* PGT-SR using NGS and haplotype analysis with microsatellite markers to distinguish normal from balanced Robertsonian translocation carrier embryo

Introduction Robertsonian translocations are among the most common balanced structural rearrangements seen in general population with a frequency in newborn surveys of about 1 in 1000. Rob (13q14q) accounts for around 75% of all Robertsonian translocations. Because of the trivalent formations during meiosis, phenotypically normal carriers of Robertsonian translocation may produce gametes with unbalanced products, which can cause repeated miscarriages and male infertility. Preimplantation genetic testing for structural rearrangement (PGT-SR) by next generation sequencing (NGS) offers an effective way to minimize the risk of abnormal pregnancy caused by unbalanced segregation. However, NGS alone is unable to distinguish normal embryos from balanced translocation carrier, nor detection of uniparental disomy (UPD). In this study, we try to distinguish normal embryos from balanced translocation carrier embryos by linkage analysis with microsatellite markers. Material and methods Delineate the haplotype of translocation chromosomeA 36-year-old patient with her husband's karyotype 45,XY,rob(13;14)(q10;q10), presented for PGT-SR. Haplotype of translocation chromosome was established by single sperm haplotype analysis. Twenty single sperms were isolated and transferred into PCR tubes. Whole genome amplification (WGA, Qiagen) were performed followed by haplotype analysis. Microsatellite markers near centromeres of chromosome 13 and 14 with heterozygosity value >0.7 were chosen. Firstly, reference sperms with disomic signals on chromosome 13/14 were used to delineate the haplotype of translocation chromosomes. Then haploid sperms were used to cross check and to delineate the haplotypes of normal homologs. According to the recombination events detected on sperms, a set of microsatellite markers within 7.9Mb and 4.9 Mb from centromeres on chromosomes 13 and 14 respectively were selected to be used in PGT-SR.PGT-SRPGT-SR was performed by WGA followed by NGS (VeriSeq-PGS). Haplotype analysis was performed on the same WGA DNA. Euploid embryo determined by NGS with haplotype of the translocation chromosomes indicated a balanced translocation carrier. Disomy detected by NGS but homozygous signal on haplotype analysis indicated UPD. Results Upon linkage analysis, three sperms were disomy 14, eight sperms carried normal homologs 13 and 14; nine sperms carried translocated chromosome only. In the PGT-SR cycle, 18 oocytes were retrieved, trophectoderm biopsy was performed on 8 blastocysts. Haplotype analysis was successfully performed in all the blastocysts. No aneuploidy was detected in 6 blastocysts in which 5 were balanced translocation carriers and one was normal. No UPD was detected. At the time of abstract submission, frozen-thawed embryo transfer has not been performed yet. Recombination was detected on one blastocyst on chromosome 13. Conclusions PGT-SR by NGS, together with haplotype analysis, can be used to distinguish normal embryo from balanced translocation carrier. UPD can also be detected, if any. A panel of microsatellite markers on both translocated chromosomes should be used in order to minimize the possibility of misdiagnosis due to recombination.