- Email: [email protected]
Optimization of PGD for recurrent t(11;22) carrier
RBMO VOLUME 38 ISSUE S1 2019
option that does not compromise the clinical results and perinatal outcome in a PGT-A program. The annual data collection provide an important resource for data mining and for following trends in PGT practice, the same way ESHRE Consortium and ASRM recommend. doi: 10.1016/j.rbmo.2019.03.075
29. WHY DAY 3 BIOPSY MOSAICISM IS BLAMED FOR THE LACK OF SUCCESS OF PGD-A?
P. Eibes1, E. Francis1, R. Chaturvedi1, M. Nawaz1, S. Shyju1, D. Thulasidas1, A. Hellani1 1
Viafet Genomics Laboratory, Al Shafar Building 7Suite 208Al Wasl Road, Dubai, UAE
Introduction: Preimplantation Genetic Diagnosis- Aneuploidy Screening (PGD-A) is a technique commonly used in conjunction with IVF treatments. PGD-A is intended to identify those embryos with greater likelihood to implant. It is offered to patients at increased risk of producing chromosomally abnormal embryos (increased maternal age, Recurrent miscarriage, IVF failure and severe male factor). Technical related Error rates of PGD-A have fallen from 7% when using FISH to less than 1% when using modern techniques such as Next Generation Sequencing (NGS). Another source of error that is related to the physiology of the embryos is mosaicism described as the presence of two or more cells lines within an embryo. The advantage of NGS made the detection of mosaicism easier due to the i) higher sensitivity in aneuploidy detection and ii) the reliability of the technique making non-mosaicism related error minimal. The objective of our study is to assess the mosaicism rate and the misdiagnosis rate due to euploid/ aneuploid mosaic embryos on day 3 biopsy.
Material and Methods: Embryos from patients undergoing PGD-A were biopsied on day 3 and sent to our laboratory for Aneuploidy Screening by NGS. Blind Reanalysis ofembryonic cells from embryos diagnosed as aneuploid but with good morphology and development was performed. Rebiopsy was carried out either on day 4, day 5 or day 6. Both, original analysis and reanalysis were performed by NGS (MySeq, Illumina platform). Result(s): PGD-A was performed in our laboratory on 29309 embryos at cleavage stage from 2016 to date. Rebiopsy for 173 embryos, originally diagnosed as aneuploidy, were blindly reanalyzed. There was 118 out of 173 showed aneuploidy (91.3%) while 15 out of 173 were found euploids (8.7%). The aneuploid embryos were divided into 3 categories: first where 118 embryos out of 173 (68.2%) showed the same initial result; second, 37 of the 173 (21.4%) showed at least one aneuploidy that was present in the initial result and third 3 out 173 (1.7%) showed a different an inverted figure of deletion/duplication for the same chromosome. Conclusions: This study shows a very high confirmation rate (more than 90%) for day 3 biopsies. Our misdiagnosis rate after day 3 biopsy is 8.7%, due to mosaicism. Our data support the usefulness of day 3 biopsy PGD-A by NGS in places where day 5 biopsy is not possible. The mosaic rate on a relatively high number of embryos observed in this study is in line with other similar reported data. Our reanalysis was based on rebiopsies on embryos showing a good division after the first biopsy which might explain the low mosaic rate. Despite the high accuracy in day 3 biopsy diagnosis, implantation/pregnancy rate is still in the range of 50-60% implicating the necessity of finding more embryonic
e47
viability markers allowing a better outcome after IVF/PGD-A. Keywords: Cleavage stage, PGD-AS, NGS, Mosaicism
doi: 10.1016/j.rbmo.2019.03.076
30. OPTIMIZATION OF PGD FOR RECURRENT T(11;22) CARRIER
Takema Kato1, Maki Kato1, Yasuko Shinkai1, Haruki Nishizawa2, Toshiaki Endo3, Chikako Kani4, Masanori Ochi4, Shin Hayashi5, Johji Inazawa5, Hiroki Kurahashi1 1
Division of Molecular Genetics, Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi 470-1192, Japan 2 Department of Obstetrics and Gynecology, Fujita Health University, Toyoake, Aichi 470-1192, Japan 3 Department of Obstetrics and Gynecology, Sapporo Medical University, Genome and Transcriptome Analysis Center, Fujita Health University, SapporoToyoake, HokkaidoAichi 060-8543470-1192, JapanJapan 4 Ochi Yume Clinic Nagoya, Aichi 460-0002, Japan 5 Department of Molecular Cytogenetics, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan
Introduction: t(11;22)(q23;q11) is the most common reciprocal translocation in humans. Carriers of the balanced t(11;22) are phenotypically normal, but they often manifest problems with reproduction such as infertility, recurrent pregnancy loss, or the birth of unbalanced offspring with the Emanuel syndrome. Preimplantation genetic diagnosis (PGD) is the one of the solution for such reproductive problems. In general, comprehensive chromosomal analyses by microarray-based comparative genomic hybridization (aCGH) or next generation sequence (NGS) are applied for PGD. In addition, since the breakpoints of both chromosomes 11 and 22 are localized within a small region, translocationspecific PCR might be applied to the PGD for t(11;22). Material and Methods: To set up optimal PGD method for t(11;22), we compared various cytogenetics analyses using whole genome amplified DNA from lymphoblastoid
e48
RBMO VOLUME 38 ISSUE S1 2019
cell line from a patient with Emanuel syndrome having trisomy at the regions distal to 11q23 and proximal to 22q11. Results: NGS is suited for determination of the copy number of the unbalanced translocation region better than aCGH or SNP array analyses. CNV-seq method improves detection sensitivity further. However, since the chromosome 22 breakpoint region includes a considerable number of segmental duplications, all methods have low sensitivity to detect copy number change at the proximal 22q11 region. Since translocationspecific PCR can detect the translocation chromosomes in whole genome amplified DNA, combination of NGS and this PCR system is useful to predict the segregation pattern of translocated chromosomes. Conclusions: Translocation-specific PCR is a simple, rapid and costeffective method in the PGD for tt(11;22) ranslocation. Keyword: St(11;22), Translocation-specific PCR, Palindrome
doi: 10.1016/j.rbmo.2019.03.077
31. CLINICAL OUTCOME: THE RELATIONSHIP BETWEEN MOSAICISM AND ADVANCED MATERNAL AGE WITH THE USE OF NEXT GENERATION SEQUENCING (NGS)
M.H. Yeoh, J.J. Chen, E. Sinthamoney, P.S. Wong Sunfert International Fertility Centre, Unit 2-2Level 2 Nexus7Jalan Kerinchi, Bangsar South, Kuala Lumpur 59200, Wilayah Persekutuan Kuala Lumpur Corresponding Authors: Aaron, Chen; Eeson, Sinthamoney; Pak Seng, Wong
Introduction: Chromosomal mosaicism refers to the presence of two or more cell lines with a different chromosome content in an individual or tissue sample. The implementation of high resolution NGS for PGS has provided more information that it is not only
used for embryonic aneuploidy screening, butdetects the presence of mosaicism in trophectoderm biopsies. Recent studies have shown that transfer of mosaic embryos resulted in lower implantation rates and higher miscarriage rates as compared to euploid transfer. This study aims to evaluate whether there is an association between chromosomal mosaicism and advanced maternal age. It also aims to compare the clinical pregnancy and implantation rate between euploid and whole chromosomal mosaic embryos transfer. Materials and Method: Retrospective observational study of 533 blastocyststage embryos analysed by NGS from IVF cycles from Sunfert International Fertility Centre over the course of 2015 to 2016. Of those analysed, 197 blastocysts were transferred. The clinical outcome obtained after transfer of whole chromosomal mosaic embryos was compared with those resulting from a control group of 190 euploid blastocysts. Blastocyst-stage embryos underwent biopsy and trophectoderm cells are extracted for aneuploidy screening using IIlumina NGS protocol. Mosaic information for each chromosome was obtained. For this study, only whole chromosomal mosaicism is considered; segmental mosaicism were categorized under euploid embryos. These embryos were further stratified by maternal age group: <35 year old (yo), 36 to 38 yo, 39 to 40 yo, and >40 yo. Result: Results are summarized in Figure 1. The result showed that mosaicism rates did not vary with advancing maternal age, staying at an average of 10.14%. Euploid and aneuploidy embryos, however, did show changes in regards to age factor as the former displayed a descending trend whereas the latter displayed an increasing trend.
Transfer of mosaic embryos resulted in higher clinical pregnancy rate (60% vs 57.5%) and implantation rate (57% vs 52.9%) than euploid embryos. No incidence of miscarriage was reported. Conclusion: In accordance to most of the studies published, our findings showed that mosaicism is not associated with advanced maternal age. Unlike aneuploidy which arises from meiotic error, mosaicism, on the other hand is caused by impaired mitotic chromosome segregation which does not appear to increase with advancing maternal age. Therefore, while there is a direct relationship between advanced maternal age and aneuploidy, age is not a contributor to mosaicism. On the contrary, our findings showed conflicting result in the implantation rate of mosaic embryos. Similar rate of implantation as compared to euploid transfer embryos was found in our clinical study whereas in general, transfer of mosaic embryos have lower implantation rate. This might be the consequence of small sample size of mosaic embryos transfer (<10% of total blastocysts transferred) in this clinical study. Furthermore, no incidence of miscarriage was reported. doi: 10.1016/j.rbmo.2019.03.078
32. IS EUPLOIDY WHAT EMBRYOS NEED FOR IMPLANTATION AND LIVE BIRTH? WE ALSO NEED QUALITY
Amparo Mifsud1, Amparo Mercader1, Laura Escrich1, Damiá Castelló1, Fernanda Insua1, Alberto Tejera1, Diana Beltrán1, Arantzazu Delgado1, Pilar Buendía1, María José de los Santos1 1
IVIRMA-VLC
Introduction: To have an euploid embryo is one of the main
option that does not compromise the clinical results and perinatal outcome in a PGT-A program. The annual data collection provide an important resource for data mining and for following trends in PGT practice, the same way ESHRE Consortium and ASRM recommend. doi: 10.1016/j.rbmo.2019.03.075
29. WHY DAY 3 BIOPSY MOSAICISM IS BLAMED FOR THE LACK OF SUCCESS OF PGD-A?
P. Eibes1, E. Francis1, R. Chaturvedi1, M. Nawaz1, S. Shyju1, D. Thulasidas1, A. Hellani1 1
Viafet Genomics Laboratory, Al Shafar Building 7Suite 208Al Wasl Road, Dubai, UAE
Introduction: Preimplantation Genetic Diagnosis- Aneuploidy Screening (PGD-A) is a technique commonly used in conjunction with IVF treatments. PGD-A is intended to identify those embryos with greater likelihood to implant. It is offered to patients at increased risk of producing chromosomally abnormal embryos (increased maternal age, Recurrent miscarriage, IVF failure and severe male factor). Technical related Error rates of PGD-A have fallen from 7% when using FISH to less than 1% when using modern techniques such as Next Generation Sequencing (NGS). Another source of error that is related to the physiology of the embryos is mosaicism described as the presence of two or more cells lines within an embryo. The advantage of NGS made the detection of mosaicism easier due to the i) higher sensitivity in aneuploidy detection and ii) the reliability of the technique making non-mosaicism related error minimal. The objective of our study is to assess the mosaicism rate and the misdiagnosis rate due to euploid/ aneuploid mosaic embryos on day 3 biopsy.
Material and Methods: Embryos from patients undergoing PGD-A were biopsied on day 3 and sent to our laboratory for Aneuploidy Screening by NGS. Blind Reanalysis ofembryonic cells from embryos diagnosed as aneuploid but with good morphology and development was performed. Rebiopsy was carried out either on day 4, day 5 or day 6. Both, original analysis and reanalysis were performed by NGS (MySeq, Illumina platform). Result(s): PGD-A was performed in our laboratory on 29309 embryos at cleavage stage from 2016 to date. Rebiopsy for 173 embryos, originally diagnosed as aneuploidy, were blindly reanalyzed. There was 118 out of 173 showed aneuploidy (91.3%) while 15 out of 173 were found euploids (8.7%). The aneuploid embryos were divided into 3 categories: first where 118 embryos out of 173 (68.2%) showed the same initial result; second, 37 of the 173 (21.4%) showed at least one aneuploidy that was present in the initial result and third 3 out 173 (1.7%) showed a different an inverted figure of deletion/duplication for the same chromosome. Conclusions: This study shows a very high confirmation rate (more than 90%) for day 3 biopsies. Our misdiagnosis rate after day 3 biopsy is 8.7%, due to mosaicism. Our data support the usefulness of day 3 biopsy PGD-A by NGS in places where day 5 biopsy is not possible. The mosaic rate on a relatively high number of embryos observed in this study is in line with other similar reported data. Our reanalysis was based on rebiopsies on embryos showing a good division after the first biopsy which might explain the low mosaic rate. Despite the high accuracy in day 3 biopsy diagnosis, implantation/pregnancy rate is still in the range of 50-60% implicating the necessity of finding more embryonic
e47
viability markers allowing a better outcome after IVF/PGD-A. Keywords: Cleavage stage, PGD-AS, NGS, Mosaicism
doi: 10.1016/j.rbmo.2019.03.076
30. OPTIMIZATION OF PGD FOR RECURRENT T(11;22) CARRIER
Takema Kato1, Maki Kato1, Yasuko Shinkai1, Haruki Nishizawa2, Toshiaki Endo3, Chikako Kani4, Masanori Ochi4, Shin Hayashi5, Johji Inazawa5, Hiroki Kurahashi1 1
Division of Molecular Genetics, Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi 470-1192, Japan 2 Department of Obstetrics and Gynecology, Fujita Health University, Toyoake, Aichi 470-1192, Japan 3 Department of Obstetrics and Gynecology, Sapporo Medical University, Genome and Transcriptome Analysis Center, Fujita Health University, SapporoToyoake, HokkaidoAichi 060-8543470-1192, JapanJapan 4 Ochi Yume Clinic Nagoya, Aichi 460-0002, Japan 5 Department of Molecular Cytogenetics, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan
Introduction: t(11;22)(q23;q11) is the most common reciprocal translocation in humans. Carriers of the balanced t(11;22) are phenotypically normal, but they often manifest problems with reproduction such as infertility, recurrent pregnancy loss, or the birth of unbalanced offspring with the Emanuel syndrome. Preimplantation genetic diagnosis (PGD) is the one of the solution for such reproductive problems. In general, comprehensive chromosomal analyses by microarray-based comparative genomic hybridization (aCGH) or next generation sequence (NGS) are applied for PGD. In addition, since the breakpoints of both chromosomes 11 and 22 are localized within a small region, translocationspecific PCR might be applied to the PGD for t(11;22). Material and Methods: To set up optimal PGD method for t(11;22), we compared various cytogenetics analyses using whole genome amplified DNA from lymphoblastoid
e48
RBMO VOLUME 38 ISSUE S1 2019
cell line from a patient with Emanuel syndrome having trisomy at the regions distal to 11q23 and proximal to 22q11. Results: NGS is suited for determination of the copy number of the unbalanced translocation region better than aCGH or SNP array analyses. CNV-seq method improves detection sensitivity further. However, since the chromosome 22 breakpoint region includes a considerable number of segmental duplications, all methods have low sensitivity to detect copy number change at the proximal 22q11 region. Since translocationspecific PCR can detect the translocation chromosomes in whole genome amplified DNA, combination of NGS and this PCR system is useful to predict the segregation pattern of translocated chromosomes. Conclusions: Translocation-specific PCR is a simple, rapid and costeffective method in the PGD for tt(11;22) ranslocation. Keyword: St(11;22), Translocation-specific PCR, Palindrome
doi: 10.1016/j.rbmo.2019.03.077
31. CLINICAL OUTCOME: THE RELATIONSHIP BETWEEN MOSAICISM AND ADVANCED MATERNAL AGE WITH THE USE OF NEXT GENERATION SEQUENCING (NGS)
M.H. Yeoh, J.J. Chen, E. Sinthamoney, P.S. Wong Sunfert International Fertility Centre, Unit 2-2Level 2 Nexus7Jalan Kerinchi, Bangsar South, Kuala Lumpur 59200, Wilayah Persekutuan Kuala Lumpur Corresponding Authors: Aaron, Chen; Eeson, Sinthamoney; Pak Seng, Wong
Introduction: Chromosomal mosaicism refers to the presence of two or more cell lines with a different chromosome content in an individual or tissue sample. The implementation of high resolution NGS for PGS has provided more information that it is not only
used for embryonic aneuploidy screening, butdetects the presence of mosaicism in trophectoderm biopsies. Recent studies have shown that transfer of mosaic embryos resulted in lower implantation rates and higher miscarriage rates as compared to euploid transfer. This study aims to evaluate whether there is an association between chromosomal mosaicism and advanced maternal age. It also aims to compare the clinical pregnancy and implantation rate between euploid and whole chromosomal mosaic embryos transfer. Materials and Method: Retrospective observational study of 533 blastocyststage embryos analysed by NGS from IVF cycles from Sunfert International Fertility Centre over the course of 2015 to 2016. Of those analysed, 197 blastocysts were transferred. The clinical outcome obtained after transfer of whole chromosomal mosaic embryos was compared with those resulting from a control group of 190 euploid blastocysts. Blastocyst-stage embryos underwent biopsy and trophectoderm cells are extracted for aneuploidy screening using IIlumina NGS protocol. Mosaic information for each chromosome was obtained. For this study, only whole chromosomal mosaicism is considered; segmental mosaicism were categorized under euploid embryos. These embryos were further stratified by maternal age group: <35 year old (yo), 36 to 38 yo, 39 to 40 yo, and >40 yo. Result: Results are summarized in Figure 1. The result showed that mosaicism rates did not vary with advancing maternal age, staying at an average of 10.14%. Euploid and aneuploidy embryos, however, did show changes in regards to age factor as the former displayed a descending trend whereas the latter displayed an increasing trend.
Transfer of mosaic embryos resulted in higher clinical pregnancy rate (60% vs 57.5%) and implantation rate (57% vs 52.9%) than euploid embryos. No incidence of miscarriage was reported. Conclusion: In accordance to most of the studies published, our findings showed that mosaicism is not associated with advanced maternal age. Unlike aneuploidy which arises from meiotic error, mosaicism, on the other hand is caused by impaired mitotic chromosome segregation which does not appear to increase with advancing maternal age. Therefore, while there is a direct relationship between advanced maternal age and aneuploidy, age is not a contributor to mosaicism. On the contrary, our findings showed conflicting result in the implantation rate of mosaic embryos. Similar rate of implantation as compared to euploid transfer embryos was found in our clinical study whereas in general, transfer of mosaic embryos have lower implantation rate. This might be the consequence of small sample size of mosaic embryos transfer (<10% of total blastocysts transferred) in this clinical study. Furthermore, no incidence of miscarriage was reported. doi: 10.1016/j.rbmo.2019.03.078
32. IS EUPLOIDY WHAT EMBRYOS NEED FOR IMPLANTATION AND LIVE BIRTH? WE ALSO NEED QUALITY
Amparo Mifsud1, Amparo Mercader1, Laura Escrich1, Damiá Castelló1, Fernanda Insua1, Alberto Tejera1, Diana Beltrán1, Arantzazu Delgado1, Pilar Buendía1, María José de los Santos1 1
IVIRMA-VLC
Introduction: To have an euploid embryo is one of the main