Non-invasive Preimplantation Genetic Testing Research Articles

NGS-Based Application for Routine Non-Invasive Pre-Implantation Genetic Assessment in IVF.

Although non-invasive pre-implantation genetic testing for aneuploidy (NIPGT-A) is potentially appropriate to assess chromosomal ploidy of the embryo, practical application of it in a routine IVF centre have not been started in the absence of a recommendation. Our objective in this study was to provide a comprehensive workflow for a clinically applicable strategy for NIPGT-A based on next-generation sequencing (NGS) technology with the corresponding bioinformatic pipeline. In a retrospective study, we performed NGS on spent blastocyst culture media of Day 3 embryos fertilised with intracytoplasmic sperm injection (ICSI) with quality score on morphology assessment using the blank culture media as background control. Chromosomal abnormalities were identified by an optimised bioinformatics pipeline applying copy number variation (CNV) detecting algorithm. In this study, we demonstrate a comprehensive workflow covering both wet- and dry-lab procedures supporting a clinically applicable strategy for NIPGT-A that can be carried out within 48 h, which is critical for the same-cycle blastocyst transfer. The described integrated approach of non-invasive evaluation of embryonic DNA content of the culture media can potentially supplement existing pre-implantation genetic screening methods.

Cell-free DNA in spent culture medium effectively reflects the chromosomal status of embryos following culturing beyond implantation compared to trophectoderm biopsy.

This prospective study evaluated the accuracy of non-invasive preimplantation genetic testing for aneuploidy (niPGT-A) using cell-free DNA in spent culture medium, as well as that of preimplantation genetic testing for aneuploidy (PGT-A) using trophectoderm (TE) biopsy after culturing beyond implantation. Twenty frozen blastocysts donated by 12 patients who underwent IVF at our institution were investigated. Of these, 10 were frozen on day 5 and 10 on day 6. Spent culture medium and TE cells were collected from each blastocyst after thawing, and the embryos were cultured in vitro for up to 10 days. The outgrowths after culturing beyond implantation were sampled and subjected to chromosome analysis using next-generation sequencing. Chromosomal concordance rate, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), false-positive rate (FPR), and false-negative rate (FNR) of niPGT-A and PGT-A against each outgrowth were analyzed. The concordance rate between the niPGT-A and outgrowth samples was 9/16 (56.3%), and the concordance rate between the PGT-A and outgrowth samples was 7/16 (43.8%). NiPGT-A exhibited 100% sensitivity, 87.5% specificity, 88.9% PPV, 100% NPV, 12.5% FPR, and 0% FNR. PGT-A exhibited 87.5% sensitivity, 77.8% specificity, 87.5% PPV, 75% NPV, 14.3% FPR, and 22.2% FNR. NiPGT-A may be more accurate than PGT-A in terms of ploidy diagnostic accuracy in outgrowths.

The Technological Advances in Embryo Selection and Genetic Testing: A Look Back at the Evolution of Aneuploidy Screening and the Prospects of Non-Invasive PGT

Since the birth of the first IVF baby, Louise Brown, in 1978, researchers and clinicians have sought ways to improve pregnancy outcomes through embryo selection. In the 1990s, blastomere biopsy and fluorescence in situ hybridization (FISH) were developed in human embryos for the assessment of aneuploidy and translocations. Limitations in the number of chromosomes that could be assayed with FISH lead to the development of comparative genomic hybridization (CGH); however, pregnancy rates overall were not improved. The later development of trophectoderm biopsy with comprehensive chromosome screening (CCS) technologies, as well as the subsequent development of next-generation sequencing (NGS), have shown much greater promise in improving pregnancy and live birth rates. Recently, many studies are focusing on the utilization of non-invasive preimplantation genetic testing (niPGT) in an effort to assess embryo ploidy without exposing embryos to additional interventions.

Non-invasive preimplantation genetic testing: a literature review.

Non-invasive preimplantation genetic testing emerged from the discovery of embryonic DNA in spent embryo culture medium. Considering that such methodology would be an important advance in assisted reproduction, this study aimed to evaluate the current scientific evidence, based on the reliability of non-invasive chromosome screening, through a literature review. We analyzed 14 original research papers in PubMed and SciELO, in English and Portuguese, published between 2016 and 2021 related to the topic. The agreement rate for ploidy compared to the traditional method ranged from 3.5% - 93.8% raising the discussion about the possible causes of this large variation, which may be due to the day of collection, spent culture media contamination, amplification methodology or the cytogenetic method used by each author. We concluded that the non-invasive test has many advantages over the traditional method, but that clinical replacement is not yet possible, and further studies are needed in order to have an accurate clinical test with the non-invasive methodology.

Embryonic Cell-free DNA in Spent Culture Medium: A Non-invasive Tool for Aneuploidy Screening of the Corresponding Embryos.

Preimplantation genetic testing (PGT) for chromosomal screening, based on embryo biopsy, has significant limitations. Cell-free DNA (cf-DNA) has been detected in spent culture medium (SCM), opening new horizons for the development of non-invasive PGT (ni-PGT). In this study, we evaluated the diagnostic performance of ni-PGT for aneuploidy (niPGT-A), comparing the results of trophectoderm biopsies (TE) and respective SCM from individually cultured embryos via Next Generation Sequencing (NGS). Forty fresh embryos were analyzed. TE and SCM from blastocysts were collected and analyzed. We detected cfDNA in 100% of samples tested. The overall concordance rate between the ni-PGT-A and PGT-A was 27/33 (81.8%). The full concordance rate was 21/33 (63.6%). The aneuploidy agreement was 91.66%, and the euploidy agreement was 76.19%. We found a good accordance between TE and SCM analysis, suggesting that niPGT-A could be a reliable alternative for chromosomal abnormalities assessment of in vitro cultured embryos.

Consistent results of non-invasive PGT-A of human embryos using two different techniques for chromosomal analysis

Research questionAre discordances in non-invasive preimplantation genetic testing for aneuploidies (niPGT-A) results attributable to the technique used for chromosomal analysis? DesignA prospective blinded study was performed (September 2018 to December 2019). In total 302 chromosomal analyses were performed: 92 trophectoderm PGT-A biopsies and their corresponding spent embryo culture medium (SCM) evaluated by two methods (n = 184), negative controls (n = 8), and trophectoderm and inner cell mass biopsies from trophectoderm-aneuploid embryos (n = 18). Trophectoderm analyses were carried out using Veriseq (Illumina), and SCM was analysed using Veriseq and NICS (Yikon). ResultsGenetic results were obtained for 96.8% of trophectoderm samples versus 92.4% for both SCM techniques. The mosaicism rate was higher for SCM regardless of the technique used: 30.4% for SCM-NICS and 28.3% for SCM-Veriseq versus 14.1% for trophectoderm biopsies (P = 0.013, P = 0.031, respectively). No significant differences in diagnostic concordance were seen between the two SCM techniques (74.6% for SCM-NICS versus 72.3% for SCM-Veriseq; P = 0.861). For embryos biopsied on day 6, these rates reached 92.0% and 86.5%, respectively. On reanalysing trophectoderm-aneuploid embryos, the discrepancies were shown to be due to maternal DNA contamination (55.6%; 5/9), embryo mosaicism (22.2%; 2/9) and low resolution in SCM-NICS (11.1%; 1/9) and in both SCM techniques (11.1%; 1/9). ConclusionsThis is the first study evaluating the consistency of different chromosomal analysis techniques for niPGT-A. In conclusion, the diagnostic concordance between PGT-A and niPGT-A seems independent of the technique used. Optimization of culture conditions and medium retrieval provides a potential target to improve the reliability of niPGT-A.

Noninvasive preimplantation genetic testing in assisted reproductive technology: current state and future perspectives

Invasive genetic screening of pre-implantation embryos via biopsied trophectoderm (TE) cells has been in use for more than 20 years, while its benefits in selecting euploid embryos remain controversial. Recent advances in the ability to process embryonic cell-free DNA (cfDNA) from blastocoel fluid (BF) and spent culture media (SCM) of blastocysts in a manner similar to that of a biopsied TE sample provide a potential alternative holding great promise for obtaining cytogenetic information of the embryos without intrusive biopsy of traditional biopsy-based pre-implantation genetic testing (PGT). Several studies have reported even higher diagnostic accuracy in non-invasive PGT (ni-PGT) than conventional PGT. However, there are still several technical challenges to be overcome before ni-PGT can be accepted as a reliable genomic information source of embryo. In this review, we have summarized the emergence and current state of ni-PGT, and discussed our own perspectives on their limitations and future prospect. There is still a long way to go before truly wide clinical application of ni-PGT.

Preimplantation Genetic Testing – A small walk through – A review

Application of Preimplantation Genetic Testing technique for the diagnosis of monogenic disorder in the embryos of couples who were at risk of transferring genetic disorder to their child began in the mid 1980’s. In the mid-1990s, this method was used as a method for embryo selection in patients undergoing in vitro fertilization, by analyzing maximum number of chromosomes possible, with anticipation of selecting chromosomally normal embryo which may lead to higher implantation and decrease pregnancy loss. We discuss here the evolution of Preimplantation Genetic Testing method, the challenges faced during this process of development, to evaluate its efficiency and drawbacks of different methods and to summarize the recent progress of the new methods and its potential application in increasing the live birth rate. All relevant articles addressing Preimplantation Genetic Testing, Preimplantation Genetic Screening or Diagnosis published from 1980’s to 2019’s were reviewed and analyzed. Preimplantation Genetic Testing has helped any couples in having a healthy baby and though biopsy technique is considered invasive due to the removal of blastomere from the embryo, blastocyst biopsy is considered as the most reliable among the biopsy methods available. Non-invasive preimplantation genetic testing are considered to replace conventional biopsy technique but their limitations despite their advantage needs to be addressed and require validation for clinical application.

Diagnostic efficiency of blastocyst culture medium in noninvasive preimplantation genetic testing

ObjectiveTo evaluate the diagnostic efficiency of spent blastocyst culture medium (BCM) in noninvasive preimplantation genetic testing (niPGT) by comparing the karyotype concordance with corresponding inner cell mass (ICM) among initial trophectoderm (TE) biopsy, TE re-biopsy, and BCM sampling.DesignRe-analysis aneuploid/mosaic blastocysts donated for research by couples.SettingInstitutional in vitro fertilization center.Patient(s)A total of 12 couples donated their blastocysts, which had previously been diagnosed as aneuploid or mosaic by initial TE-biopsy preimplantation genetic testing for aneuploidy (PGT-A) for research.Intervention(s)A total of 26 frozen−thawed blastocysts were re-analyzed by TE re-biopsy, ICM biopsy, and the collection of spent BCM.Main Outcome Measure(s)Karyotype concordance rates.Result(s)For 23 embryos diagnosed as aneuploid by initial TE biopsy, 78.3% of initial TE samples, 87.0% of TE re-biopsies samples, and 78.3% of BCM samples were concordant with corresponding ICM samples, and for three mosaic embryos, the concordance rates with ICM of these three groups were 0%, 100%, and 100%, respectively. With the corresponding ICM result as the true result, sensitivity of both niPGT-A and initial TE were 100%; however, the false-positive rate (FPR) of initial TE was higher than that of niPGT-A (100% vs. 0).Conclusion(s)niPGT-A using BCM had diagnostic efficiency similar to that of TE-biopsy PGT-A. In the case of mosaic embryos, niPGT-A using BCM may be more reliable for predicting karyotypes of ICM than initial TE biopsy.

DIAGNOSTIC EFFICIENCY OF BLASTOCYST CULTURE MEDIUM IN NONINVASIVE PREIMPLANTATION GENETIC TESTING (NIPGT)

To evaluate the diagnostic efficiency of spent blastocyst culture medium (BCM) in noninvasive preimplantation genetic testing (niPGT) by comparing the karyotype concordance with corresponding inner cell mass (ICM) among initial trophectoderm (TE) biopsy, TE re-biopsy and BCM sampling. Re-analysis aneuploid/mosaic blastocysts donated for research by couples. A total of 26 blastocysts with aneuploid (n=23) or mosaic diagnosis (n=3) were donated for research from 12 couples with indications of preimplantation genetic testing for chromosomal structural rearrangements (PGT-SR) or preimplantation genetic testing for aneuploidy (PGT-A), at Reproductive Medicine Research Center of the Sixth Affiliated Hospital of Sun Yat-Sen University, from July 2018 to July 2019. TE and ICM were biopsied (5-10 cells) in a routine procedure; TE was re-biopsied (more than 10 cells) at other sites of trophectoderm compartment. The MALBAC single-cell whole genome amplification (WGA) method was used to amplify DNA from all cells and BCM samples. Embryos were diagnosed as euploid, aneuploid or mosaic by NGS results. Embryos were classified as mosaic if the level of mosaicism ranged from 40% to 70%, while less than 40% was labeled as euploid and more than 70% as aneuploid. But for chr13, chr16, chr18 and chr21 chromosomes, the criterion for mosaicism was set to be 30%. The primary outcomes of this study, concordance rates with ICM biopsies, were compared among groups (initial TE biopsies, TE re-biopsies and BCM samples). The karyotype concordance of each two groups was defined as the presence of the same abnormal chromosome, especially in the target chromosome in different testing results compared. The clinical concordance of all four samples was defined as the concordance in embryo diagnosis, such as uniform aneuploid or not. To assess the diagnostic efficiency of BCM sample, false positive rate (FPR), false negative rate (FNR), sensitivity, specificity, accuracy, positive predictive value (PPV) and negative predictive value (NPV) were calculated according to the corresponding ICM results. Statistical analyses were performed with the Chi-Square test, Yates' correction, or Fisher's exact test accordingly when comparing frequencies or proportions. All statistical analysis was performed using SPSS 22.0. P<0.05 was considered as significant. For 23 embryos diagnosed as aneuploid by initial TE biopsy, 78.3% of initial TE samples, 87.0% of TE re-biopsies and 78.3% of BCM samples were concordant with corresponding ICM samples (P>0.05); but for 3 mosaic embryos, the concordance rates with ICM of these three groups were 0%, 100% and 100% (P<0.05), respectively. With the corresponding ICM result as the true result, sensitivity of both niPGT-A and initial TE were 100%; but the FPR of initial TE was higher than that of niPGT-A (100% vs. 0, P=0.100). NiPGT-A using spent BCM had similar diagnostic efficiency as TE-biopsy PGT-A. And in case of mosaic embryos, niPGT-A using BCM may be more reliable for predicting the karyotypes of ICM than initial TE biopsy.

THE VALUE OF NONINVASIVE PREIMPLANTATION GENETIC TESTING FOR ANEUPLOIDY ON HUMAN CELL-FREE DNA FROM BLASTOCOELE FLUID WITH GENOME SEQUENCING: A PROSPECTIVE COHORT STUDY

Noninvasive preimplantation genetic testing for aneuploidy (niPGT-A) on cell-free DNA in blastocoele fluid (BF) is a promising but controversial method for selecting euploid embryos in assisted reproductive technologies.

NONINVASIVE PREIMPLANTATION GENETIC TESTING FOR ANEUPLOIDY EXHIBITS HIGH RATES OF DNA AMPLIFICATION FAILURE AND LACKS CONSISTENCY WITH PGT-A USING TROPHOECTODERM BIOPSY

Preimplantation genetic testing for aneuploidy (PGT-A) is currently performed via analyses of trophectoderm (TE) biopsies. Concerns regarding the safety of TE biopsy and the technical skill required to perform the procedure have led to increased interest in noninvasive preimplantation genetic testing for aneuploidy (niPGT-A). The noninvasive testing modality assesses an embryo’s genetic composition indirectly by analyzing spent culture media. This study begins the validation process of a commercially available assay by investigating: 1) the prevalence of DNA amplification failure with niPGT-A compared to TE biopsy; 2) factors affecting amplification failure with niPGT-A, and 3) the frequency of discrepant results between niPGT-A and traditional PGT-A.

Is cell-free DNA in spent embryo culture medium an alternative to embryo biopsy for preimplantation genetic testing? A systematic review.

Preimplantation genetic testing (PGT) is increasingly used worldwide. It currently entails the use of invasive techniques, i.e. polar body, blastomere, trophectoderm biopsy or blastocentesis, to obtain embryonic DNA, with major technical limitations and ethical issues. Evidence suggests that invasive PGT can lead to genetic misdiagnosis in the case of embryo mosaicism, and, consequently, to the selection of affected embryos for implantation or to the destruction of healthy embryos. Recently, spent culture medium (SCM) has been proposed as an alternative source of embryonic DNA. An increasing number of studies have reported the detection of cell-free DNA in SCM and highlighted the diagnostic potential of non-invasive SCM-based PGT for assessing the genetic status of preimplantation human embryos obtained by IVF. The reliability of this approach for clinical applications, however, needs to be determined. In this systematic review, published evidence on non-invasive SCM-based PGT is presented, and its current benefits and limitations compared with invasive PGT. Then, ways of optimizing and standardizing procedures for non-invasive SCM-based PGT to prevent technical biases and to improve performance in future studies are discussed. Finally, clinical perspectives of non-invasive PGT are presented and its future applications in reproductive medicine highlighted.

Noninvasive Preimplantation Genetic Testing for Aneuploidy (niPGT-A): The first Brazilian baby.

Recently, a new technology known as the Noninvasive Preimplantation Genetic Testing for Aneuploidy (niPGT-A) emerged, using cell-free DNA present in the spent culture media of human blastocysts. Unlike PGT-A, in which only trophectoderm cells are used, niPGT-A reflects the ploidy state of these cells and internal cell mass, suggesting that this new technology may be less prone to error, being more reliable than the invasive test. The aim of the present study was to report the first occurrence of childbirth following niPGT-A in Brazil.

Relationship between age and blastocyst chromosomal ploidy analyzed by noninvasive preimplantation genetic testing for aneuploidies (niPGT-A).

Objective:To assess the relationship between human blastocyst chromosomal ploidy established by niPGT-A and increasing age.Methods:This is a prospective multicenter study carried out by ten assisted reproduction centers after their embryologists acquired training and validated their results with the previous use of niPGT-A. A total of 94 couples with indication for niPGT-A due to increase maternal age, male factor, repeated implantation failures, recurrent abortion or because they requested niPGT-A were included in this study. The couples had no karyotype abnormalities. After ICSI, the embryos were cultured until blastocyst stage using one or two step culture systems, single or sequential media respectively, at 37ºC in an atmosphere of 6-7% CO2 and 5-20% O2 incubators. On day 3, we re-evaluated cleavage embryos to complete cumulus cells removal. The embryos were then cultured in individual well, with 20µl of medium under oil until they reached blastocyst stage. The blastocysts were vitrified and stored in liquid nitrogen. After that, the spent blastocyst culture medium (20µl) was transferred to a PCR tube and sent for analysis in the genetic laboratory, where it was stored at -80ºC until sequencing. A total of 243 samples of spent blastocyst culture medium were collected on the 5th/6th day. Cell-free DNA secreted on culture medium was amplified using NICS Sample Preparation Kit (Yikon Genomics), based on the MALBAC technology. After whole genome amplification, the DNA was measured using a Qubit 2.0 fluorometer and subjected to next generation sequencing (NGS) using Illumina MiSeq® platform. The data were analyzed using the ChromGo® software (Yikon Genomics).Results:The mean age of the patients was 38±4.08 years with an interval of 20-44 years. The euploid was diagnosed in 36.4% (80/220) of cases, aneuploidy in 31.3% (69/220), and mosaicism in 32.3% (71/220; with ≥60% aneuploidy) of blastocysts. Mosaic values ranged from 29.8% to 33.8% in different age groups. Individually, the most frequent chromosomal abnormality was XXY (Klinefelter Syndrome) occurring in 18 cases, followed by chromosome 21 (trisomy/monosomy) in 8 cases. The niPGT-A data showed a ≥60% incidence of aneuploid cells in all cases of chromosomal mosaicism (n=71).Conclusion:A high degree of mosaicism with aneuploidy cells was detected, and some hypotheses were suggested for this data (niPGT-A sensitivity in detecting the self-correction of chromosomal abnormalities phenomenon). However, it did not vary remarkably with age. On the other hand, euploidy levels had a negative correlation with age and aneuploidy levels had a positive relationship. This is the first report in the literature to relate chromosomal ploidy in blastocysts using niPGT-A and increasing patient age.

Noninvasive preimplantation genetic testing for aneuploidies (niPGT-A) and the principle of primum non nocere

Perhaps with the intention of obtaining larger amounts of free-DNA, some groups are routinely postponing and establishing free-DNA collection in culture medium for Noninvasive preimplantation genetic testing for aneuploidies (niPGT-A) to day 6 for all blastocysts. A meta-analysis served as the basis for such decision, since statistically similar live birth rates were observed when the transfers of euploid blastocysts were performed on day 5 versus day 6 However, the euploidy analysis was conducted in only two studies However, after including the two more studies we performed a new meta-analysis that clearly showed the risks of losing live births with the decision of adopting the 6th day as the endpoint for gathering free-DNA. We would be losing 1.71x more live births.

Non-invasive preimplantation genetic testing (niPGT): the next revolution in reproductive genetics?

Preimplantation genetic testing (PGT) encompasses methods that allow embryos to be tested for severe inherited conditions or for chromosome abnormalities, relevant to embryo health and viability. In order to obtain embryonic genetic material for analysis, a biopsy is required, involving the removal of one or more cells. This invasive procedure greatly increases the costs of PGT and there have been concerns that embryo viability could be compromised in some cases. The recent discovery of DNA within the blastocoele fluid (BF) of blastocysts and in spent embryo culture media (SCM) has led to interest in the development of non-invasive methods of PGT (niPGT). This review evaluates the current scientific evidence regarding non-invasive genetic assessment of preimplantation embryos. The success of different PGT methodologies in collecting and analysing extra-embryonic DNA is evaluated, and consideration is given to the potential biological and technical hindrances to obtaining a reliable clinical diagnosis. Original research and review papers concerning niPGT were sourced by searching PubMed and Google Scholar databases until July 2019. Searches comprised the keywords: 'non-invasive'; 'cell-free DNA'; 'blastocentesis'; 'blastocoel fluid'; 'spent culture media'; 'embryo culture medium'; 'preimplantation genetic testing'; 'preimplantation genetic diagnosis'; 'preimplantation genetic screening'; and 'aneuploidy'. Embryonic DNA is frequently detectable in BF and SCM of embryos produced during IVF treatment. Initial studies have achieved some success when performing cytogenetic and molecular genetic analysis. However, in many cases, the efficiency has been restricted by technical complications associated with the low quantity and quality of the DNA. Reported levels of ploidy agreement between SCM/BF samples and biopsied embryonic cells vary widely. In some cases, a discrepancy with respect to cytogenetic data obtained after trophectoderm biopsy may be attributable to embryonic mosaicism or DNA contamination (usually of maternal origin). Some research indicates that aneuploid cells are preferentially eliminated from the embryo, suggesting that their DNA might be over-represented in SCM and BF samples; this hypothesis requires further investigation. Available data suggest that BF and SCM samples frequently provide DNA templates suitable for genetic analyses, offering a potential means of PGT that is less expensive than traditional methods, requires less micromanipulation skill and poses a lower risk to embryos. Critically, DNA isolation and amplification protocols must be optimised to reproducibly obtain an accurate clinical diagnosis, whilst minimising the impact of confounding factors such as contamination. Further investigations are required to understand the mechanisms underlying the release of embryonic DNA and to determine the extent to which this material reflects the true genetic status of the corresponding embryo. Currently, the clinic al potential of niPGT remains unknown.

Embryo culturing conditions and the development of non-invasive preimplantation genetic testing for aneuploidy detection (NI-PGT-A)

The absence of standardised culturing conditions or molecular testing methodologies, including whole genome amplification (WGA) used for non-invasive preimplantation genetic testing of spent embryo culture media 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 impact the accumulation of embryonic and contaminating DNA in spent embryo culture media and optimisation of either the culturing conditions, molecular testing methodologies, or both, should yield the highest level of concordant results for NI-PGT-A. This study examined rates of ploidy concordance between spent embryo culture media and embryo biopsies to evaluate the impact of culturing conditions on NI-PGT-A results. 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. Equivalent volumes of spent embryo culture media samples from 10μl-60μl culture droplets, from either continuous (n=4 labs) or two-step cultures (n=4 labs) were whole genome amplified using DOPlify® kit reagents (PerkinElmer). WGA DNA yield was assessed by gel electrophoresis and high sensitivity quantification using a Qubit® instrument (Thermo Fisher® Scientific). Next generation sequencing libraries were generated according to the PG-Seq™ kit 48 sample protocol and sequencing was performed on a MiSeq® instrument (Illumina®). Data was bioinformatically aligned to hg19, and WGA DNA yield, NGS metrics, and whole chromosome aneuploidy concordance with the PGT-A result for the embryo biopsy were determined. Whole genome amplification using the 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 29-75% for autosomal chromosomes and 47-94% for sex chromosomes using a single-step culturing system (n=4), compared with concordance rates of 67-90% and 50-97% respectively when media was changed during the 5-6 day culture (n=4). DNA yield was not affected by embryo culture media droplet volume, or continuous or two-step culture. Sex chromosome concordance varied between individual labs, suggesting that embryological processes are important in NI-PGT-A testing. Further statistical analysis during the ongoing larger scale collaborative study will determine quality control parameters for acceptance of NI-PGT-A results. 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.

Diseases Have No Borders, Neither Should Research!

In response to recent anti-Chinese sentiment in the US, Sunney Xie uses his own experiences to assert that American ideals should not be replaced by nationalism and populism and that everybody wins in Sino-US scientific collaborations, contrary to what Americans are led to believe: that China is the sole beneficiary.

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.