Correction of disease-causing mutations in human embryos holds the potential to reduce the burden of inherited genetic disorders and improve fertility treatments for couples with disease-causing ...mutations in lieu of embryo selection. Here, we evaluate repair outcomes of a Cas9-induced double-strand break (DSB) introduced on the paternal chromosome at the EYS locus, which carries a frameshift mutation causing blindness. We show that the most common repair outcome is microhomology-mediated end joining, which occurs during the first cell cycle in the zygote, leading to embryos with non-mosaic restoration of the reading frame. Notably, about half of the breaks remain unrepaired, resulting in an undetectable paternal allele and, after mitosis, loss of one or both chromosomal arms. Correspondingly, Cas9 off-target cleavage results in chromosomal losses and hemizygous indels because of cleavage of both alleles. These results demonstrate the ability to manipulate chromosome content and reveal significant challenges for mutation correction in human embryos.
Display omitted
•Cas9-mediated DSB induction and repair by end joining occurs within hours•End joining provides an efficient way to restore reading frames without mosaicism•Unrepaired DSBs persist through mitosis and result in frequent chromosome loss•Off-target effects of Cas9 cause indels as well as chromosome loss
CRISPR-Cas9 gene editing in early human embryos leads to frequent loss of the targeted chromosome, indicating that human germline gene editing would pose a substantial risk for aneuploidy and other adverse genetic consequences
For over 2 decades preimplantation genetic testing (PGT) has been in clinical use to reduce the risk of miscarriage and genetic disease in patients with advanced maternal age and risk of transmitting ...disease. Recently developed methods of genome-wide genotyping and machine learning algorithms now offer the ability to genotype embryos for polygenic disease risk with accuracy equivalent to adults. In addition, contemporary studies on adults indicate the ability to predict polygenic disorders with risk equivalent to monogenic disorders. Existing biobanks provide opportunities to model the clinical utility of polygenic disease risk reduction among sibling adults. Here, we provide a mathematical model for the use of embryo screening to reduce the risk of type 1 diabetes. Results indicate a 45-72% reduced risk with blinded genetic selection of one sibling. The first clinical case of polygenic risk scoring in human preimplantation embryos from patients with a family history of complex disease is reported. In addition to these data, several common and accepted practices place PGT for polygenic disease risk in the applicable context of contemporary reproductive medicine. In addition, prediction of risk for PCOS, endometriosis, and aneuploidy are of particular interest and relevance to patients with infertility and represent an important focus of future research on polygenic risk scoring in embryos.
Preimplantation genetic testing commonly employs simplistic copy-number analyses to screen for aneuploidy in blastocyst trophectoderm biopsies. Interpreting intermediate copy number alone as evidence ...of mosaicism has led to suboptimal estimation of its prevalence. Because mosaicism originates from mitotic nondisjunction, utilizing SNP microarray technology to identify the cell-division origins of aneuploidy might provide a more accurate estimation of its prevalence. The present study develops and validates a method of determining the cell-division origin of aneuploidy in the human blastocyst by using both genotyping and copy-number data in parallel. The concordance of predicted origins with expected results was demonstrated in a series of truth models (99%–100%). This included determination of X chromosome origins from a subset of normal male embryos, determination of the origins of translocation chromosome-related imbalances via embryos from couples with structural rearrangements, and prediction of either mitotic or meiotic origins via multiple rebiopsies of embryos with aneuploidy. In a cohort of blastocysts with parental DNA (n = 2,277), 71% were euploid, 27% were meiotic aneuploid, and 2% were mitotic aneuploid, indicating a low frequency of bona fide mosaicism in the human blastocyst (mean maternal age: 34.4). Chromosome-specific trisomies in the blastocyst were also consistent with observations previously established in products of conception. The ability to accurately identify mitotic-origin aneuploidy in the blastocyst could benefit and better inform individuals whose IVF cycle results in all aneuploid embryos. Clinical trials with this methodology might also help provide a definitive answer regarding the reproductive potential of bona fide mosaic embryos.
Validation of a genetic-testing strategy involving SNP-array-based preimplantation distinguished between meiotic and mitotic aneuploidy in the human blastocyst. Results demonstrated that mitotic origin aneuploidy accurately predicts mosaicism in the remaining embryo and that bona fide mosaicism is more rare than previous methods have estimated.
Infertility affects approximately 10-15% of couples of reproductive age. Assisted reproductive technologies (ART), particularly in vitro fertilization (IVF), has been able to significantly improve ...live birth rates in couples struggling with infertility. For some couples, the use of preimplantation genetic testing for aneuploidy (PGT-A) may be warranted to transfer a single chromosomally normal (euploid) embryo, increasing the chances of a successful pregnancy outcome. However, even with the use of PGT-A, pregnancy loss and adverse pregnancy outcomes may persist, with some couples experiencing recurrent pregnancy loss (RPL). This thesis focuses on two markers that may help evaluate embryonic competence to provide the greatest chance of a successful IVF outcome. The first is a new genetic marker that predisposes couples to the risk of early and late miscarriages known as the M2 haplotype, and the second allows prediction of the origins of aneuploidy. The M2 haplotype consists of four point mutations in the core promoter of the Annexin A5 gene which decreases Annexin A5 protein (ANXA5) levels. Decreased ANXA5 increases the risk for thrombophilia in the placenta leading to placental mediated pregnancy complications. Forty-four percent of couples attempting in vitro fertilization (IVF) are carriers of the M2 haplotype compared to 15% in the general population. Importantly, evidence suggests the M2 haplotype is embryonal, and that the risks of pregnancy complications originate with the carrier status of the embryo. A novel qPCR based genotyping method was developed and validated to screen IVF patients as well as preimplantation genetic embryos for the M2 haplotype with an accuracy of 100% and 97-100%, respectively. In addition, using large data banks, population wide phenotypic associations were made to characterize the M2 haplotype. Imputation methods were validated to infer the M2 genotypes of the UK Biobank (UKBB) data set with an accuracy of 97%. Validated imputation methods were applied to the UKBB and severe pre-eclampsia was reported in M2 carriers at a frequency of 31% compared to 22% in non-carriers. The utility of PGT in carrier patients will be assessed as a means for embryo selection as opposed to conventional low molecular weight heparin (LMWH) treatment. A clinical trial recruited 166 couples, 23 of those couples elected for PGT for M2 to select non carrier embryos, and 11 IVF outcomes have been reported. In addition to screening for the M2 Haplotype, improvements to PGT-A may help increase the success of IVF. The use of PGT-A has become increasingly common over the last few years. However, most methods of PGT-A are limited to copy number analyses that can lead to false positive diagnosis of mosaicism. Mosaicism, defined as the presence of mitotic errors in the embryo, has been difficult to classify and report with current PGT-A methods and is currently being overreported by many PGT laboratories. The second focus of this thesis is to improve PGT-A methods using both copy number and SNP genotyping data to predict the parental and cell division origins of aneuploidy. This may lead to determining the true prevalence of mosaic embryos and provide more accurate diagnoses of preimplantation embryos. After validating a method to predict the origins of trisomies with 99%-100% accuracy on truth models it was applied to a cohort of embryos where parental DNA was available and meiotic aneuploidy was responsible for 27% of aneuploidy and mitotic aneuploidy only 2%. With the development and validation of both methodologies, embryonic competence may be evaluated providing patients with a higher chance of IVF success.
Preimplantation genetic testing (PGT) has been successfully applied to reduce the risk of miscarriage, improve IVF success rates, and prevent inheritance of monogenic disease and unbalanced ...translocations. The present study provides the first method capable of simultaneous testing of aneuploidy (PGT-A), structural rearrangements (PGT-SR), and monogenic (PGT-M) disorders using a single platform. Using positive controls to establish performance characteristics, accuracies of 97 to >99% for each type of testing were observed. In addition, this study expands PGT to include predicting the risk of polygenic disorders (PGT-P) for the first time. Performance was established for two common diseases, hypothyroidism and type 1 diabetes, based upon availability of positive control samples from commercially available repositories. Data from the UK Biobank, eMERGE, and T1DBASE were used to establish and validate SNP-based predictors of each disease (7,311 SNPs for hypothyroidism and 82 for type 1 diabetes). Area under the curve of disease status prediction from genotypes alone were 0.71 for hypothyroidism and 0.68 for type 1 diabetes. The availability of expanded PGT to evaluate the risk of polygenic disorders in the preimplantation embryo has the potential to lower the prevalence of common genetic disease in humans.
To develop a test for evaluating the annexin A5 M2 haplotype in in vitro fertilization patients and preimplantation embryos.
Test performance was measured by comparing Sanger sequencing of parental ...blood DNA and quantitative real-time polymerase chain reaction (qPCR) of saliva DNA, 3 fibroblast cell line 7-cell aliquots and their corresponding purified DNA, 123 trophectoderm biopsy samples, and DNA isolated from 1 embryonic stem cell line along with the Mendelian inheritance expectations, embryo Sanger sequencing, and single-nucleotide polymorphism (SNP) microarray-based linkage analysis.
Preimplantation genetic testing laboratory research on IVF patient and embryo DNA.
An assay was developed for the detection of the M2 haplotype on saliva samples of 6 in vitro fertilization patients. In addition, 13 patients who underwent preimplantation genetic testing with data on parental and embryo biopsy DNA available for research use were evaluated.
None.
The concordance rates between Sanger sequencing, SNP array-based linkage analysis, and Mendelian inheritance expectations with qPCR.
The concordance rate between Sanger sequencing and qPCR was 100% on parental blood DNA and saliva DNA. The sample concordance rate between all replicates of 7-cell aliquots was 100%. The sample concordance rate between 3 cell lines used to prepare 7-cell aliquots and purified genomic DNA was 100%. The concordance rate between qPCR and Sanger sequencing results from a single trophectoderm biopsy and isolated embryonic stem cell line was 100%. The concordance rate of trophectoderm biopsy qPCR results and expectations from Mendelian inheritance rules was 97%; however, when SNP array-based linkage analysis was included, the concordance rate reached 100%.
This study resulted in the development of a convenient saliva collection method and qPCR-based genotyping method to screen for the M2 haplotype. In addition, a novel method for testing preimplantation embryos has been established, providing an alternative to the use of low molecular weight heparin, through selection of embryos without the M2 haplotype.
PDF
