Using the CytoSure™ Embryo Screen array to identify aneuploidies and large structural imbalances in Polar Bodies 1 and 2

Thursday 23 July 2015
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K.R. Held1 , S. Knebel1 , V. Baukloh2

1. Reprogenetics Germany GmbH, 2. MVZ Fertility Center Hamburg GmbH

In this whitepaper learn how a team from Reprogenetics Germany and the MVZ Fertility Center, Hamburg have demonstrated the effective use of polar body screening as a method to detect large chromosomal abnormalities and show how parallel processing of 14 samples on one slide offers benefits to high-throughput laboratories over more traditional BAC arrays.

Introduction

Chromosome abnormalities are extremely common in human embryos particularly those generated during in vitro fertilisation (IVF). Embryos containing the wrong number of chromosomes (aneuploidy) or chromosomes with large structural abnormalities are known to be a major factor of pregnancy failure. Most anueploidies are maternal in origin and are more prevalent in older women. To maximise the chance of successfulI IVF, it is important that only chromosomally normal embryos are selected for implantation. Historically embryos have been screened using fluorescence in situ hybridisation (FISH); however, this is somewhat limited due to the availability of only five fluorochromes, meaning that a maximum of five chromosomes can be examined at the same time. The introduction of single cell amplification and array comparative genomic hybridisation (aCGH) for embryo screening has enabled the whole genome to be screened for aberrations at a resolution of several megabases as well as for whole chromosome anueploidies. But using aCGH is not without its challenges, for example mosaicism can affect embryos during cleavage stage and can lead to an incorrect result. To overcome this limitation, embryos have been tested earlier in development by screening polar bodies (PB) and later in development using the trophectoderm.

Screening PBs is an indirect way of examining the embryo. PBs form early in embryo development, with the first PB being formed at the completion of the first meiotic division, and contains a single copy of the maternal chromosomes. Errors in this division can be seen by analysing PB1, for example two copies of a chromosome in PB1 indicates that this chromosome will be missing in the oocyte. PB2 is formed after fertilisation and the completion of the second meiotic division. Each chromosome is split into two chromatids and one set of chromatids is passed to the second polar body. Examination of PB2 can reveal errors in the second meiotic division.

During the IVF process, the health and development of the embryo is critical. Once formed PBs are no longer required by the embryo and will spontaneously degrade. Therefore it is thought that PB biopsy is less invasive than blastomere biopsy and so has a lower impact on embryo development. In addition the process by which the PBs form ensures that there is no possibility that the results are distorted by mosaicism. The major application of polar body analysis is the detection of maternally derived chromosomal aneuploidies and translocations in oocytes — paternally acquired abnormalities cannot be identified. In addition, abnormalities acquired after the second meiotic division are not detected; however, research1 shows that as the majority of abnormalities arise during the first or second meiotic division, the analysis of PBs is an accurate way of identifying genetic abnormalities in embryos.

Aneuploid oocytes

Figure 1: Aneuploid oocytes due to chromatid predivision in meiosis I (left) or failure of chromatid separation in meiosis II (right).

The aim of this study is to evaluate the CytoSure Embryo Screen array from Oxford Gene Technology (OGT) for ease of use in a high-throughput lab, and its ability to correctly identify aneuploidies and large structural aberrations in both PB1 and PB2.

Materials and Methods

PB1 and PB2 biopsies were performed on 61 embryos and the DNA amplified using the PicoPlex™ WGA Kit (Rubicon Genomics). The amplified material was then labelled using the CytoSure HT Genomic DNA Labelling Kit (OGT, cat. no. 500040). Both reference and PB samples were labelled with Cy3 and Cy5. Following purification, the DNA concentration and dye incorporation rate was determined using a NanoDrop™ spectrophotometer (Thermo Fisher Scientific). Samples were then combined as shown in Figure 2 and hybridised to a CytoSure Embryo Screen Array (OGT, cat. no. 020044). Detailed protocols for sample preparation, amplification and labelling and array setup can be obtained from OGT (www.ogt.com). All results were compared against those obtained using an alternative BAC-based array according to the standard manufacturers’ protocol.

Reference and PB sample layout on the CytoSure Embryo Screen arrayFigure 2: Reference and PB sample layout on the CytoSure Embryo Screen array. The array allows the simultaneous analysis of up to fourteen samples.

Following hybridisation the slides were washed and scanned according to the standard OGT protocol. The resulting image files were extracted using standard image analysis software (Agilent, Feature Extraction Software v.11.0.1.1). The resultant text file was analysed using CytoSure Interpret Software v4.7.16 using the recommended settings for preimplantation genetic screening (PGS).

Results

In total 112 samples were run on 7 CytoSure Embryo Screen array slides. These 112 samples comprised of 8 reference samples (2 per hybridisation) and 104 PB samples which had been selected from the 61 different embryos, note that both PB1 and PB2 were not always available from each embryo. In general, with the exception of 3 samples which were excluded from the study, the sample quality was good, therefore the data from 101 samples were analysed (Table 1). The number of chromosome and chromatid gains and losses were very similar between platforms; however, chromatid gains and losses were more frequent than whole chromosome gains and losses. In addition a higher frequency of chromatid gains and losses were observed in the second polar body compared to the first.

Table 1: Results of aberrations detected on the CytoSure Embryo Screen array platform. Table 1: Results of aberrations detected on the CytoSure Embryo Screen array platform.

CytoSure Embryo Screen array data were analysed using CytoSure Interpret Software (Figure 3) and specific chromosomal gains and losses were identified in each sample.

Screenshot from CytoSure Interpret Software showing the dedicated PGS analysis tabFigure 3: Screenshot from CytoSure Interpret Software showing the dedicated PGS analysis tab which summarises the results of eight samples. The detailed view of sample 4 clearly shows a significant gain of chromosome 15.

The results for each sample from CytoSure Interpret Software were compared to previous findings on the BAC-based array platform. In total, 7 out of 101 samples showed slight differences in the aberrations reported (Table 2). All but one of the discrepancies observed occurred in the first polar body of oocytes considered to be aneuploid irrespective of the observed discrepancy. As such, the resultant conclusion for the oocyte would therefore not differ between platforms. Recent improvements to CytoSure Interpret Software, which now calculates the call against both male and female references, have enhanced analysis of X chromosome aberrations. Further confirmatory studies would be required to confirm which platform was correct.

Discrepancies detected between the BAC-based array platform and OGT’s CytoSure Embryo Screen arrays platformTable 2: Discrepancies detected between the BAC-based array platform and OGT’s CytoSure Embryo Screen arrays platform . # = whole chromosome, sc = single chromatid.

The correct differentiation between chromosome and chromatid malsegregation is of major importance as a chromatid malsegregation in meiosis I can be corrected in meiosis II, whereas a chromosome malsegregation will usually result in an aneuploid oocyte.

A broad range of aberration size and complexity was detected from whole chromosome gains and losses down to structural aberrations of several megabases (Figure 4).

Polar body 1: top panel BAC-based array, bottom panel OGT CytoSure Embryo Screen arrayPolar body 2: top panel BAC-based array, bottom panel OGT CytoSure Embryo Screen array

Figure 4: An example of a translocation t(8;21)(p10;q10) that was observed in both polar bodies on both platforms. (A) Polar body 1: top panel BAC-based array, bottom panel OGT CytoSure Embryo Screen array. (B) Polar body 2: top panel BAC-based array, bottom panel OGT CytoSure Embryo Screen array.

Discussion

Working with PBs ideally requires the processing of both PB1 and PB2 from each embryo, which means that, even with a relatively low number of embryos (typically between 6 and 10) being generated per IVF cycle, the number of samples that require analysis can quickly build-up. Working with the 8x60k multiplex format of the CytoSure Embryo Screen array and the optimised CytoSure HT Genomic DNA Labelling Kit ensured that processing of 112 samples is straightforward and easily completed over several days by only one or two laboratory personnel. A key component of the streamlining process is the ability to run the arrays in a single colour mode. This allows 14 PBs to be tested on a single array and only 2 reference samples need to be used. This reduces the number of labelling reactions per sample and minimises the volume of reagents required for hybridisation and washing as only 7 slides require processing. This is in contrast to the BAC platform, which would require over 20 slides to be processed. As well as reducing the amount of reagents needed, processing only 7 slides also reduces the time taken for scanning, which at 15 minutes per slide (depending on the scanner used), is a time consuming step in the process. Overall, the practical improvements made to the labelling, hybridisation, washing and scanning steps resulted in a noticeable reduction in hands-on time and improved efficiency.

The study demonstrated good concordance between the CytoSure Embryo Screen array and the traditional BAC-based array platform. The majority of discrepancies observed would not alter the conclusion on embryo euploid/aneuploid state. All the differences pertain to the number of chromatids and were observed more often in duplications rather than deletions due to the fact that the shift in the amplitude is smaller in duplications than in deletions. Further confirmatory studies would be required to confirm which platform was correct. The detection of both whole chromosome aneuploidies and smaller structural aberrations (~10Mb) was easily achieved using CytoSure Interpret Software with its dedicated PGS functionality. PGS-specific features include automatic loading and processing, an optimised smoothing algorithm and a simplified view of the embryo status. The reporting of PGS data has also been tailored to the needs and requirements of IVF laboratories, with the facility to report the results of multiple embryos in a single document, providing results of the overall treatment rather than specific samples. A pre-requisite for robust embryo screening is the analysis of both polar bodies and a sufficient number of fertilised oocytes (5-8). As was shown in the study, the occurrence of poorquality samples is not uncommon, so it is also possible to reflect this in the final report with the option to designate samples as “not tested” thus providing a more complete picture of the screening.

In conclusion, the CytoSure Embryo Screen array proved to be a reliable, accurate and easy-to-use platform suited to a high-throughput IVF clinic screening embryos for the correct genetic content utilising polar bodies. Pregnancy rates and births can be increased per transfer and per cycle by polar body analysis (unpublished data). In countries where pre-implantation genetic diagnosis (PGD) or screening (PGS) via blastomere or trophectoderm biopsy is not permitted, screening of polar bodies may offer a viable alternative.

References

  1. Christopikou, D. et al (2013) Polar body analysis by array comparative genomic hybridization accurately predicts aneuplodies of maternal meiotic origin in cleavage stage embryos of women of advanced maternal age. Human Reproduction, Vol.28, No. 5, 1426-1434

CytoSure: For research use only; not for use in diagnostic procedures

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