubileo, 11, 00138 Rome, Italy

bReproductive Medicine, European Hospital, Via Portuense, 700, 00149 Rome, Italy

*Corresponding Author:

Francesco Fiorentino, PhD,

GENOMA Group - Molecular Genetics Laboratories

Via di Castel Giubileo, 11 00138 Rome, Italy.

Tel.:+39068811270 Fax: +390685344693

E-mail:

Running title: Chromosomal mosaicism influences IVF outcome

Supplementary data

IVF and embryo biopsy procedure

Patients enrolled in this study were treated with a stimulation protocol and intracytoplasmic sperm injection (ICSI), as previously described (1). On day 3 of embryo development, a hole was made through the zona pellucida of all cleaving embryos using a laser (Research Instruments, Cornwall TR11 4TA, UK) to facilitate blastocyst hatching. All embryos were individually cultured at 37°C, 6.0% CO2, and 5.0% O2 in droplets of sequential culture media (Quinn’s Advantage Medium; SAGE, USA) under oil and graded every day until blastocyst stage. A medium changeover was performed on day 3 and on day 5. All blastocysts reaching at least an expansion of grade 3, with a distinct inner cell mass and an adequate cellular trophectoderm (TE) (2), were biopsied. The remaining slower growing embryos were reassessed on day 6 and on day 7 for possible TE biopsy with subsequent vitrification.

Approximately 8–10 TE cells were aspirated with a biopsy pipette (COOK, Ireland Ltd, Limerick, Ireland) and removed with the use of a laser. All biopsy procedures were performed in droplets of buffered medium (HEPES, Sage In-Vitro Fertilization, Inc., Trumbull, CT, USA) overlaid with mineral oil on the heated stage of a Nikon IX-70 microscope, equipped with micromanipulation tools. After biopsy, the TE cells were washed in sterile phosphate-buffered saline (PBS) solution (Cell Signalling Technologies, Beverly, MA, USA), placed in 0.2 ml PCR tubes containing 2 µl PBS, and then transferred to the GENOMA laboratory to be processed by NGS analysis.

The transfer of mosaic embryos detected by NGS analysis was offered to a consecutive non-selected series of women in which no euploid embryos were available. All mosaic blastocysts were transferred after cryopreservation in a subsequent natural cycle, in order to have time to perform an appropriate genetic counselling in which the patients were informed about the PGS results achieved and the potential consequences related to the transfer of each specific mosaic embryo. All transfer procedures were performed with the use of a catheter (Wallace; Smiths Medical, Dublin, Ireland) under direct ultrasound guidance. Vitrification and warming were carried out at room temperature according to the Kuwayama protocol with Cryotop (3). The media employed were vitrification and warming kits (Kitazato Vitrification Kit; BioPharma, Shizouka, Japan).

Single-cell isolation and reconstitution experiments

Fetal cells derived from amniotic fluids were cultured under standard conditions. G-banding assay for conventional karyotype and array-CGH (4) were performed on the cell lines to confirm the presence or absence of aneuploidies and verify the stability of each cell line. The cell lines had the following karyotypes: 46, XY, 47, XX,+21, 47,XX,+18. Cells were dissociated using trypsin-EDTA at 37°C for 3 minutes. The resulting cells were subsequently washed with phosphate buffered saline buffer (PBS, SIGMA) and sorted with a flow sorter FACS Aria II SE (BD Biosciences). A defined number of euploid cells were added to a 96 well plate, and the plate was then washed three times with PBS. Finally, aneuploid cells were added to the same 96 well plate to obtain different ratios of euploid/aneuploid cells. Plates containing only euploid or aneuploid cells were included, in order to obtain the individual default copy number for a disomic or trisomic status, respectively. A total of 100 experiments were performed for samples containing euploid cells only, in order to define the thresholds for euploid call To obtain a condition closer to a trophectoderm biopsy, a sample size of 10 cells was initially chosen to set the mosaicism reconstruction experiments. However, experiments with a total of 100 and 1000 cells were also performed in order to minimize the risk of bias due to cell damage. A total of 228 samples in which the euploid/aneuploid ratios were 10:0; 9:1; 8:2; 7:3; 6:4; 5:5; 4:7; 3:7; 2:8; 1:9 and 0:10, respectively, were prepared in triplicate (Table S1 and S2).

Whole genome amplification

For WGA, cells were first lysed and genomic DNA was randomly fragmented and amplified using the SurePlex DNA Amplification System (Illumina Inc., San Diego, USA), according to the manufacturer’s protocol. This proprietary single tube technology is based on random fragmentation of genomic DNA and subsequent amplification by PCR utilizing flanking universal priming sites, as previously described (5-7).

Briefly, biopsies collected in 2.5 μL of 1x PBS were lysed using 2.5 μL of SurePlex cell extraction buffer and 5μL of the SurePlex Extraction cocktail master mix with incubation at 75°C for 10 minutes followed by incubation at 95°C for 4 minutes. The random fragmentation of genomic DNA was carried out by adding 5 μL of SurePlex Pre-amplification cocktail to the lysed biopsy samples or to genomic DNA controls and incubating the mixture according to the following protocol: one cycle of 95°C for 2 min, followed by 12 cycles of 95°C for 15s, 15°C for 50s, 25°C for 40s, 35°C for 30s, 65°C for 40s and 75°C for 40s, followed by a hold at 4°C. Thereafter, 60 μL of freshly prepared Sureplex Amplification cocktail was added to 15 μL of synthesis product in each reaction tube. Resulting mixtures were amplified according to the following thermal cycler program: one cycle of 95°C for 2 min, followed by 14 cycles of 95°C for 15s, 65°C for 1 min and 75°C for 1 min, followed by a hold at 4°C. To assess the success of the amplification, 5μL of each amplified sample plus 5μL gel loading buffer were examined by electrophoresis on a 1.5 % agarose 1x TBE gel. WGA products were then quantified using the Qubit® dsDNA HS Assay Kit (Life Technologies Corporation, Grand Island, NY, USA).

Array-CGH analysis

WGA products were processed with 24sure V3 microarrays (Illumina, Inc.), according to the manufacturer’s protocol. Briefly, amplified samples, controls and some reference DNAs (Illumina, Inc.) were labelled with Cy3 and Cy5 fluorophores using random primers of the 24sure V3 Pack (Illumina, Inc.), which contains the reagents needed to perform an assay, including: 24sure V3 arrays, Fluorescent Labelling System [dCTP] and Cot Human DNA. Every batch of biopsied samples requires hybridization of four labelled reference DNA samples; two male and two female. These were compared in silico with the intensities from biopsied sample hybridizations run at the same time in the same batch. The resulting labelling mixes were combined and co-precipitated with Cot Human DNA in preparation for hybridization. Labelled DNA was resuspended in dextran sulphate hybridization buffer and hybridized under cover slips to 24sure V3 slides (8-10). Thereafter, the labelled products were hybridized to 24sure V3 slides and washed to remove unbound labelled DNA. A laser scanner was used to excite the hybridized fluorophores read and store the resulting images of the hybridization, as described elsewhere (8,9). BlueFuse Multi software was developed to enable the analysis of the 24sure V3 experiments, including the automated creation of a reference database, using a single batch import file. The analysis of 24sure single channel experiments was fully automated and proceeded in a similar way to all BlueGnome microarrays. The software automatically combines the data from the single channel sample experiments with both male and female references from the hybridized reference subarrays, to produce a single fused result compared with a sex matched and a mismatched reference. Once a specific amplification was observed (i.e., low autosomal noise), autosomal profiles were assessed for gain or loss of whole chromosomal ratios using a 3 x SD assessment, greater than ±0.3 log2 ratio call, or both. To pass hybridization quality control, female samples hybridized with a male reference DNA (sex mismatch) had to show a consistent gain on the X chromosome and a consistent loss of the Y chromosome (6,11).

NGS analysis

Libraries were prepared using the VeriSeq PGS workflow (Illumina, Inc.). DNA ‘indexing’ (12) was performed in order to simultaneously analyze embryos from different patients, using the Veriseq Index Kit-PGS (Illumina, Inc.). During the library preparation step, the input DNA is tagmented (tagged and fragmented) by the NexteraTM XT transposome. The Nextera transposome simultaneously fragments the input dsDNA and adds adapter sequences to the ends, allowing amplification by PCR in subsequent steps. A limited-cycle PCR reaction uses these adapter sequences to amplify the insert DNA. The PCR reaction also adds index sequences on both ends of the DNA, thus enabling dual-indexed sequencing. One nanogram of quantified dsDNA template at 0.2 ng/µl was added to 5 µl of Amplicon Tagmentation Mixture (ATM) and 10 µl of Tagmentation DNA Buffer (TD). The tagmented DNA was amplified via a limited-cycle PCR. PCR product clean-up used AM Pure XP beads (BeckamCoulter, Brea, CA, USA) to purify the library DNA. Purified libraries were eluted with 50 µl of the Nextera XT Resuspension Buffer.

Each indexed library was normalized by beads and then multiplexed in 24-plex library pools.

Single-end, dual index 36 base pair reads (1 × 36 double index ) sequencing was performed using the Illumina v3 chemistry workflow on a MiSeq sequencer (Illumina, Inc.) with the MiSeq Reagent Kit v3-PGS (Illumina, Inc.), which contains the ready to load on-board clustering and SBS chemistry reagents. A sample sheet, used by both the MiSeq system and Bluefuse software, was generated using BlueFuse Workflow Manager. Reads were demultiplexed and aligned to the human genome hg19 by the on-instrument MiSeq Control Software (MCS v2.5). BAM files from the MiSeq system are imported directly into the BlueFuse Multi (4.1) analysis software (Illumina, Inc.) using the prepared sample sheet. BlueFuse Multi (4.1) analysis software processes and displays the data to provide genomic profiles of each sample in a run. The samples acceptance criteria was a number of total reads > 700,000 with a number of reads passing filter > 500,000.

As previously described (8,9) the count data in each bin was normalized using GC content and in silico reference data in order to remove bias, and copy numbers were determined using of a combination of a Gaussian probability function (PDF; with copy number states 0–4 and a standard deviation of 0.33) and thresholding. The copy number state with the highest probability for a chromosome was used unless the distance to the next most probable copy number was >0.011. In that case, the median value of the most likely copy number states of all bins of a chromosome was used, set to a gain when >2.5 and to a loss when <1.5.

Concordance analysis.

Copy number calls automatically generated by the NGS or array-CGH pipeline and BlueFuse Multi were assessed manually. The results were compared to the karyotype of cells and the concordance was calculated with the use of the classifications true positive (TP; gain or loss detected), true negative (TN; euploidy status confirmed), false negative (FN; gain or loss missed), and false positive (FP; additional gain or loss called).

Evaluation of Sensitivity and Specificity of Aneuploidy Screening by NGS.

To assess the reliability of NGS for aneuploidy detection, the sensitivity, and specificity, of the test were calculated as follow:

Specificity: no. of true negatives/(no. of true negatives + no. of false positives)

Sensitivity: no. of true positives/(no. of true positives + no. of false negatives).

Sensitivity defines the probability that the aneuploidy call will be positive when aneuploidy is present (true positive rate). Specificity defines the probability that the aneuploidy call will be negative when aneuploidy is not present (true negative rate).

A sample was classified as euploid when all chromosomes showed a copy number value within the normal ploidy line. A sample was classified as aneuploid when an automatic call for one or more chromosome was recorded. A sample was classified as diploid/aneuploid when chromosomal mosaicism and no aneuploidy on other chromosomes was detected in the same sample.

Statistical Analysis

Results were reported as average ± standard deviation (SD) from at least three replicated experiments for each group of interest. Chromosome copy number values for each percentage of mosaicism in different sets of experiments were compared using T-test with corresponding P values for each comparison made. Clinical outcome between different groups of mosaic embryos or between euploid and mosaic embryos was compared using a chi-square test or Fisher exact test for significance. P values were determined to be significant at p<0.05 using PRISM software (GraphPad Software, Inc., San Diego, CA).

Results

NGS and array-CGH analysis of reconstructed mosaic samples revealed that for each ratio of euploid to aneuploid cells, there was a linear increase in abnormal chromosome copy number with increasing number of trisomic cells or a decrease in chromosome copy number with an increase in monosomic cells (Supplemental Figure 1).

In total, 228 samples were assessed for mosaicism detection, 162 chromosomal mosaics (true positive) and 30 aneuploid and 36 euploid (true negative) samples. All euploid and aneuploidy and 144/162 of mosaic samples were correctly classified with NGS. The 18 false negative results obtained with NGS were from samples with 10% mosaicism. Sensitivity was 90% (95% confidence interval [95% CI]:84.66% to 93.96%) and specificity was 100% (95% confidence interval [95% CI]: 73.54% to 100.00%). This study indicated a limit of mosaicism detection of ≥20%.

Array-CGH identified 192/228 samples with a sensitivity and specificity for mosaic call of 84.21% (95% CI 78.82% -88.69%) and 100% (95% CI 90.26%-100.00%) respectively. The 36 samples produced discordant results with array-CGH were samples with < 20% mosaicism. These results demonstrated that both PGS methodologies are capable to detect mosaicism but showed different sensitivity. NGS has higher sensitivity compared to array-CGH.

References

1.  Greco E, Litwicka K, Ferrero S, Baroni E, Sapienza F, Rienzi L, et al. GnRH antagonists in ovarian stimulation for ICSI with oocyte restriction: a matched, controlled study. Reprod Biomed Online 2007;14:572–578.