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Non-invasive preimplantation genetic screening using array comparative genomic hybridization on spent culture media: a proof-of-concept pilot study
Reproductive BioMedicine Online
Aneuploidy is the leading cause of implantation failure. Currently, the ability to identify euploid embryos from an available cohort is the Holy Grail in reproductive genetics. Currently, the standard practice for preimplantation genetic testing involves blastomere or trophoectoderm biopsy. Disadvantages include not only extra time, effort and expense for embryo biopsy, but also a concern for harming the embryo. Spent culture media has always attracted interest as a readily available sample which could reflect embryonic status. Feichtinger and co-workers report the use of spent culture media for assessment of embryonic euploidy by array comparative genomic hybridization in a proof of concept study. An interesting and potentially productive line of research, which could simplify preimplantation genetic screening and increase its uptake.
The aim of this pilot study was to assess if array comparative genomic hybridization (aCGH), non-invasive preimplantation genetic screening (PGS) on blastocyst culture media is feasible. Therefore, aCGH analysis was carried out on 22 spent blastocyst culture media samples after polar body PGS because of advanced maternal age. All oocytes were fertilized by intracytoplasmic sperm injection and all embryos underwent assisted hatching. Concordance of polar body analysis and culture media genetic results was assessed. Thirteen out of 18 samples (72.2%) revealed general concordance of ploidy status (euploid or aneuploid). At least one chromosomal aberration was found concordant in 10 out of 15 embryos found to be aneuploid by both polar body and culture media analysis. Overall, 17 out of 35 (48.6%) single chromosomal aneuploidies were concordant between the culture media and polar body analysis. By analysing negative controls (oocytes with fertilization failure), notable maternal contamination was observed. Therefore, non-invasive PGS could serve as a second matrix after polar body or cleavage stage PGS; however, in euploid results, maternal contamination needs to be considered and results interpreted with caution.
Keywords: NIPGS, PGS, Non-invasive, PGD, Culture-medium.
Over the past decades, childbearing in the western world has been increasingly delayed. The number of embryonic aneuploidies, however, accumulate with advanced maternal age, and contribute to high infertility rates observed in this group (Hassold and Hunt, 2001).
In those patients undergoing assisted reproduction techniques, the transfer of one single euploid embryo to achieve pregnancy and avoid multiples was associated with favourable pregnancy outcomes (Forman et al, 2014 and Thurin et al, 2004). Methodologies to screen for embryonic aneuploidies with full accuracy and precision to select one healthy embryo, however, have not yet been validated. Invasive approaches, such as biopsy of cleavage-stage embryos or trophectoderm cells and less invasive techniques, such as blastocoel fluid aspiration, polar body biopsy, time-lapse analysis and metabolomics or proteomics testing, have been used for the selection of one healthy embryo for transfer (Botros et al, 2008, Handyside et al, 1990, McReynolds et al, 2011, Meseguer et al, 2011, Palini et al, 2013, and Verlinsky et al, 1990). Most commonly used, invasive techniques offer a direct genetic evaluation of the embryo. Still, misleading results can occur as a result of possible mosaicism, and embryo impairment caused by the removal of one or several cells cannot be excluded (Cohen et al, 2007 and Fragouli et al, 2013). Non-invasive methods, such as time-lapse, or metabolomics or proteomics analysis of the culture medium, on the other hand, lack accuracy owing to their indirectness (Gardner et al., 2015). Polar body analysis as a minimally invasive technique was suggested to improve live birth rates in patients of advanced maternal age; however, as polar bodies only mirror the oocyte, no paternally derived or mitotic defects can be analysed (Capalbo et al, 2013 and Feichtinger et al, 2015).
DNA within the blastocoel was confirmed to show high concordance with embryonic ploidy (Magli et al, 2016 and Palini et al, 2013). Similar to blastocoel fluid, embryo culture medium was suggested to harbour DNA that can be used for further analysis (Palini et al, 2013 and Scaruffi et al, 2011). Therefore, the possibility of non-invasive preimplantation genetic screening (PGS) was discussed as a potential future technique for embryo assessment (Cohen et al, 2013, Hammond et al, 2016, and Lu et al, 2016).
In the present study, the feasibility of diagnosing embryonic ploidy status through spent culture media at the blastocyst stage was assessed and ploidy results compared with polar-body analysis results.
Materials and methods
Twenty-two blastocysts from advanced maternal age patients (>35 years) were selected after polar-body-based preimplantation genetic screening (PGS). After intracytoplasmic sperm injection (ICSI), all oocytes underwent array comparative genomic hybridization (aCGH) polar body PGS on 23 chromosomes. All patients gave their informed consent to participate in the study.
Pooled polar body biopsy and aCGH has previously been described in detail (Feichtinger et al., 2015). Hyaluronidase was used to dissociate the cumulus cells and mechanically remove them before ICSI to prevent maternal cell contamination. Assisted hatching was carried out using a Saturn™ Active 5 Laser system (Research Instruments, Falmouth, United Kingdom) 14–18 h after ICSI fertilization. The zona pellucida was opened close to the polar bodies, and both polar bodies were biopsied.
Thereafter, both polar bodies were placed together in a 0.2-ml micro tube with 2.5 µl phosphate-buffered saline. the Illumina SurePlex CGH Amplification System protocol (SurePlex; Illumina, Chicago, USA) was used to extract DNA. A total of 5-µl aliquots of the amplified probes were separated by gel electrophoresis for quality control and 1-µl aliquots were used for DNA quantification using a Qubit 2.0 Fluorometer (Life Technologies, Vienna, Austria). For aCGH, 8 µl of amplified DNA was labelled according to the 24sure V3 protocol (Illumina, Chicago, USA). Cy3 dCTP or Cy5 dCTP nucleotides were incorporated into the DNA using random primers and Klenow enzyme. After a labelling reaction of 2 h in a thermal cycler at 37°C, the samples were dried. The probes were then incubated with 25 µl of COT human DNA (1 µg/µl) and the volume was reduced to 3 µl at 75°C in a thermal cycler (Concentrator plus/Vacufuge plus, Eppendorf, Hamburg, Germany) at 220 g for 30 min. DNA denaturation was carried out in 21 µl of hybridization buffer containing 15% dextran sulphate for 10 min at 75°C. A total of 18 µl of DNA solution were used for bacterial artificial chromosome array hybridization at 4–16 h at 47°C.
After washing and drying according to the Illumina 24sure V3 protocol, the slides were scanned using a DNA Microarray Scanner (Agilent Technologies, Santa Clara, USA) at 10 µm resolution. Signals were called with Illumina software (BlueFuse Multi analysis software version 3.0), with adjustment for first polar body analysis as published previously (Feichtinger et al., 2015). Embryos were cultured in single 25-µl droplets using a single step medium (G-TL, Vitrolife, Gothenburg, Sweden) from fertilization until day 5/6 at 37°C with 20% O2 and 6% CO2 in a Galaxy Mini-A incubator (Eppendorf, Hamburg, Germany).
Five days after fertilization, 5 µl of culture medium were transferred into a 200-µl microtube and centrifuged (60 s, 4°C at 16,000 g) and stored at −20°C. DNA amplification was carried out according to the SurePlex CGH amplification system protocol (SurePlex; Illumina, San Diego, USA). After DNA amplification, 5-µl aliquots of the products were separated by gel electrophoresis as a quality control measure, and 1-µl aliquots were used for DNA quantification with a Qubit 2.0 Fluorometer (Life Technologies, Vienna, Austria).
Array CGH was carried out according to the 24 sure protocol (Illumina, San Diego, USA). Chromosomal aberrations were analysed using the Blue-Fuse Multi software (Illumina, San Diego, USA) as described above. Blastocysts were then scored by morphological criteria according to the Istanbul consensus based on the criteria by Gardner et al. (Alpha Scientists in Reproductive Medicine and ESHRE Special Interest Group of Embryology, 2011). Grading was from 1 to 4 according to their developmental stage (1 for an early stage blastocyst, 4 for hatched blastocysts), 1–3 according to inner cell mass morphology and 1–3 according to the trophectoderm morphology (1 = good quality, 3 = poor quality) (Gardner and Schoolcraft, 1999).
After aCGH analysis, concordance of polar body analysis and culture media genetic results was assessed. After concluding the experiments on blastocysts, negative controls were added to control for maternal contamination. Four oocytes after ICSI with fertilization failure and six samples of fresh culture media out of the bottle were used as negative controls. According to the protocol described above, oocytes after fertilization failure were consecutively cultured in the same medium and culture conditions as the study samples for 5 days and all control samples were amplified according to the protocol described above.
This study was approved by the Ethics Commission of the Medical University of Vienna on 16 February 2016 (reference number: 1978/2015).
Eighteen out of 22 culture media samples could be amplified (81.8%), with a mean DNA yield of 21.33 ng/µl (range 6.7–41); these samples were used for further analysis (Table 1). Thirteen out of 18 amplified samples (72.2%) revealed concordance of general ploidy status (euploid or aneuploid). In 11 out of 15 cases of polar body aneuploidy, the culture medium concordantly was evaluated as aneuploid (73.3%) (Figure 1 and Figure 2). In two out of three euploid tested polar bodies, the culture medium revealed a concordant euploid result (66.7%) (Figure 3). In 10 out of 15 polar body aneuploidy cases, at least one of the appropriate chromosomal aberrations of the polar body could be diagnosed in the culture medium as well (66.7%). Generally, 17 out of 35 single chromosome aneuploidies of the polar body were concordant in the amplified culture medium (48.6%). These results suggest a sensitivity of 73.3% of culture medium detecting aneuploidy based on polar body biopsy and a specificity of 66.7% with a positive predictive value of 91.7% and a negative predictive value of 33.3%. In the polar body samples, 35 aneuploidies could be observed whereas, in the culture medium; 34 could be diagnosed. In four cases, culture medium provided a euploid result after aneuploid polar bodies (22.2%). Of the negative controls, culture media derived from oocytes with fertilization failure revealed four out of four positive DNA amplifications and all six fresh culture media samples amplified out of the bottle were negative.
Day 5 embryo score: amplified DNA from culture media and ploidy status revealed by polar body and culture media analysis.
|Embryo||Day 5 embryo score||Culture medium DNA yield (ng/µl)||Polar body ploidy||Culture medium ploidy|
|1||2/2–2||25.2||Aneuploid −15, +16||Aneuploid +15, −16|
|2||2/2–2||22.2||Aneuploid −9, −15,+16||Aneuploid +9, +15, −22|
|3||Arrested||27.2||Aneuploid −19||Aneuploid +19|
|6||Compacted||17.1||Aneuploid −6||Aneuploid +6, +21|
|7||Compacted||9,3||Aneuploid +1,+2, −9,+21||Aneuploid +9, +20|
|8||Compacted||21.6||Aneuploid −13, −16,+18,+21||Aneuploid −1, +13|
|9||Compacted||17.7||Aneuploid −8, −9,+10, +15, −18||Aneuploid +9, −10|
|10||Early Blastocyst||14.5||Aneuploid +1, −14,+15, −18||Aneuploid −1, +8, −12 ,+14, −15, +18, −22|
|12||2/2–2||14.4||Aneuploid −21||Aneuploid −18|
|13||2/2–2||6.7||Aneuploid +6, −13||Aneuploid −2, +13, +14, −16, −17, −18, +19, +X|
|15||Early Blastocyst||41||Aneuploid −16, +22||Euploid|
|17||4/2–1||27.8||Aneuploid −6, +21, −22||Aneuploid −16, −21, +22|
|19||Arrested||Aneuploid +4, −9, +14, +22, −X||No amplification|
|20||2/2–2||Aneuploid +8, +12, +17, +22, +X||No amplification|
|21||Arrested||Aneuploid +19||No amplification|
Array comparative genomic hybridization (aCGH) plot showing a concordant aneuploidy result in polar body and culture medium aCGH analysis (Embryo number 3).
Array comparative genomic hybridization (aCGH) plot showing a partly concordant aneuploidy result in polar body and culture medium aCGH analysis (Embryo number 10).
Array comparative genomic hybridization (aCGH) plot showing a concordant euploid result in polar body and culture medium aCGH analysis (Embryo number 16).
In the present pilot study, we investigated the possibility of carrying out non-invasive aCGH ploidy evaluation on blastocyst culture medium. Genomic DNA could be amplified from blastocyst culture media in more than 80% of cases. Ploidy status of the embryo diagnosed by polar-body biopsy corresponded with the diagnosis of the culture media in most cases. Maternal contamination, however, has to be considered and is likely to be the reason for false euploid results.
During its preimplantation development, the embryo undergoes rapid dynamic transformation resulting in high gain and loss of cells (Hardy et al., 1989). Generally, extensive cell-fragmentation is associated with reduced embryo quality and IVF outcome (Chavez et al, 2012 and Ebner et al, 2001). In blastocysts however, extensive cell fragmentation and apoptosis even takes place in genetically healthy embryos (Hardy et al., 2003). Fragmented cells are expelled into the blastocoel fluid and the perivitelline space, potentially analysable material entering the culture medium after hatching (Sathananthan et al., 2003).
The possibility of amplifying cell-free DNA from culture medium to test for X-linked diseases, cystic fibrosis or alpha-thalassemia has been investigated (Assou et al, 2014, Galluzzi et al, 2015, and Wu et al, 2015). In the present study, however, we were able to amplify whole genomes, making it likely that whole trophectoderm or inner cell mass cells expelled into the culture medium had been analysed. In fact, previous studies observed blastocysts extruding whole cells into the perivitelline space (Alikani et al., 1999). In another study investigating mitochondrial and genomic DNA content in culture media (Stigliani et al., 2013), more than 99% of day 2 and 3 embryos had DNA in their culture medium, and the amount of amplified DNA was correlated with maternal age and embryo morphology. In this study, however, ploidy status of the embryos was not investigated. Recently, several groups have investigated the feasibility of PGS on culture medium. Shamonki et al. (2016) presented data in a proof of concept study that nuclear DNA can be amplified from culture media. This group, however, reported reliable amplification results only in a few amplified cases and did not provide results on concordance levels between the culture medium and the embryo (Shamonki et al., 2016). Xu et al. (2016) applied next-generation sequencing techniques on previously vitrified donated embryos, showing high sensitivity and specificity when non-invasive chromosome screening on culture media was applied. Despite these promising results, a recent study reported mitochondrial and nuclear DNA in spent culture media to be of mixed origin owing to maternal cell contamination (Hammond et al., 2017).
In our study, DNA amplification could be carried out in good- and bad-quality embryos, and our results suggest that expelled embryonic cells might reflect general embryo ploidy status in most cases. It should be considered, however, that embryonic cells in the culture medium might reflect aneuploid cells actively eliminated by the embryo. One previous study on induced mosaicism in mouse embryos has shown that mosaic embryos possess certain self-correcting mechanisms eliminating aneuploid cells through apoptosis during blastocyst development (Bolton et al., 2016). Our data, however, could show that, in two-thirds of embryos with (meiotic) aneuploidies detected by polar-body analysis, at least one chromosomal aberration diagnosed in the polar bodies could be detected in the appropriate culture media sample, making it unlikely that embryonic cells in the culture media consist solely of extruded aneuploid cells not fully representative of the mosaic embryo. Similar to cleavage stage blastomeres, amplified culture media DNA mirrors chromosome aneuploidies detected in the polar bodies: chromosome trisomies of the embryo and embryonic cells in the culture medium appear as monosomies in the polar bodies and vice versa.
Biopsy of cleavage-stage embryos and trophectoderm cells has been associated with reduced implantation potential possibly caused by embryonic damage (Cohen et al, 2007 and Zhang et al, 2016). All embryos in our study underwent ICSI, polar-body biopsy and assisted hatching; no additional invasive procedure potentially impairing the embryo has been applied in this study. The artificial opening in the zona pellucida could be the reason why single whole cells or cell-free DNA reach the culture medium during embryonic development.
In the present study, in 72.2% of aneuploid embryos diagnosed by polar body analysis, the clinical euploidy or aneuploidy diagnosis could be confirmed in the culture medium, with 48.6% of concordant aneuploidies per chromosome. In a previous study by Capalbo et al. (2013), 61.7% of polar body diagnosed aneuploidies in a trophectoderm biopsy could be detected, with a concordance rate of 38.1% per chromosome; and 20% of blastocysts tested euploid even though polar body biopsy revealed an aneuploid result. The present study shows similar concordance rates between the polar body and culture media analyses.
When applying PGS to culture medium, contamination with maternal cells could lead to false negative results, hence precautions against maternal cell contamination should be taken, such as ICSI fertilization and removal of cumulus cells. In cases of detection of euploidy by NIPGS after aneuploidy detection by polar body biopsy, however, maternal contamination cannot be ruled out completely at present, even though thorough precautions have been taken to prevent parental cell contamination. Our results suggest that a significant amount of DNA found in the culture media might reflect maternal DNA as observed in cultured oocytes with fertilization failure serving as negative controls. Out-of-the-bottle culture media yielded no amplifiable DNA; therefore, maternal (and not culture media inherent) contamination may be hypothesized.
A present, therefore, culture medium analysis cannot be recommended without combining it with other techniques to either control for contamination or in combination with established PGS techniques.
All embryos analysed in the present study were cultured until day 5 in a single-step culture medium. In contrast, the study by Xu et al. (2016) used culture media of vitrified-warmed cleavage stage embryos cultured until blastocyst stage with very promising sensitivity and specificity rates. Whether exchanging culture media at one or several occasions could reduce maternal contamination remains to be investigated. The favourable results obtained in the study by Xu et al. (2016), however, might have been derived from exchanging culture media at day 3 and by using next-generation sequencing. In their study, only one out of four false-positive embryos was female, suggesting low rates of maternal contamination. On the other hand, in contrast to the study by Xu et al. (2016), through the opening of the zona pellucida, we suspected a higher amount of analysable DNA to be present in the culture medium. By culturing the embryos in a single-step medium until the blastocyst stage, we assumed that, over time, a higher number of cells could move from the embryo to the culture medium. Future studies are necessary to investigate if sampling culture medium at earlier time points will offer similar results and amplification rates. In the study by Xu et al. (2016), 100% of samples could be analysed; our results suggest that even by culturing embryos until the blastocyst stage, and by carrying out assisted hatching, 20% of embryos do not extrude a sufficient amount of cells into the culture medium and amplification fails. Additionally, in our cohort of embryos, several culture media samples of bad-quality embryos were analysed, even though they would normally not be selected for transfer or even for trophectoderm biopsy.
All patients included in this study received polar-body analysis for preimplantation genetic screening, the standard technique for PGS used at our institution, in accordance with legislative regulations. Using trophoblast biopsy as a second control was not possible because of legislation restricting embryo donation for science and PGS on embryos. By using polar-body PGS, only maternally derived meiotic aneuploidies can be diagnosed, whereas mitotic aneuploidies, mosaicism and paternally derived aneuploidies, which are thought to contribute to 3–4% of chromosomal aberrations, are not detected by this method (Hassold and Hunt, 2001). Additionally, possible pitfalls of polar body analysis, such as trisomic rescue or reciprocal aneuploidy, resulting in euploid embryos after aneuploid tested polar bodies, have to be considered when comparing polar body and culture media genetic results (Forman et al., 2013). Up to 20% of embryos shown to be aneuploid by polar-body biopsy have been shown to be euploid after PGS analysis at the blastocyst stage, possibly caused by postzygotic rescue mechanisms (Capalbo et al, 2013, Christopikou et al, 2013, and Handyside et al, 2012). Therefore, in practice, after maternal contamination is ruled out, culture medium analysis could serve as a second matrix to allow higher accuracy of embryo ploidy evaluation and prevent discarding embryos that underwent rescue mechanisms in their cleavage state. In the case of good embryonic development, the culture media of these embryos shown to be aneuploid by polar-body biopsy could be analysed and embryos cryopreserved if showing a euploid karyotype. In the case of euploid results after culture medium-based, PGS, maternal contamination could be ruled out by short tandem repeat analysis; however, these additional tests would impair the overall cost-efficiency of non-invasive PGS.
These findings demand additional data and reason for caution in the application of culture medium based PGS for embryo aneuploidy. At present, maternal contamination in the case of euploid results after aneuploid polar-body testing cannot be ruled out even if maximum precautions against parental contaminations have been made. In case of aneuploidy, culture media analysis had a 91.7% positive predictive value and a sensitivity of 73.3% predicting the aneuploidy diagnosed in the polar body analysis. Specificity and negative predictive value, however, were low with 66.7% and 33.3%, respectively, most likely caused by maternal cell contamination.
In conclusion, this pilot study suggests the possibility of carrying out aCGH-based ploidy evaluation on blastocyst culture medium. Genomic DNA can be amplified from blastocyst culture media, possibly after cell expulsion from the blastocyst into the perivitelline space and the culture medium. Culture media contamination, however, needs to be considered and derived euploid results interpreted with caution. Larger studies are needed to confirm these results and to investigate diagnostic accuracy of this technique.
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Michael Feichtinger is a resident and PhD candidate at the Department of Obstetrics and Gynaecology at the Medical University of Vienna, Austria. His research focus is preimplantation genetic screening, reproductive epidemiology and endocrinology. In spring 2016, he undertook a scientific research fellowship at the Reproductive Unit of Karolinska University Hospital in Stockholm.
Ploidy evaluation on blastocyst culture medium based on array-comparative genomic hybridization seems possible. Genomic DNA can be amplified from blastocyst culture media, possibly after cell expulsion from the blastocyst into the perivitelline space and the culture medium. Culture media contamination needs to be considered, and euploid results interpreted with caution.
a Wunschbaby Institut Feichtinger, Lainzerstrasse 6, 1130 Vienna, Austria
b Department of Obstetrics and Gynaecology, Division of Gynecologic Endocrinology and Reproductive Medicine, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria
c IVF Unit, Cervesi Hospital Cattolica, Cattolica, 47841 Rimini, Italy
* Corresponding author.
© 2017 Reproductive Healthcare Ltd., Published by Elsevier B.V.