Towards comprehensive PGT

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RBMO VOLUME 38 ISSUE S1 2019

Results: In preliminary studies of naturally conceived pregnancies undergoing testing for autosomal recessive conditions, fetal genotypes predicted by cSMART assay were concordant with invasive results in 4 of 4 cases (100%) of Wilson disease, 18 of 18 cases (100%) of PKU, 73 of 80 cases (91%) of autosomal recessive hearing loss and 98 of 100 cases (98%) of beta thalassemia. There was a strong association of low fetal DNA fraction and poor quality plasma samples with discordant NIPT results. In a further study, we performed NIPT follow up for a complicated PGT-M case for Fanconi Anemia with HLA matching. The case involved a couple who were both carriers of an identical FANCG deletion mutation. Following embryo testing by mutation analysis and linked STR analysis, 2 HLA matched and disease free (normal and carrier) embryos were transferred, resulting in a twin pregnancy. At 15 weeks gestation, cSMART analysis of the pregnancy plasma determined fetal DNA fractions of 14.2% and 6.6% for twin 1 and 2, respectively. The maternal plasma FANCG mutation ratio was measured at 46.2% (50% minus half the fetal fraction of twin 2), which was consistent with the presence of a carrier fetus (twin 1) and a normal fetus (twin 2). Additional retrospective studies of the WGA products from the transferred embryos using single molecule sequencing also confirmed the FANCG genotypes of the transferred embryos and a HLA match to the sick sibling. Conclusions: In proof of concept studies for autosomal recessive conditions, we demonstrate that NIPT can be successfully used to reliably and accurately determine fetal genotypes. NIPT has clinical utility as a safe alternative to invasive testing for confirmation of PGT-M cases that result in an ongoing pregnancy. doi: 10.1016/j.rbmo.2019.03.003

PGT AS QUALITY CONTROL FOR ART METHODS

Santiago Munne, PhD CooperGenomics, Livingston, NJ, USA; Overture Life, New York, NY, USA; Dept. Ob. Gyn. And Reprod Med. at Yale University, New Heaven, CT, USA

Between 20% to 80% of ART generated embryos are chromosomally abnormal, increasing with advancing maternal age, embryo dysmorphism but also caused by iatrogenic factors. Aneuploidy has been shown to increase with advancing maternal age but not mosaicism. Mosaicism was extensively investigated using FISH on entire embryos showing that it occurs through post-zygotic malsegregation and increases with cleavage-stage dysmorphism, but not with advancing maternal age. With newer PGT molecular techniques, biopsied cells from a TE biopsy are analyzed as a group, and not individually. Mosaicism was barely detectable from TE biopsies with some molecular techniques such as aCGH, qPCR, or SNP arrays but with the advent of Next Generation Sequencing (hr-NGS), which has a higher dynamic range and offer much higher resolution, we can now detect mosaicism in TE biopsies at a 20- 80% abnormal cell range or is 1/5 to 4/5 abnormal cells. We recently showed that chromosome abnormalities in egg donors, a group of patients quite homogeneous, are ART-induced ranging from 20% in some centers to 60% in others. Several reasons for these changes were investigated, and we found differences between doctors of the same center, pointing to hormonal stimulation and/or other treatment related factors. One being investigated is differences between follicular size of eggs retrieved. On the other hand, changes in Ph, Temperature, volatile compounds, and culture media have been reported to affect chromosome abnormalities. As such, PGT can be used as a faster QC

method to determine best practices and methods instead of waiting for pregnancy outcome, while selecting good prognosis embryos even within a sub-optimal ART set up. doi: 10.1016/j.rbmo.2019.03.004

TOWARDS COMPREHENSIVE PGT

J.R. Vermeesch Centre for Human Genetics, KULeuven, Leuven, Belgium

Large scale whole genome and exome sequencing is uncovering a plethora of novel mutations that cause highly penetrant, early-onset, severe, or later-onset life-threatening dominant and recessive disorders. For couples who are known carriers of mutant alleles, preimplantation genetic diagnosis enables the detection of genetic disorders in embryos that have been fertilized in vitro, thereby avoiding their transmission to offspring. Traditional PGD methods require a mutation and family specific work-up. We and others have developed generic methods that can be readily applied for all transmitted genetic disorders. They are termed karyomapping and haplarithmisis. The methods reconstructs genomewide haplotype architectures as well as the copy-number and segregational origin of those haplotypes byemploying phased parental genotypes and deciphering WGA-distorted SNP B-allele fractions. I will present the principles, the use and the results of its implementation since clinical implementation. The introduction of genome wide screening of embryo's raised novel ethical questions. The principles guiding embryo selection and prioritization that are applied at our center according to the chromosomal content and mutational load of the embryos, will be presented. I will present novel technical developments broadening the scope of use. In addition, I will present new insights in the origins

RBMO VOLUME 38 ISSUE S1 2019

and mechanisms leading to early meiotic and mitotic errors as well as on the origin of mixoploid and chimaeric embryos. doi: 10.1016/j.rbmo.2019.03.005

PREIMPLANTATION GENETIC TESTING (PGT) FOR NONTRADITIONAL INDICATIONS

Svetlana Rechitsky, PhD, Anver Kuliev, MD, PhD Reproductive Genetic Innovations, LLC, 2910 MacArthur Boulevard, Northbrook, IL 60062, USA

Since the introduction of PGT, the indications for PGD are gradually expanding. Initially performed mainly for childhood-onset genetic conditions with clearly defined penetrance, severity and disease phenotypes, PGT is now applied to common late onset conditions determined by genetic predisposition, which may be realized with age or not realized in a lifetime. We present here the experience of PGT for these borderline indications in our series of over 17,000 PGT cases, which includes 5,780 PGT-M Of the521 conditions tested, the borderline indications included 71 cycles for cardiac conditions, 461 cycles for HLA matching and 702 cycles for cancer predisposition. Combined, these 1,163 cycles represent 20.1% of the overall cases. The most common cardiac genetic condition was Dilated Cardiomyopathy (DCM), 5causatives genes (over 30 different mutations), as well as Hypertrophic Cardiomyopathy (HCM) with 6 causative genes (over 16 different mutations). These cycles resulted in birth of 32 cardiac disease predisposition free children. The largest group for non-traditional indications was PGT-M for 23 cancer predisposition syndromes (642 cases overall), including BRCA1 and BRCA2, accounting for more than a third of the cases (32.7%;

210/642). These cycles resulted in birth of 271children free of cancer predisposition genes, which is the world's largest series. The number of cases for cancer predisposition syndromes has increased steadily since 1999, after we performed the world's fist PGT. The other group of PGT for non-traditional indications is PGT for HLA matching, which was requested either in combination with PGT-M (73.5% (339/461), or exclusively for HLA typing (26.5% (122/461), with or without PGT-A. Referrals for PGT are becoming increasingly complex as the improving testing technologies identify a wide genetic heterogeneity, variable severity and penetrance of adult onset conditions, for which the application of PGT may present importance ethical problems. However, our experience shows the increasing referral for PGT of such conditions, which was extremely accurate and efficient, allowing couples at risk to produce the offspring free of genes predisposing to common conditions of later life. doi: 10.1016/j.rbmo.2019.03.006

PGT FOR STRUCTURAL REARRANGEMENTS (PGT-SR)

Tomas Escudero Background: Patients with structural chromosome rearrangements encounter a variety of reproductive obstacles as a result of the high frequency of unbalanced gametes in these individuals, including low pregnancy rates, increase in spontaneous abortion (SAB) rate and an increased risk for unbalanced, genetically abnormal offspring1. The most common structural chromosome abnormality is the reciprocal translocation, seen in about 0.16% of the general population. A reciprocal translocation is the exchange of genetic material between two chromosomes; it is estimated

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that approximately 70% of embryos generated by a parent with a reciprocal translocation are abnormal. The next two most common structural chromosome abnormalities are Robertsonian translocations (0.1% of the general population) and inversions (0.02% of the general population). Other cases of structural chromosome abnormalities are less common, more complex, and supposedly precipitate the chances to have a successful outcome. Due to the scarcity of these cases these structural abnormalities have been poorly studied. For this study we include the chromosomal structural abnormalities classified as insertions and complex chromosome rearrangements (CCRs). Insertions are the relocation of a region of a chromosome to another location of the genome (whether the same chromosome or other chromosome), and CCRs are structural chromosome abnormalities involving three or more chromosomes. This study aims to determine the proportion of unbalanced embryos and reproductive risk to patients carrying insertions or CCR. Materials and methods: This study collects data from insertion and CCR cases analyzed by PGD techniques available at the time the case was processed. These techniques are fluorescence in situ hybridization (FISH), array comparative genome hybridization (aCGH), and next generation sequencing (NGS). CCR cases were classified further depending of the type of abnormalities of which the CCR is compounded (Table 1). 32 patients were included in this studies, from which 58 cycles were performed, and 517 embryos were analyzed