- Email: [email protected]
29. CHROMOSOMAL COPY NUMBER ANALYSIS OF CHORIONIC VILLUS FROM SPONTANEOUS ABORTION BY NEXT GENERATION SEQUENCING
e44
RBMO VOLUME 39 ISSUE s1 2019
need for direct mutation detection (Handyside et al. 2010). Although suitable for most couples undergoing PGT-M, the use of a fixed set of SNPs may provide inadequate information in certain genetic regions or in consanguineous couples (Konstantinidis et al. 2015). Whole genome sequencing (WGS) has the potential to individualise informative SNP detection and perform concurrent direct mutation detection. However, no studies have evaluated the potential for application of the karyomapping algorithm to WGS data.
common genes requiring PGT-M demonstrated an average 32x increase in the number of targets available for analysis in a PGT-M cycle. Analysis of 15x coverage WGS data in a model of embryo biopsy demonstrated an average 279,851 informative SNPs detected using standard variant calling and an average 285,107 using AI-based variant calling. The median distance between informative SNPs was reduced from 26,782 nucleotides to 1,212 nucleotides when comparing WGS-based karyomapping to SNP array-based karyomapping.
Material and Methods: The Platinum Genomes dataset (https://sapac. illumina.com/platinumgenomes. html) was analysed as a model for the application of karyomapping to WGS data. Variant call format (VCF) files were analysed according to the principles of karyomapping to create a map of informative biallelic SNPs in each grandparental haplotype. A model of embryo biopsy was created by diluting DNA from one of the family members (NA12878) to 30pg and whole genome amplification was performed with the Repli-G single cell kit (Qiagen, Hilden, Germany) as a model of trophectoderm biopsy. Whole genome sequencing was performed in triplicate on a high output NextSeq 2500 run (Illumina, California, USA) at average 15x coverage and the number of informative SNPs detected compared to the karyomapping SNP microarray. Comparison of variant calling methods was performed using the Genome Analysis Toolkit v3.4 (Broad Institute, Massachusetts, USA) and DeepVariant (Google, California, USA).
Conclusions: This study provides preclinical validation for the application of the karyomapping algorithm to WGS data. The markedly increased number of informative SNPs will allow successful feasibility studies for all couples presenting for PGT-M. The decreased distance between informative SNPs detected in a model embryo biopsy will improve diagnostic accuracy, particularly in detecting small double crossover events and reducing the number of cycles in which inadequate informative markers have been detected in the embryo sample. Finally, the use of WGS for diagnostic purposes, especially for rare and undiagnosed genetic disease, will allow the creation of a high resolution digital karyomap for couples with an affected child and improve the transition to IVF and PGT-M.
Results: Analysis of WGS data from the Platinum Genomes family revealed a total of 2,338,158 informative SNPs distributed evenly between grandparental haplotypes, of which 85,053 (3.6%) would have been detectable using the karyomapping SNP array. Analysis of the karyomapping window around
Keywords: Karyomapping; whole genome sequencing; AI
doi: 10.1016/j.rbmo.2019.04.081
29. CHROMOSOMAL COPY NUMBER ANALYSIS OF CHORIONIC VILLUS FROM SPONTANEOUS ABORTION BY NEXT GENERATION SEQUENCING
Y. Tamura1, M. Santo1, Y. Araki1, H. Matsubayashi2, Y. Takaya2, M. Doshida3, K. Sakaguchi3, K. Yamaguchi3, S. Mizuta2, N. Kim2,
K. Okuno2, K. Kitaya2, T. Takeuchi3, T. Ishikawa2 1 Nippon
Reprogenetics Inc., Maebashi, Japan Clinic Osaka, Kita-ku, Osaka, Japan Clinic Tokyo, Minato-ku, Tokyo, Japan
2 Reproduction
3 Reproduction
Introduction: Chromosomal abnormalities are the most common reason for spontaneous abortion. Conventional cytogenetic analysis by G-banded karyotyping is generally performed for chromosomal analysis, but it has a problem with lowresolution, and is needed long term cell culture and enough experience for diagnosis. More recently, next generation sequencing has been introducing and improving for chromosomal analysis as an accurate, high resolution and throughput method. In this study we aimed to compare the consistency between conventional G-banding and NGSbased chromosomal copy number analysis for chorionic villus from spontaneous abortion. In addition, the frequency of each chromosomal aneuploidy was evaluated. Materials and methods: From February, 2018, to April, 2018, chromosomal analysis for 7 chorionic villus samples from spontaneous abortions (from 7 to 9 weeks) were carried out both conventional G-banding and NGS (VeriSeqPGS, Illumia). The frequency of each chromosomal aneuploidy was investigated for 110 cases from February, 2018 to December, 2018. Results: NGS was able to analyze all 7 cases, but G-banding was able to detect 6 cases, and one case was cell growth failure. In 6 cases analyzed by G-banding, the results of 5 cases were consistent with the results of NGS, but one case suspected maternal cell contamination. Among 7 cases analyzed by the NGS, 2 cases were normal male karyotype (46, XY) and 5 cases were autosomal trisomy, implying that there were no cases suspected of maternal cell contamination.
Among 110 cases, chorionic villus was not observed under microscope from 18 samples, NGS result was obtained from 92 cases. Seventy-one cases were found to have abnormal chromosomes (71/92, 77.2%), and 21 cases were normal karyotype (21/92, 22.8%). Aneuploidy of chromosome 22 (21.8%), chromosome 16 (17.9), chromosome 15 (11.9%), chromosome 21 (10.3%) and Chromosome X (10.3%) were more frequently, consistent with previous report. Conclusions: Chromosome analysis using NGS not only obtained comparable results to conventional G-banding, but also is able to analyze more accurately and quickly. Keywords: Chorionic villus; Karyotyping; Next generation sequencing; Spontaneous abortion
doi: 10.1016/j.rbmo.2019.04.082
30. LIVE BIRTHS FOLLOWING DAY 7 BLASTOCYST TRANSFER AFTER PREIMPLANTATION GENETIC TESTING FOR ANEUPLOIDY (PGT-A)
C.W. Chan, Y.X. Lim, M.W. Lim, C.S.S. Lee, C.S. Tan Alpha International Women's Wpecialists SDN BHD, Kuala Lumpur, Malaysia
Introduction: Extended culture to blastocyst stage is being adopted in many IVF clinics, with the aim to improve embryo selection and promoting single embryo transfer. Selection of these blastocysts usually occurs up to Day 6 of embryo culture. However, a study (Su et al., 2016) has revealed that Day 7 blastocysts can be euploid and can result in healthy live births. In this study, we evaluate the euploidy rate of Day 7 blastocysts and the clinical outcome of transferring these euploid Day 7 vitrified-warmed blastocysts in Alpha Fertility Centre. Materials & Methods: Forty-five (45) patients had utilisable blastocysts only on Day 7 of culture between
RBMO VOLUME 39 ISSUE s1 2019
Aug-2015 and Nov-2018. A total of 58 blastocysts which were at least fair graded (Gardner, 1999) were biopsied on Day 7 and analysed using Next-Generation Sequencing (Life Technologies, USA). Vitrification was done after biopsy using Cryotec method (Cryotec, Japan). Clinical outcome was assessed following the transfer of euploid blastocysts. All successful pregnancies were monitored until delivery. Results: The mean maternal age was 36.0 (age range:22-44). Of the 58 blastocysts analysed, 46.6% (27/58) blastocysts were euploid, 39.6% (23/58) blastocysts were aneuploid and 13.8% (8/58) blastocysts were mosaic. Subsequently, 10 patients had frozen blastocyst transfer: 9 had single blastocyst transfer (SBT); 1 had double blastocysts transfer (DBT). All blastocysts survived intact post-warmed. The clinical pregnancy rate was 70% (7/10) with an implantation rate of 63.6% (7/11). Two (2) of these patients each delivered a healthy baby weighing 2.81 kg and 3.08 kg respectively, while 1 pregnancy is ongoing at 30+3 weeks of gestation at the time of writing. Unfortunately, the remaining 4 pregnancies resulted in miscarriages at 8+1, 8+2, 7+5 and 9+4 weeks of gestation respectively. Conclusions: Our study suggests that Day 7 blastocysts can indeed be euploid and yield healthy live births albeit the seemingly higher miscarriage rate. Therefore, extended culture of embryos to Day 7 should be considered as a standard practice in IVF laboratories for patients with no utilisable blastocyst on Day 5 or Day 6 of culture. Keywords: Live birth; Day 7 blastocyst;
Preimplantation Genetic Testing for Aneuploidy (PGT-A); Frozen blastocyst transfer
REFERENCE Su, Y., Li, J.J., Wang, C., Haddad, G., Wang, W.H. Aneuploidy analysis in day 7 human blastocysts produced by in vitro fertilization.
e45
Reproductive biology and endocrinology: RB&E 2016; 14: 20
doi: 10.1016/j.rbmo.2019.04.083
31. CHROMOSOMAL MOSAICISM AS A RISK FACTOR FOR INCORRECT RESULTS OF PGT-A
V. Kaimonov1, E. Musatova1, T. Khryapenkova2, J. Zinina2, K. Ilyin2, E. Pomerantseva1 1
"GENETICO", Moscow, Russian Federation GMS IVF Clinic - Family Planning Center, Moscow, Russian Federation 2
Introduction: Mosaicism may cause discordance between the result of PGT-A and the karyotype of cells outside the biopsy area. How likely is the risk of incorrect results of PGT-A due to chromosomal mosaicism? The aim of present study was comparison of molecular karyotypes of trophectoderm cells and inner cell mass. The study presents the results of re-analysis of the molecular karyotypes of 10 embryos by the comparative genomic hybridization on microarrays (aCGH). Material & methods: Initially, the trophectoderm biopsy was performed for all 10 embryos for PGT-A using the aCGH analysis (24sure, Illumina). All 10 trophectoderm samples were found to be aneuploid. Corresponding embryos were not recommended for transfer and were included in this study per patients’ informed consent. All embryos were divided into 2 parts: trophectoderm (TE) and the inner cell mass (ICM). Each part was analyzed separately. For 2 embryos blastomeres (B) not included in the blastocyst were also available (for embryos №2 and №3). Re-analysis was performed using the aCGH analysis (24sure, Illumina). Results: Molecular karyotypes of TE cells and ICM 7 of 10 embryos coincided with PGT-A results. Molecular karyotypes of 3 samples demonstrated incomplete concordance with the PGT-A results. We detected
RBMO VOLUME 39 ISSUE s1 2019
need for direct mutation detection (Handyside et al. 2010). Although suitable for most couples undergoing PGT-M, the use of a fixed set of SNPs may provide inadequate information in certain genetic regions or in consanguineous couples (Konstantinidis et al. 2015). Whole genome sequencing (WGS) has the potential to individualise informative SNP detection and perform concurrent direct mutation detection. However, no studies have evaluated the potential for application of the karyomapping algorithm to WGS data.
common genes requiring PGT-M demonstrated an average 32x increase in the number of targets available for analysis in a PGT-M cycle. Analysis of 15x coverage WGS data in a model of embryo biopsy demonstrated an average 279,851 informative SNPs detected using standard variant calling and an average 285,107 using AI-based variant calling. The median distance between informative SNPs was reduced from 26,782 nucleotides to 1,212 nucleotides when comparing WGS-based karyomapping to SNP array-based karyomapping.
Material and Methods: The Platinum Genomes dataset (https://sapac. illumina.com/platinumgenomes. html) was analysed as a model for the application of karyomapping to WGS data. Variant call format (VCF) files were analysed according to the principles of karyomapping to create a map of informative biallelic SNPs in each grandparental haplotype. A model of embryo biopsy was created by diluting DNA from one of the family members (NA12878) to 30pg and whole genome amplification was performed with the Repli-G single cell kit (Qiagen, Hilden, Germany) as a model of trophectoderm biopsy. Whole genome sequencing was performed in triplicate on a high output NextSeq 2500 run (Illumina, California, USA) at average 15x coverage and the number of informative SNPs detected compared to the karyomapping SNP microarray. Comparison of variant calling methods was performed using the Genome Analysis Toolkit v3.4 (Broad Institute, Massachusetts, USA) and DeepVariant (Google, California, USA).
Conclusions: This study provides preclinical validation for the application of the karyomapping algorithm to WGS data. The markedly increased number of informative SNPs will allow successful feasibility studies for all couples presenting for PGT-M. The decreased distance between informative SNPs detected in a model embryo biopsy will improve diagnostic accuracy, particularly in detecting small double crossover events and reducing the number of cycles in which inadequate informative markers have been detected in the embryo sample. Finally, the use of WGS for diagnostic purposes, especially for rare and undiagnosed genetic disease, will allow the creation of a high resolution digital karyomap for couples with an affected child and improve the transition to IVF and PGT-M.
Results: Analysis of WGS data from the Platinum Genomes family revealed a total of 2,338,158 informative SNPs distributed evenly between grandparental haplotypes, of which 85,053 (3.6%) would have been detectable using the karyomapping SNP array. Analysis of the karyomapping window around
Keywords: Karyomapping; whole genome sequencing; AI
doi: 10.1016/j.rbmo.2019.04.081
29. CHROMOSOMAL COPY NUMBER ANALYSIS OF CHORIONIC VILLUS FROM SPONTANEOUS ABORTION BY NEXT GENERATION SEQUENCING
Y. Tamura1, M. Santo1, Y. Araki1, H. Matsubayashi2, Y. Takaya2, M. Doshida3, K. Sakaguchi3, K. Yamaguchi3, S. Mizuta2, N. Kim2,
K. Okuno2, K. Kitaya2, T. Takeuchi3, T. Ishikawa2 1 Nippon
Reprogenetics Inc., Maebashi, Japan Clinic Osaka, Kita-ku, Osaka, Japan Clinic Tokyo, Minato-ku, Tokyo, Japan
2 Reproduction
3 Reproduction
Introduction: Chromosomal abnormalities are the most common reason for spontaneous abortion. Conventional cytogenetic analysis by G-banded karyotyping is generally performed for chromosomal analysis, but it has a problem with lowresolution, and is needed long term cell culture and enough experience for diagnosis. More recently, next generation sequencing has been introducing and improving for chromosomal analysis as an accurate, high resolution and throughput method. In this study we aimed to compare the consistency between conventional G-banding and NGSbased chromosomal copy number analysis for chorionic villus from spontaneous abortion. In addition, the frequency of each chromosomal aneuploidy was evaluated. Materials and methods: From February, 2018, to April, 2018, chromosomal analysis for 7 chorionic villus samples from spontaneous abortions (from 7 to 9 weeks) were carried out both conventional G-banding and NGS (VeriSeqPGS, Illumia). The frequency of each chromosomal aneuploidy was investigated for 110 cases from February, 2018 to December, 2018. Results: NGS was able to analyze all 7 cases, but G-banding was able to detect 6 cases, and one case was cell growth failure. In 6 cases analyzed by G-banding, the results of 5 cases were consistent with the results of NGS, but one case suspected maternal cell contamination. Among 7 cases analyzed by the NGS, 2 cases were normal male karyotype (46, XY) and 5 cases were autosomal trisomy, implying that there were no cases suspected of maternal cell contamination.
Among 110 cases, chorionic villus was not observed under microscope from 18 samples, NGS result was obtained from 92 cases. Seventy-one cases were found to have abnormal chromosomes (71/92, 77.2%), and 21 cases were normal karyotype (21/92, 22.8%). Aneuploidy of chromosome 22 (21.8%), chromosome 16 (17.9), chromosome 15 (11.9%), chromosome 21 (10.3%) and Chromosome X (10.3%) were more frequently, consistent with previous report. Conclusions: Chromosome analysis using NGS not only obtained comparable results to conventional G-banding, but also is able to analyze more accurately and quickly. Keywords: Chorionic villus; Karyotyping; Next generation sequencing; Spontaneous abortion
doi: 10.1016/j.rbmo.2019.04.082
30. LIVE BIRTHS FOLLOWING DAY 7 BLASTOCYST TRANSFER AFTER PREIMPLANTATION GENETIC TESTING FOR ANEUPLOIDY (PGT-A)
C.W. Chan, Y.X. Lim, M.W. Lim, C.S.S. Lee, C.S. Tan Alpha International Women's Wpecialists SDN BHD, Kuala Lumpur, Malaysia
Introduction: Extended culture to blastocyst stage is being adopted in many IVF clinics, with the aim to improve embryo selection and promoting single embryo transfer. Selection of these blastocysts usually occurs up to Day 6 of embryo culture. However, a study (Su et al., 2016) has revealed that Day 7 blastocysts can be euploid and can result in healthy live births. In this study, we evaluate the euploidy rate of Day 7 blastocysts and the clinical outcome of transferring these euploid Day 7 vitrified-warmed blastocysts in Alpha Fertility Centre. Materials & Methods: Forty-five (45) patients had utilisable blastocysts only on Day 7 of culture between
RBMO VOLUME 39 ISSUE s1 2019
Aug-2015 and Nov-2018. A total of 58 blastocysts which were at least fair graded (Gardner, 1999) were biopsied on Day 7 and analysed using Next-Generation Sequencing (Life Technologies, USA). Vitrification was done after biopsy using Cryotec method (Cryotec, Japan). Clinical outcome was assessed following the transfer of euploid blastocysts. All successful pregnancies were monitored until delivery. Results: The mean maternal age was 36.0 (age range:22-44). Of the 58 blastocysts analysed, 46.6% (27/58) blastocysts were euploid, 39.6% (23/58) blastocysts were aneuploid and 13.8% (8/58) blastocysts were mosaic. Subsequently, 10 patients had frozen blastocyst transfer: 9 had single blastocyst transfer (SBT); 1 had double blastocysts transfer (DBT). All blastocysts survived intact post-warmed. The clinical pregnancy rate was 70% (7/10) with an implantation rate of 63.6% (7/11). Two (2) of these patients each delivered a healthy baby weighing 2.81 kg and 3.08 kg respectively, while 1 pregnancy is ongoing at 30+3 weeks of gestation at the time of writing. Unfortunately, the remaining 4 pregnancies resulted in miscarriages at 8+1, 8+2, 7+5 and 9+4 weeks of gestation respectively. Conclusions: Our study suggests that Day 7 blastocysts can indeed be euploid and yield healthy live births albeit the seemingly higher miscarriage rate. Therefore, extended culture of embryos to Day 7 should be considered as a standard practice in IVF laboratories for patients with no utilisable blastocyst on Day 5 or Day 6 of culture. Keywords: Live birth; Day 7 blastocyst;
Preimplantation Genetic Testing for Aneuploidy (PGT-A); Frozen blastocyst transfer
REFERENCE Su, Y., Li, J.J., Wang, C., Haddad, G., Wang, W.H. Aneuploidy analysis in day 7 human blastocysts produced by in vitro fertilization.
e45
Reproductive biology and endocrinology: RB&E 2016; 14: 20
doi: 10.1016/j.rbmo.2019.04.083
31. CHROMOSOMAL MOSAICISM AS A RISK FACTOR FOR INCORRECT RESULTS OF PGT-A
V. Kaimonov1, E. Musatova1, T. Khryapenkova2, J. Zinina2, K. Ilyin2, E. Pomerantseva1 1
"GENETICO", Moscow, Russian Federation GMS IVF Clinic - Family Planning Center, Moscow, Russian Federation 2
Introduction: Mosaicism may cause discordance between the result of PGT-A and the karyotype of cells outside the biopsy area. How likely is the risk of incorrect results of PGT-A due to chromosomal mosaicism? The aim of present study was comparison of molecular karyotypes of trophectoderm cells and inner cell mass. The study presents the results of re-analysis of the molecular karyotypes of 10 embryos by the comparative genomic hybridization on microarrays (aCGH). Material & methods: Initially, the trophectoderm biopsy was performed for all 10 embryos for PGT-A using the aCGH analysis (24sure, Illumina). All 10 trophectoderm samples were found to be aneuploid. Corresponding embryos were not recommended for transfer and were included in this study per patients’ informed consent. All embryos were divided into 2 parts: trophectoderm (TE) and the inner cell mass (ICM). Each part was analyzed separately. For 2 embryos blastomeres (B) not included in the blastocyst were also available (for embryos №2 and №3). Re-analysis was performed using the aCGH analysis (24sure, Illumina). Results: Molecular karyotypes of TE cells and ICM 7 of 10 embryos coincided with PGT-A results. Molecular karyotypes of 3 samples demonstrated incomplete concordance with the PGT-A results. We detected