65. SUCCESSFUL STRATEGY OF COMPREHENSIVE PRE-IMPLANTATION GENETIC TESTING FOR BETA-THALASSEMIA-HEMOGLOBIN E DISEASE AND CHROMOSOME BALANCE USING KARYOMAPPING

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64. CAN FISH PROBE STRATEGIES INFLUENCE MOSAIC EMBRYO RATES IN ROBERTSONIAN TRANSLOCATION PGD?

R. Zenagui, C. Ciabrini, B. Izabel, T. Anahory Montpellier Hospital, Montpellier, France

Introduction: Robertsonian translocation (RT) results from centric fusion of two acrocentric chromosomes. RT carriers are phenotypically normal, but they are known to be at increased risk of repeated miscarriages or of pregnancies resulting in the birth of a child with congenital anomalies, mental retardation. Preimplantation Genetic Diagnosis (PGD) is therefore a solution for RT carriers. A suitable probe strategy on cleavage stage allows us to differentiate balanced embryos, unbalanced embryos, mosaic and chaotic embryos. According to ESHRE recommendations, only balanced embryos are considered for transfer. However, transfer of some mosaic embryos has resulted in live births. We performed the first comparative analysis between a two or a three probes FISH strategies, in order to enhance the safety and security of embryos transferred and to establish the best reliable and low-cost strategy for RT PGD management. Material and methods: Retrospective analysis of 253 preimplantation genetic diagnosis (PGD) cycles were performed on 91 men and 42 women carriers of different Robertsonian translocations t(13;14), t(14;21), t(14;15), t(15;21), t(13;15), t(13;21), t(14;22), t(15;22), t(21;22), t(13;13). The first strategy involved two relevant probes use. Each probe is located within the terminal long arm. The second strategy required a third probe addition on one of the two translocated chromosomes. The additional probe is located close to centromeric regions.

Result(s): Regardless of probe strategies, 253 PGD cycles were performed in the aim to exclude imbalances in both carriers. On a total of 1,549 biopsied cells, 1,458 were successfully analyzed. Among these, 232 were transferred. Clinical pregnancy was achieved in 108 patients, and 85 healthy babies were delivered. No significant difference was observed in the balanced embryo rates (40.5% and 40.4% in a two probes and a three probe strategies respectively). Among these balanced embryos, diagnostic established on single or two mononucleate cells, wasn't affected by the probe strategies. However, a significant difference (p <=0.0001) was observed in mosaic embryo rates. Mosaic embryo rate decreased with three probes strategy in comparison with two probes strategy. On the other hand, chaotic embryo rate increased with three probes strategy in comparison with two probes strategy. Conclusion(s): According to the present findings, concerning balanced embryo rates, the two probes strategy seems more suitable for Robertsonian translocation PGD analysis, firstly because it led to similar results as the three probes strategy. Secondly, this study also highlighted an increase mosaic embryo rate observed with the two probes strategy. Recent data indicated that mosaic embryos may represent a second category to transfer after balanced embryos. Obviously, balanced embryos must be transferred in priority. However in absence of balanced embryos, transfer of a mosaic embryo should be considered. In this case, genetic counseling about risks and potential benefits is of utmost importance to ensure informed decision-making by patients. Keywords: PGD; embryo

development; implantation; viability; robertsoniantranslocation

doi: 10.1016/j.rbmo.2019.04.117

65. SUCCESSFUL STRATEGY OF COMPREHENSIVE PREIMPLANTATION GENETIC TESTING FOR BETATHALASSEMIA-HEMOGLOBIN E DISEASE AND CHROMOSOME BALANCE USING KARYOMAPPING

W. Piyamongkol1, S. Mongkolchaipak2, S. Piyamongkol3 1 

Department of Obstetrics and Gynaecology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand 2  Beloved Fertility Center, Phyathai Sriracha Hospital, Chon Buri, Thailand 3  Department of Pharmaceutical Sciences, Faculty of Pharmacy, Chiang Mai University, Chiang Mai, Thailand

Introduction: Thalassemia syndrome and hemoglobinopathy are the world commonest monogenic disease and cause health and economic burden worldwide. Preimplantation genetic testing (PGT) is an alternative to traditional prenatal diagnosis (PND). However, the nature of a vast variety of mutations makes molecular genetic testing sophisticated and labor intensive. Modern haplotyping using SNP array (aSNP) and Karyomapping algorithm would omit molecular analysis development process and provide chromosome balance information at the same time. This study applied Karyomapping for PGT-M of betathalassemia-hemoglobin E (Hb E) disease in 2 clinical PGT cycles in comparison to PCR testing techniques. Material and methods: Two families at risk of having beta-thalassemiaHb E disesase offspring decided to join the project following thoroughly counselling and inform consent was obtained. The patients underwent routine IVF procedures. Embryo biopsy was performed on Day-5 post-fertilization and the biopsied trophectoderm underwent whole genome amplification. SNP array with Karyomapping analysis was carried out for haplotyping as well as copy number variation (CNV). Multiplex PCR with minisequencing was performed



alongside for confirmation standard molecular mutation analysis. Informative polymorphic marker was also included for contamination identification Results: Thirteen and nine embryos with good morphology from families YW and CS were chosen for PGT, respectively. Karyomapping results of family YW (beta–thalassemia (c.17A>T)-Hb E (c.26G>A) disease) revealed four normal, two betathalassemia trait, one Hb E trait and six affected with beta-thalassemiaHb E disease. Standard mutation analysis using multiplex fluorescent PCR and mini-sequencing confirmed haplotyping results in all embryos. In addition, Karyomapping demonstrated three embryos with chromosome unbalanced, i.e. 45,XY, -19 (affected), 47,XY, +16 (betathalassemia trait) and 47,XX, +21 (beta-thalassemia trait). Therefore, four normal (three male and one female) and one Hb E trait embryos were fulfilled for transfer. One normal male embryo was chosen for transfer and one normal male baby was delivered. Prenatal and postnatal DNA testing confirmed PGT results. Karyomapping results of family CS (beta–thalassemia (c.17A>T)-Hb E (c.26G>A) disease) revealed six Hb E trait and three affected with beta-thalassemia-Hb E disease. Standard mutation analysis using multiplex fluorescent PCR and minisequencing confirmed haplotyping results in all embryos. Additionally, Karyomapping demonstrated two embryos with chromosome unbalanced, i.e. 45,XY, -21 (affected) and 45,XY, -22 (Hb E trait). Therefore, five Hb E trait embryos were fulfilled for transfer. One Hb E trait embryo was chosen for transfer and one ongoing pregnancy was resulted. Polymorphic marker analysis revealed the absence of extraneous DNA contamination. Conclusions: Two clinical PGT-M cycles using Karyomapping were performed for two families at

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risk of having beta–thalassemia (c.17A>T)-Hb E (c.26G>A) disease offspring. Additional standard multiplex PCR with mini-sequencing analysis confirmed haplotyping results of Karyomapping. However, aSNP provides the benefit of extra information of chromosome balance. This study demonstrated that kryomapping can omit the risk of transfer chromosomally unbalanced embryos, termination of abnormal chromosome pregnancy later and the birth of abnormal chromosome babies. Therefore, Karyomapping provides an accurate, quick, time saving for protocol development, universal PGT-M method for every monogenic disease, and also the advantage of CNV information which is common in pre-implantation embryos. Keywords: beta-thalassaemia-hemoglobin

E disease; embryo selection; haplotyping; karyomapping; pre-implantaton genetic testing for monogentic disease (PGT-M)

doi: 10.1016/j.rbmo.2019.04.118

66. THE DETECTION OF MITOCHONDRIAL DNA IN THE BASTOCOELIC FLUID OF EXPANDED BLASTOCYSTS

F. Carano1, M.C. Magli1, S. De Fanti2, G. Terzuoli1, S. Azzena1, C. Albanese1, L. Gianaroli1 1  S.I.S.Me.R.

Reproductive Medicine Unit, Bologna, Italy of Biological, Geological and Environmental Sciences. University of Bologna, Bologna, Italy 2  Department

Introduction: The pivotal role of mitochondria in ATP production, as well as the high mutation rate of their genome (mtDNA) are well known. Although variants fixation within the population depends to the phenotype functionality, some recurrent variants with deleterious effect survive to natural selection. We already described a relation between mitochondrial haplogroups and aneuploidy susceptibility. Since the segregation process is strictly dependent on adequate ATP provision, the mutation load

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analysis of mtDNA in blastocysts may be informative of embryo viability. The aim of this study was to verify whether mtDNA analysis could be conducted on the fluid extracted from the blastocoelic cavity through a procedure that is moderately invasive compared with the conventional forms of biopsy. Material and Methods: In the first part of the study, the blastocoelic fluid (BF) retrieved from 10 expanded blastocysts previously inferred as aneuploid by a-CGH was amplified for the mitochondrial D-loop region and sequenced by Sanger. Maternal age of the 10 patients ranged between 37 and 41 years. In the second part of the study, D-loop sequencing was carried out in five additional sets of sequential biopsies including both polar bodies (PB I; PB II), BF, trophectoderm (TE) and the corresponding whole embryo (EM), accounting for a total of 25 samples. Also in this case, the 5 sets had been diagnosed as aneuploid by chromosome analysis on PBs. All sequences were aligned to mtDNA reference sequence prior to haplogroup inference. Results: In the first part of the study, the complete D-loop amplification and sequencing was successful for all the BF samples. Haplogroups were inferred with 60% of lineages belonging to Macro-haplogroup H, while the remaining 40% was represented by the lineages I, W and K. These haplogroups are characteristic of the European population.In the second part of the study, D-loop amplification and sequencing was possible in all samples except for the PBs from one sample set. The intra-group sequence comparison highlighted full correspondence in 4 sets, while the remaining set showed a heteroplasmic position in one PB I. Conclusions: The feasibility to amplify and sequence mtDNA