- Email: [email protected]
PGT as Quality Control for ART methods
e2
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
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