Aberrant landscapes of maternal meiotic crossovers contribute to aneuploidies in human embryos.

Meiotic recombination is crucial for human genetic diversity and chromosome segregation accuracy. Understanding its variation across individuals and the processes by which it goes awry are long-standing goals in human genetics. Current approaches for inferring recombination landscapes rely either on population genetic patterns of linkage disequilibrium (LD)-capturing a time-averaged view-or on direct detection of crossovers in gametes or multigeneration pedigrees, which limits data set scale and availability. Here, we introduce an approach for inferring sex-specific recombination landscapes using data from preimplantation genetic testing for aneuploidy (PGT-A). This method relies on low-coverage (<0.05×) whole-genome sequencing of in vitro fertilized (IVF) embryo biopsies. To overcome the data sparsity, our method exploits its inherent relatedness structure, knowledge of haplotypes from external population reference panels, and the frequent occurrence of monosomies in embryos, whereby the remaining chromosome is phased by default. Extensive simulations show our method's high accuracy, even at coverages as low as 0.02×. Applying this method to PGT-A data from 18,967 embryos, we mapped 70,660 recombination events with ∼150 kbp resolution, replicating established sex-specific recombination patterns. We observed a reduced total length of the female genetic map in trisomies compared with disomies, as well as chromosome-specific alterations in crossover distributions. Based on haplotype configurations in pericentromeric regions, our data indicate chromosome-specific propensities for different mechanisms of meiotic error. Our results provide a comprehensive view of the role of aberrant meiotic recombination in the origins of human aneuploidies and offer a versatile tool for mapping crossovers in low-coverage sequencing data from multiple siblings.

Aberrant landscapes of maternal meiotic crossovers contribute to aneuploidies in human embryos.

Meiotic recombination is crucial for human genetic diversity and chromosome segregation accuracy. Understanding its variation across individuals and the processes by which it goes awry are long-standing goals in human genetics. Current approaches for inferring recombination landscapes either rely on population genetic patterns of linkage disequilibrium (LD)-capturing a time-averaged view-or direct detection of crossovers in gametes or multi-generation pedigrees, which limits dataset scale and availability. Here, we introduce an approach for inferring sex-specific recombination landscapes using data from preimplantation genetic testing for aneuploidy (PGT-A). This method relies on low-coverage (<0.05×) whole-genome sequencing of in vitro fertilized (IVF) embryo biopsies. To overcome the data sparsity, our method exploits its inherent relatedness structure, knowledge of haplotypes from external population reference panels, as well as the frequent occurrence of monosomies in embryos, whereby the remaining chromosome is phased by default. Extensive simulations demonstrate our method's high accuracy, even at coverages as low as 0.02×. Applying this method to PGT-A data from 18,967 embryos, we mapped 70,660 recombination events with ~150 kbp resolution, replicating established sex-specific recombination patterns. We observed a reduced total length of the female genetic map in trisomies compared to disomies, as well as chromosome-specific alterations in crossover distributions. Based on haplotype configurations in pericentromeric regions, our data indicate chromosome-specific propensities for different mechanisms of meiotic error. Our results provide a comprehensive view of the role of aberrant meiotic recombination in the origins of human aneuploidies and offer a versatile tool for mapping crossovers in low-coverage sequencing data from multiple siblings.

Personalization of IVF-ICSI workflow based on patient characteristics improves IVF laboratory outcomes and embryo ploidy by PGT-A.

BACKGROUND: Intracytoplasmic sperm injection (ICSI) has become a common method of fertilization in assisted reproduction worldwide. However, there are still gaps in knowledge of the ideal IVF-ICSI workflow including the optimal duration of time between induction of final oocyte maturation, oocyte denudation and ICSI. The aim of this study was to examine outcomes following different workflow protocols in IVF-ICSI procedures in blastocysts that have undergone undisturbed incubation and preimplantation genetic testing for aneuploidy (PGT-A) prior to transfer. METHODS: Retrospective secondary analysis of 113 patients (179 IVF cycles, 713 embryos), all of whom have gone through IVF-ICSI and PGT-A using undisturbed culture. Predictive test variables were the length of time from: trigger to OPU, OPU to denudation, and denudation to ICSI. Outcome metrics assessed were: maturation, fertilization, blastulation and euploid rates. Generalized Estimated Equations Linear Model was used to examine the relationship between key elements of a given cycle and continuous outcomes and LOESS curves were used to determine the effect over time. RESULTS: In a paired multi-regression analysis, where each patient served as its own control, delaying OPU in patients with unexplained infertility improved both maturation and blastulation rates (b = 29.7, p < 0.0001 and b = 9.1, p = 0.06, respectively). Longer incubation with cumulus cells (CCs) significantly correlated with improved ploidy rates among patients under 37, as well as among patients with unexplained infertility (r = 0.22 and 0.29, respectively), which was also evident in a multiple regression analysis (b = 6.73, p < 0.05), and in a paired analysis (b = 6.0, p < 0.05). Conversely, among patients with a leading infertility diagnosis of male factor, longer incubation of the denuded oocyte prior to ICSI resulted in a significantly higher euploid rate (b = 15.658, p < 0.0001). CONCLUSIONS: In this study we have demonstrated that different IVF-ICSI workflows affect patients differently, depending on their primary infertility diagnosis. Thus, ideally, the IVF-ICSI workflow should be tailored to the individual patient based on the primary infertility diagnosis. This study contributes to our understanding surrounding the impact of IVF laboratory procedures and highlights the importance of not only tracking "classic" IVF outcomes (maturation, fertilization, blastulation rates), but highlights the importance that these procedures have on the ploidy of the embryo.

Is there a correlation between paternal age and aneuploidy rate? An analysis of 3,118 embryos derived from young egg donors.

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.

Minimally Invasive Cell-Free Human Embryo Aneuploidy Testing (miPGT-A) Utilizing Combined Spent Embryo Culture Medium and Blastocoel Fluid -Towards Development of a Clinical Assay.

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.

Evaluation of a novel non-invasive preimplantation genetic screening approach.

OBJECTIVE: To assess whether embryonic DNA isolated from blastocyst culture conditioned medium (BCCM) combined with blastocoel fluid (BF) could be used for blastocyst stage non-invasive preimplantation genetic testing for chromosomal aneuploidy (non-invasive preimplantation genetic screening, NIPGS). PATIENTS: 47 embryos from 35 patients undergoing IVF. INTERVENTIONS: DNA analysis of combined BCCM plus BF in comparison with trophectoderm (TE) biopsy and/or whole blastocyst (WB)using next generation sequencing (NGS). RESULTS: Embryonic DNA was successfully amplified in 47/47 NIPGS samples (28 frozen-thawed and 19 fresh culture samples) ranging from 6.3 to 44.0 ng/µl. For frozen-thawed embryos, the concordance rate for whole chromosome copy number per sample was equivalent between NIPGS vs. TE biopsy, NIPGS vs. WB and TE vs. WB samples taken from the same embryo was 87.5%; 96.4% and 91.7% respectively (P>0.05), and the rate of concordance per single chromosome was 99.3%, 99.7% and 99.7%, respectively (P>0.05). In fresh cases (Day 4 to Day 5/6 culture), the concordance rate for whole chromosome copy number per sample between NIPGS vs. TE samples taken from the same embryo was 100%, and the rate of concordance per single chromosome was 98.2% (P>0.05). CONCLUSIONS: A combination of BCCM and BF contains sufficient embryonic DNA for whole genome amplification and accurate aneuploidy screening. Our findings suggest that aneuploidy screening using BCCM combined with BF could potentially serve as a novel NIPGS approach for use in human IVF.

Expression and regulation of miR-17a and miR-430b in zebrafish ovarian follicles.

MicroRNAs (miRNAs) are small noncoding RNAs that post-transcriptionally regulate gene expression and control many developmental and physiological processes. Oocyte maturation in fish is mainly regulated by luteinizing hormone (LH) and maturation-inducing hormone (MIH). In addition, growth factors, including members of the transforming growth factor ß (TGF-ß) superfamily, have also been shown to play important roles in regulating oocyte maturation. In this study, we determined the expression and regulation of two miRNAs, miR-17a and miR-430b, which potentially target signalling molecules in the TGF-ß pathway, in zebrafish ovarian follicles. Using real-time PCR, we observed that miR-17a and miR-430b levels in follicular cells were significantly lower in late vitellogenic and full grown follicles than in early vitellogenic follicles. Treatment with a LH analog, human chorionic gonadotropin, significantly down-regulated miR-17a and miR-430b expression in follicular cells but had no effect on their expression in oocytes. Forskolin also inhibited follicular cell miR-430b expression; however, no significant changes in miR-17a levels were observed after Forskolin treatment. Finally, MIH did not affect the expression of these miRNAs either in follicular cells or oocytes at the time points tested. These findings suggest that miR-17a and miR-430b may be involved in the regulation of follicle development and oocyte maturation in zebrafish.