Non-invasive Preimplantation Genetic Testing Research Articles

48. OBSERVING THE IMPACT OF EMBRYO CULTURING CONDITIONS ON NON-INVASIVE PREIMPLANTATION GENETIC TESTING FOR ANEUPLOIDY DETECTION (NI-PGT-A)

Introduction There are several options available to clinics culturing IVF embryos, including group and individual droplet culture, sequential or single-step medium culture and the possibility to renew the medium during culture. The absence of standardised culturing conditions or molecular testing methodologies, including whole genome amplification (WGA) used for non-invasive preimplantation genetic testing for aneuploidy (NI-PGT-A) may explain the variable rates of concordance reported between the spent embryo culture media and embryo biopsy results to-date. Culture conditions contribute to the accumulation of embryonic and contaminating DNA in spent embryo culture media. Optimisation of either the culturing conditions, molecular testing methodologies, or both, should yield the highest accuracy results for NI-PGT-A. This study examined rates of concordance between spent embryo culture media and embryo biopsies with the aim of evaluating the impact of culturing conditions on NI-PGT-A results. Material and methods Spent embryo culture media was collected from single embryo culture droplets following biopsy of the embryo for PGT-A, then stored at -20°C, with ethics approval. Spent embryo culture media samples from 10ul-60ul culture droplets were whole genome amplified using DOPlify® kit reagents (PerkinElmer). WGA DNA yield was assessed following gel electrophoresis and high sensitivity Qubit instrument quantification (ThermoFisher). Next generation sequencing libraries and sequencing was performed according to the standard PG-SeqTM kit 48 sample protocol on a MiSeq® instrument sequencer (Illumina). Data was bioinformatically aligned to hg19 and analysed using PG-SeqTM kit software. WGA DNA yield, NGS metrics, and whole chromosome aneuploidy concordance with the PGT-A result for the embryo biopsy were determined. Results Results were collated for each set of culturing conditions, including whether the media was renewed during culture. Whole genome amplification using DOPlify® kit reagents resulted in the amplification of 78-100% of spent embryo culture media samples (WGA failure rate 0-22%). Ploidy concordance with the embryo biopsy ranged from 33-55% for autosomal chromosomes and 47-53% for sex chromosomes using a single-step culturing system (n=3 protocols), compared with concordance rates of 60-95% and 50-97% respectively when media was changed during the 5-6 day culture (n=4 protocols). Conclusions Successful NI-PGT-A using spent embryo culture media will possibly require specific culturing conditions and/or specialised molecular methodologies for accurate and representative amplification and testing of the embryonic DNA. In a step toward this, we identified that renewing culture media during IVF improves overall concordance rates between the embryo biopsy and spent embryo culture media for NI-PGT-A.

A prospective study of non-invasive preimplantation genetic testing for aneuploidies (NiPGT-A) using next-generation sequencing (NGS) on spent culture media (SCM).

This study was to evaluate if spent culture media (SCM) of embryos could be used as a non-invasive tool to achieve aneuploidy screening. Ploidy calls, as well as concordance rates between PGT-A results from trophectoderm (TE) and SCM, were compared. Clinical outcomes of single euploid transfers were also evaluated. The study was conducted from March 2017 to June 2018 in a university-based ART center. SCM of day 3 to the day(s) of TE biopsy of all biopsied blastocysts were collected for testing. PGT-A results of SCM were compared with the standard results of TE, with clinical relevance and outcomes examined. NiPGT-A using SCM gave a sensitivity of 81.6%, specificity of 48.3%, positive predictive value of 82.6%, and negative predictive value of 46.7% in ploidy calling. The concordance rates for autosomes and sex determination were 62.1% and 82.4%, respectively. There were 14 single embryo transfer cycles of euploids as determined by TE biopsy. Clinical outcomes not only confirmed 3 false positive results from SCM but also reflected the true ploidy status of the transferred embryo in one case. If ploidy calls were dichotomized without mosaic embryos, the sensitivity and NPV would increase to 91.0% and 66.7% (p = 0.60 and p = 0.25), respectively. Cell-free DNA found in SCM could provide ploidy information of an embryo as in PGT-A from its TE. Given its potential to reflect the comprehensive chromosomal profile of the whole embryo, more research based on clinical outcomes is required to determine if SCM could be a reliable selection tool in PGT-A.

Noninvasive preimplantation genetic testing for aneuploidy in spent medium may be more reliable than trophectoderm biopsy.

Preimplantation genetic testing for aneuploidy (PGT-A) with trophectoderm (TE) biopsy is widely applied in in vitro fertilization (IVF) to identify aneuploid embryos. However, potential safety concerns regarding biopsy and restrictions to only those embryos suitable for biopsy pose limitations. In addition, embryo mosaicism gives rise to false positives and false negatives in PGT-A because the inner cell mass (ICM) cells, which give rise to the fetus, are not tested. Here, we report a critical examination of the efficacy of noninvasive preimplantation genetic testing for aneuploidy (niPGT-A) in the spent culture media of human blastocysts by analyzing the cell-free DNA, which reflects ploidy of both the TE and ICM. Fifty-two frozen donated blastocysts with TE biopsy results were thawed; each of their spent culture medium was collected after 24-h culture and analyzed by next-generation sequencing (NGS). niPGT-A and TE-biopsy PGT-A results were compared with the sequencing results of the corresponding embryos, which were taken as true results for aneuploidy reporting. With removal of all corona-cumulus cells, the false-negative rate (FNR) for niPGT-A was found to be zero. By applying an appropriate threshold for mosaicism, both the positive predictive value (PPV) and specificity for niPGT-A were much higher than TE-biopsy PGT-A. Furthermore, the concordance rates for both embryo ploidy and chromosome copy numbers were higher for niPGT-A than TE-biopsy PGT-A. These results suggest that niPGT-A is less prone to errors associated with embryo mosaicism and is more reliable than TE-biopsy PGT-A.

Non-invasive preimplantation genetic testing for aneuploidy (NIPGT-A)

Introduction Preimplantation genetic testing without trophectoderm (TE) biopsy is an attractive approach to avoid any potential risk due to an invasive procedure.The overriding goalof the non- invasive preimplantation genetic testing (NIPGT) is to utilize the cell free embryonic DNA (cfeDNA) in assessment of chromosomal status of the embryo (NIPGT-A). Clinical application of this approach still depends on the elucidation of the cfeDNA origin and degree of representation of the whole embryo. Our aim is to develop a NIPGT-A test for whole genome screening of embryos. Material & Methods The study received research ethics approval from University of Toronto (REB# 30251). Thirteen patients (median age, 37.3 years; range, 29-42)undergoing IVF-ICSI with PGT at CReATe Fertility Centre, Toronto, Canada, were included in the study. We collected cfeDNA from spent embryo culture media together with blastocoel fluid from 26 embryos (NIPGT-A samples)under strict aseptic and clean room conditions. The corresponding TE samples were used as controls. NIPGT- A samples contained cfeDNA released from the embryo within 24-48 hours in culture (after d4).Media without embryo cultured on the same dish was used as a negative control. All collected samples were frozen and stored at -80°C until tested. For PGT-A using TE biopsy and NIPGT-A samples, SurePlex kit (BlueGnome) was used for whole genome amplification (WGA) of embryonic DNA. NGS using VeriSeq PGS kit (Illumina) was used to determine the chromosomal copy number of the analyzed samples. Concordance rates for whole chromosome copy number (WCN) between NIPGT-A and corresponding TE biopsy samples were calculated and statistically evaluated by Fisher exact test (significant at P Results PGT-A results from TE biopsy were as follow: 9 euploid, 9 aneuploid and 8 mosaic. NIPGT-A results showed 11 euploid, 9 aneuploid and 6 mosaic profiles. Our results demonstrate high concordance rate per sample for whole chromosome copy number between NIPGT and TE biopsy samples in their corresponding blastocysts:1)Ploidy status (euploid or aneuploid) NIPGT-A vs TE - 100% (26/26); 2) Euploid samples NIPGT-A vs TE – 100% (17/17); 3)Whole chromosome aneuploidy NIPGT-A vs TE – 100% (9/9). However, concordance rate for mosaic abnormality between NIPGT and TE biopsy samples was 55.6% (5/9) which can be explained by an already reported discordance between initial and repeated TE biopsy results from mosaic embryos ( Maxwell et al., 2016 ) Conclusions Spent embryo culture medium combined with blastocoels fluid has great potential to be used as a source of embryonic DNA for comprehensive aneuploidy screening.

Optimisation of whole genome amplification specifically for non-invasive PGT-A using spent embryo culture media

Introduction The clinical use of non-invasive preimplantation genetic testing for aneuploidy (PGT-A) requires concordance of the spent embryo culture media result to embryo biopsy result and the ability to distinguish maternal contamination from the embryonic DNA, especially for a euploid female result. Although concordance of spent embryo culture media and trophectoderm biopsy has been reported at as high as 95% following the collection of samples at day 5-7 using DOPlify™ ( Lane et al, 2017 ), the ability to test media collected earlier in culture requires an increased level of sensitivity. Additionally, there are a number of known PCR inhibitors in culture media, including salts and proteins, which need to be overcome. Here we describe acustomised whole genome amplification (WGA) protocol designed specifically for the amplification of DNA contained in spent embryo culture media. Material and Methods Spent embryo culture media was collected by clinics with ethics approval from single embryo culture droplets and pooled prior to storage at -20°C. Pooling was used to provide a consistent test sample to analyse across a range of protocol variants to overcome sample to sample variability. To accommodate the use of a larger sample input volume into the WGA and the composition of culture media post embryo culture, samples were amplified using a re-formulated single step version of DOPlify™. The single step protocol removes the DOPlify™ lysis step and requires the addition of only one reagent mastermix into the PCR tube containing the culture media, making this protocol ideal for automation. The same pooled media samples were amplified in parallel using the standard DOPlify™ protocol. WGA DNA yield was assessed following gel electrophoresis and HS Qubit quantification (ThermoFisher) and sequenced on a MiSeq sequencer (Illumina) according to the standard PG-Seq™ 48 sample protocol. The sequencing data for each sample was bioinformatically aligned to hg19 and the sequencing QC metrics for each protocol were collated and compared. Results A total of 32 protocol variants were tested in triplicate. Outputs measured were yield, size distribution of PCR amplicons, %GC content and PG-Seq™ NGS software quality scores. With the optimal protocol, WGA DNA yield per sample increased on average 5-fold following the amplification of pooled media samples using a re-formulated version of DOPlify™ with output measures comparable to biopsy results. It is also possible to detect mitochondrial DNA from the amplified spent culture media, although with fewer reads per sample than biopsies provide. The same increased yield and acceptable QC results have since been obtained for individual culture droplets from embryos cultured in 10-60ul of media from a range of manufacturers. Conclusions Successful non-invasive PGT-A using spent embryo culture media requires a customised approach to amplify DNA found within this complex sample type. Accurate and representative amplification of the embryo DNA is essential for the success of this non-invasive approach. Further incorporation of RHS’ target sequence enrichment protocol would provide a mechanism to detect maternal DNA within a spent culture media sample.

Non-invasive preimplantation genetic testing for aneuploidy (NI PGT-A)

Since the initial report by Stigliani et al. (2013) about the presence of mitochondrial and nuclear DNA in human embryo culture medium, spent media (SM) has been extensively explored with the ultimate aim to develop non-invasive genetic testing (NI PGT) methods. Even after the first clinical application of NI PGT-A by Xu and colleagues (2016) using SM, the efficacy of the method for clinical utility remains under much debate as there are conflicting reports on its success rates as well concerns about non- embryonic DNA contamination. There are two key aspects determining the success of an NI PGT assay; 1) embryology parameters of culture conditions and 2) molecular methods used for downstream processing of SM samples. Most of the studies on NI PGT thus far have used SM samples derived from a single IVF centre and hence differences in choice of culture media, culture volumes, type of culture (fresh vs vitrified-thawed), and timing of sampling (Day3-Day5/6 or Day4-Day5/6) would have, in part, contributed to the differences in success rates obtained by different groups ( Shamonki et al., 2016 , Xu et al., 2016 , Feichtinger et al., 2017 , Lane et al., 2017 , Kuznyetsov et al., 2017 ). Hence, it is pertinent to develop a robust and versatile NI PGT method, functional under multiple embryo culture parameters. The choice of whole genome amplification (WGA) method is another major factor which might impact the success of NI PGT assay. As the embryonic DNA in spent media is most likely fragmented in nature, shorter DNA template lengths can pose challenge to chemistries of certain WGA methods ( Wang et al., 2004 ). Secondly, certain components of embryo culture media might impede the function of buffer and/or enzyme of the WGA system, thereby resulting in suboptimal or no amplification. We have tested two commercially available WGA methods on SM samples namely Sureplex and MDA in addition to a modified MDA approach (suitable for fragmented DNA) and found that the DNA amplification rates were significantly higher in the samples amplified with the modified MDA compared to those amplified by Sureplex and MDA methods. The origin of DNA present in SM can be a confounding factor for NI PGT assay. Microbial contamination has been reported to occur at Kastrop et al., 2007 ). More recently, Vera-Rodriguez et al. (2018) reported very high levels of maternal DNA contamination with only ∼8% of DNA fraction in the SM beingembryonic in origin. However with appropriate culture conditions and amplification methods, several other groups ( Xu et al., 2016 , Kuznyetsov et al., 2017 , Lane et al., 2017 ), including ours, have obtaineda high sex chromosomes as well as overall ploidy concordance rates (range: 72% - 95%) between SM and corresponding TE biopsies. Thisindicatesthat any potential contamination present in these samples was minimal and did not impede the end results; thereby highlighting the importance of adequate embryology culture practice as well appropriate downstream processing steps for a successful NI PGT-A assay. While a few NI PGT-A clinical trials have been initiated over the last two years, many crucial questions regarding the broader clinical efficacy of the method still remain to be answered. Nonetheless, it seems that we are one step closer to the dream of a completely non-invasive method for genetic testing of preimplantation embryos than before.

Clinical application of embryo aneuploidy testing by next-generation sequencing.

We review here the evolution in the field of embryo aneuploidy testing over the last 20 years, from the analysis of a subset of chromosomes by fluorescence in situ hybridisation to the transition toward a more comprehensive analysis of all 24 chromosomes. This current comprehensive aneuploidy testing most commonly employs next-generation sequencing (NGS). We present our experience in over 130 000 embryo biopsies using this technology. The incidence of aneuploidy was lower in trophectoderm biopsies compared to cleavage-stage biopsies. We also confirmed by NGS that embryo aneuploidy rates increased with increasing maternal age, mostly attributable to an increase in complex aneuploid embryos. In contrast, the number of MII oocytes retrieved or the use of oocyte vitrification did not affect aneuploidy rates. Similarly, neither maternal age, oocyte number, nor oocyte vitrification affected the incidence of mosaicism. Analysis of clinical outcomes, indications, and potential benefits of embryo aneuploidy testing revealed advanced maternal age as the most favored group, with some evidence of improved delivery rate per transfer as well as decreased miscarriage rates and time to pregnancy. Other indications are: recurrent miscarriage, repetitive implantation failure, severe male factor, previous trisomic pregnancy, and good prognosis patients mainly undergoing single embryo transfer, with the latter indication used to reduce the occurrence of multiple pregnancies without compromising cycle outcome. In conclusion, NGS has become the most appropriate technology for aneuploidy testing in trophectoderm biopsies, with accurate results, high throughput, and cost efficiency. This technology can be also applied to the analysis of the embryonic cell free DNA released to the culture media at blastocyst stage. This is a promising approach towards a non-invasive preimplantation genetic testing of aneuploidy.

Corrigendum. Non-invasive pre-implantation genetic testing of human embryos: an emerging concept.

Corrigendum. Non-invasive pre-implantation genetic testing of human embryos: an emerging concept.

Non-invasive pre-implantation genetic testing of human embryos: an emerging concept.

The accurate genetic screening of pre-implantation embryos currently entails the use of technically challenging and biologically invasive biopsies of the human embryos. Investigating a more conservative sampling approach has emerged as a timely and desired alternative. Circulating cell-free embryonic DNA is present in the blastocoel fluid and spent culture media of blastocysts, and this has lately been sought as an attractive source of genetic information. The genetic analysis of cell-free embryonic DNA has been reported, to be useful in evaluating the genetic constitution of embryos; thus, providing a potential alternative to conventional biopsy-derived pre-implantation genetic testing (PGT). In this review, we have summarized these non-invasive alternative applications of PGT and discussed their current limitations and future clinical implications.

Origin of false positives and false negatives in non-invasive preimplantation genetic testing for aneuploidies

Origin of false positives and false negatives in non-invasive preimplantation genetic testing for aneuploidies

Evaluation of a novel non-invasive preimplantation genetic screening approach.

ObjectiveTo assess whether embryonic DNA isolated from blastocyst culture conditioned medium (BCCM) combined with blastocoel fluid (BF) could be used for blastocyst stage non-invasive preimplantation genetic testing for chromosomal aneuploidy (non-invasive preimplantation genetic screening, NIPGS).Patients47 embryos from 35 patients undergoing IVF.InterventionsDNA analysis of combined BCCM plus BF in comparison with trophectoderm (TE) biopsy and/or whole blastocyst (WB)using next generation sequencing (NGS).ResultsEmbryonic DNA was successfully amplified in 47/47 NIPGS samples (28 frozen-thawed and 19 fresh culture samples) ranging from 6.3 to 44.0 ng/μl. For frozen-thawed embryos, the concordance rate for whole chromosome copy number per sample was equivalent between NIPGS vs. TE biopsy, NIPGS vs. WB and TE vs. WB samples taken from the same embryo was 87.5%; 96.4% and 91.7% respectively (P>0.05), and the rate of concordance per single chromosome was 99.3%, 99.7% and 99.7%, respectively (P>0.05). In fresh cases (Day 4 to Day 5/6 culture), the concordance rate for whole chromosome copy number per sample between NIPGS vs. TE samples taken from the same embryo was 100%, and the rate of concordance per single chromosome was 98.2% (P>0.05).ConclusionsA combination of BCCM and BF contains sufficient embryonic DNA for whole genome amplification and accurate aneuploidy screening. Our findings suggest that aneuploidy screening using BCCM combined with BF could potentially serve as a novel NIPGS approach for use in human IVF.