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Objective To investigate a possible correlation between chromosomal aberrations and paternal age, analyzing embryos derived from young oocyte donors, with available preimplantation genetic testing for aneuploidy results from day 5/6 trophectoderm biopsy obtained by next-generation sequencing for all 24 chromosomes. Design Retrospective cohort study. Setting Canadian fertility centre. Patient(s) A total of 3,118 embryos from 407 male patients, allocated into three paternal age groups: group A, ≤39 years (n = 203); group B, 40–49 years (n = 161); group C, ≥50 years (n = 43). Intervention(s) None. Main Outcome Measure(s) The primary outcomes were aneuploidy, euploidy, mosaicism, and blastocyst formation rates. Secondary endpoints were comparison of specific chromosome aneuploidy, segmental and complex (involving two chromosomes + mosaicism >50%) aneuploidy, and analysis of overall percentage of chromosomal gains and losses within each group. Result(s) The study included 437 in vitro fertilization (IVF) antagonist cycles using 302 oocyte donors in which preimplantation genetic testing for aneuploidy was performed. Overall, 70.04% of embryos were euploid, 13.9% were aneuploid, and 16.06% were mosaic. No significant differences among paternal age groups A, B, and C were found in euploidy rates (69.2%, 70.6%, 71.4%, respectively), aneuploidy rates (14.7%, 12.8%, 13.9%, respectively) or mosaicism rates (16.1%, 16.6%, 13.6%; respectively). The fertilization rate was lower in group C compared with group B (76.35% vs. 80.09%). No difference was found in blastocyst formation rate between the study groups (median 52% [interquartile range, 41%, 67%] vs. 53% [42%, 65%] vs. 52% [42%, 64%], respectively). A generalized linear mixed model regression analysis for embryo ploidy rates found older oocyte donor age to be independently associated with embryo aneuploidy (odds ratio=1.041; 95% CI, 1.009–1.074). The rate of segmental aneuploidies was significantly higher in the older versus younger paternal age group (36.6% vs. 19.4%). Conclusion(s) No association was found between paternal age and aneuploidy rates in embryos derived from IVF cycles using young oocyte donors, after adjusting for donor, sperm, and IVF cycle characteristics. Advanced paternal age ≥ 50, compared with younger paternal ages, was associated with a lower fertilization rate and increased rate of segmental aberrations.

Balanced Translocations in Embryos Balanced chromosomal rearrangements are associated with infertility and recurrent miscarriage but are difficult to detect. In this study, long-read DNA sequencing...

Preimplantation genetic testing for aneuploidies (PGT-A) using trophectoderm (TE) biopsy samples is labour intensive, invasive, and subject to sampling bias. In this study, we report on the efficacy and factors affecting accuracy of a technique we pioneered for minimally invasive preimplantation genetic testing for aneuploidy (miPGT-A). Our technique uses cell-free embryonic DNA (cfeDNA) in spent embryo culture medium (SEM) combined with blastocoel fluid (BF) to increase the amount of assayable cfeDNA. We compared miPGT-A results (n = 145 embryos) with standard PGT-A analysis of the corresponding trophectoderm biopsy. We found that accuracy of miPGT was not related to blastocyst morphological grade. The overall concordance rate per sample for euploidy/aneuploidy status between miPGT-A and TE biopsy samples was 88/90 (97.8%), and was not different between good 47/48 (97.9%) and moderate/low quality blastocysts 41/42 (97.9%) (p > 0.05). Importantly, we also discovered that for cfeDNA analysis, the SurePlex whole genome amplification (WGA) kit can be utilized without an additional cell lysis/extraction DNA step; this efficiency likely reduces the risk of maternal contamination. Regarding origin of embryonic cfeDNA, the average amount of miPGT-A WGA-DNA we obtained from blastocysts with different morphological grades, as well as the size miPGT-A WGA-DNA fragments, suggest that it is unlikely that apoptosis and necrosis are only mechanisms of DNA release from the inner cell mass (ICM) and TE into BF and SEM.

Improved embryo prioritization is crucial in optimizing the results in assisted reproduction, especially in light of increasing utilization of elective single embryo transfers. Embryo prioritization is currently based on morphological criteria and in some cases incorporates preimplantation genetic testing for aneuploidy (PGT-A). Recent technological advances have enabled parallel genomic and transcriptomic assessment of a single cell. Adding transcriptomic analysis to PGT-A holds promise for better understanding early embryonic development and implantation, and for enhancing available embryo prioritization tools. Our aim was to develop a platform for parallel genomic and transcriptomic sequencing of a single trophectoderm (TE) biopsy, that could later be correlated with clinical outcomes. Twenty-five embryos donated for research were utilized; eight for initial development and optimization of our method, and seventeen to demonstrate clinical safety and reproducibility of this method. Our method achieved 100% concordance for ploidy status with that achieved by the classic PGT-A. All sequencing data exceeded quality control metrics. Transcriptomic sequencing data was sufficient for performing differential expression (DE) analysis. All biopsies expressed specific TE markers, further validating the accuracy of our method. Using PCA, samples clustered in euploid and aneuploid aggregates, highlighting the importance of controlling for ploidy in every transcriptomic assessment.

Improved embryo selection is crucial in optimizing the results from assisted reproduction. Preimplantation genetic screening reduces time to pregnancy and miscarriages. Correlating the transcriptome of an embryo, with fertility treatments and outcomes, holds promise in improving the overall results. We developed a novel method for embryo selection in fertility treatments that integrates embryonic genomic and transcriptomic data and evaluated it in this pilot study.A total of 21 embryos donated for research were included. Three were used for the initial development and optimization of sample processing and sequencing. Thereafter, 18 embryos were used to demonstrate the clinical safety and reproducibility of our method. Two trophectoderm biopsies were taken from each embryo: one was processed as a clinical sample for genomic profiling (control, n=18), while the other biopsy (n=18) was split and utilized for independent, simultaneous genomic and transcriptomic analysis, here termed Preimplantation Genetic and Transcriptomic Testing (PGT2).High quality genomic and transcriptomic data were obtained from all analyzed samples. The concordance between genomic data obtained with PGT2 and control samples was 100% with clinical grade quality metrics. Euploid embryos showed downregulation of genes involved in anaerobic metabolism, oxidative phosphorylation, and fatty-acid oxidation. This is the first study to provide full genomic and transcriptomic profiles from a single TE biopsy from human embryos in a clinical setting unleashing the potential of improving embryo selection and outcomes in infertility treatments. Clinical trials are needed to correlate transcriptomic data with outcomes.SUMMARYDespite advances in assisted reproductive technologies, the success rate has remained relatively constant. Under the age of 35, there is a 40% chance of delivering a child per embryo transfer, which decreases with increasing maternal age. Prioritizing embryos for transfer is based on morphological assessment and, in some cases, incorporates genetic testing as well. Selection of euploid embryos for transfer shortens the time to pregnancy and reduces the risk for miscarriages. Adding the mRNA analysis to the genomic assessment of an embryo has the potential of improving the outcomes of fertility treatments.

High DNA fragmentation index (DFI) may be associated with poor outcome after IVF. Our aim was to determine whether DFI impacts blastocyst quality or clinical outcome. This retrospective study included 134 couples who underwent 177 IVF-ICSI and pre-implantation genetic screening (PGS) cycles during January 1st, 2014-March 31st, 2016 and had documented previous DFI. Group 1 (DFI>30%) encompassed 25 couples who underwent 36 cycles; Group 2 (DFI 15-30%) included 45 couples and 57 cycles; group 3 (DFI

Pax6 is a highly conserved transcription factor that controls the morphogenesis of various organs. Changes in Pax6 dosage have been shown to affect the formation of multiple tissues. PAX6 haploinsufficiency leads to aniridia, a pan-ocular disease primarily characterized by iris hypoplasia. Herein, we employ a modular system that includes null and overexpressed conditional alleles of Pax6. The use of the Tyrp2-Cre line, active in iris and ciliary body (CB) primordium, enabled us to investigate the effect of varying dosages of Pax6 on the development of these ocular sub-organs. Our findings show that a lack of Pax6 in these regions leads to dysgenesis of the iris and CB, while heterozygosity impedes growth of the iris and maturation of the iris sphincter. Overexpression of the canonical, but not the alternative splice variant of Pax6 results in severe structural aberrations of the CB and hyperplasia of the iris sphincter. A splice variant-specific rescue experiment revealed that both splice variants are able to correct iris hypoplasia, while only the canonical form rescues the sphincter. Overall, these findings demonstrate the dosage-sensitive roles of Pax6 in the formation of both the CB and the iris.