- Email: [email protected]
48. OBSERVING THE IMPACT OF EMBRYO CULTURING CONDITIONS ON NON-INVASIVE PREIMPLANTATION GENETIC TESTING FOR ANEUPLOIDY DETECTION (NI-PGT-A)
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RBMO VOLUME 39 ISSUE s1 2019
Conclusion: Karyomapping is an applicable method to PGT-M also in families with detected "de novo" mutation. Under the assumption of a known mutation and a sufficient number of embryos as well as a coverage check in the region of interest it is possible to reveal the mutation using the sequenced embryo as a reference family member. Keywords: karyomapping; monogenic disease; de novo mutation
doi: 10.1016/j.rbmo.2019.04.100
48. OBSERVING THE IMPACT OF EMBRYO CULTURING CONDITIONS ON NON-INVASIVE PREIMPLANTATION GENETIC TESTING FOR ANEUPLOIDY DETECTION (NI-PGT-A)
M. Jasper, C. Robinson PerkinElmer Health Sciences (Australia) Pty Ltd, Adelaide, Australia
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 (NIPGT-A) may explain the variable rates of concordance reported between the spent embryo culture media and embryo biopsy results todate. 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 10ul60ul 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 3355% 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 NIPGT-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. Keywords: Non-invasive; PGT-A; spent media; WGA
doi: 10.1016/j.rbmo.2019.04.101
49. MOLECULAR PGT-M FOR VUS IN THE GENOMIC ERA: TO DO OR NOT TO DO?
K. Rotshenker Olshinka, O. Weiss, N. Srebnik, S. Shaviv, O. Freireich, R. Segel, T. Eldar Geva, G. Altarescu Shaare Zedek Medical Center, Jerusalem, Israel
Introduction: The new chromosomal microarray CMA technology, allows the identification of genetic defects in many disorders that would previously have escaped detection. Alongside the benefits, it exposes us to copy number variants (CNV) whose pathogenic significance is unclear "Variants of unknown significance" (VUS). In our cohort we reviewed all files of couples that applied for PGT-M at our clinic with a finding of VUS. We aimed to characterize these VUS and the cases in which PGT-M was decided upon. Materials and methods: A retrospective cohort study in the Zohar Unit for PGT-M, Shaare Zedek medical center, 2014-2019. Files of all couples with a finding of VUS among those who applied for PGT-M with any CMA findings were reviewed. VUS were classified (likely pathogenic, VUS, likely benign) according to the American collage of medical genetics and Genomics classification at time of first consultation and to date (December 2018). Pathogenic variants were not included in the study. PGT-M was performed on blastromeres of throphoectoderm biopsy by PCR using surrounding polymorphic
RBMO VOLUME 39 ISSUE s1 2019
Conclusion: Karyomapping is an applicable method to PGT-M also in families with detected "de novo" mutation. Under the assumption of a known mutation and a sufficient number of embryos as well as a coverage check in the region of interest it is possible to reveal the mutation using the sequenced embryo as a reference family member. Keywords: karyomapping; monogenic disease; de novo mutation
doi: 10.1016/j.rbmo.2019.04.100
48. OBSERVING THE IMPACT OF EMBRYO CULTURING CONDITIONS ON NON-INVASIVE PREIMPLANTATION GENETIC TESTING FOR ANEUPLOIDY DETECTION (NI-PGT-A)
M. Jasper, C. Robinson PerkinElmer Health Sciences (Australia) Pty Ltd, Adelaide, Australia
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 (NIPGT-A) may explain the variable rates of concordance reported between the spent embryo culture media and embryo biopsy results todate. 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 10ul60ul 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 3355% 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 NIPGT-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. Keywords: Non-invasive; PGT-A; spent media; WGA
doi: 10.1016/j.rbmo.2019.04.101
49. MOLECULAR PGT-M FOR VUS IN THE GENOMIC ERA: TO DO OR NOT TO DO?
K. Rotshenker Olshinka, O. Weiss, N. Srebnik, S. Shaviv, O. Freireich, R. Segel, T. Eldar Geva, G. Altarescu Shaare Zedek Medical Center, Jerusalem, Israel
Introduction: The new chromosomal microarray CMA technology, allows the identification of genetic defects in many disorders that would previously have escaped detection. Alongside the benefits, it exposes us to copy number variants (CNV) whose pathogenic significance is unclear "Variants of unknown significance" (VUS). In our cohort we reviewed all files of couples that applied for PGT-M at our clinic with a finding of VUS. We aimed to characterize these VUS and the cases in which PGT-M was decided upon. Materials and methods: A retrospective cohort study in the Zohar Unit for PGT-M, Shaare Zedek medical center, 2014-2019. Files of all couples with a finding of VUS among those who applied for PGT-M with any CMA findings were reviewed. VUS were classified (likely pathogenic, VUS, likely benign) according to the American collage of medical genetics and Genomics classification at time of first consultation and to date (December 2018). Pathogenic variants were not included in the study. PGT-M was performed on blastromeres of throphoectoderm biopsy by PCR using surrounding polymorphic