High-level Mosaicism Research Articles

P-516 Minimizing mosaicism: impact of trophectoderm (TE) biopsy specimen size on mosaic blastocysts in preimplantation genetic testing for aneuploidy (PGT-A)

Abstract Study question Is the size of TE biopsy specimens related to the chromosomal status of the blastocysts in PGT-A? Summary answer Smaller biopsy specimens are associated with higher proportion and altered composition of mosaic blastocysts in PGT-A. What is known already Concordance between TE biopsy and inner cell mass or whole embryos is influenced by mosaicism type, degree, biopsy location, number of cells biopsied, and cell loading. Particularly, mosaic embryos and those with structural rearrangements have a higher level of inconsistency. Recent studies have demonstrated the potential reclassification of chaotic embryos as euploid, leading to successful live births. This study aimed to investigate the potential influence of TE biopsy specimen size on the chromosomal status of blastocysts undergoing PGT-A. Study design, size, duration This retrospective study, conducted from October, 2023, to January, 2024, included 246 blastocysts from patients undergoing TE biopsy and PGT-A at the blastocyst stage on days 5, 6, or 7. Next-generation sequencing (NGS) was used to obtain PGT-A results, categorizing blastocysts into euploid, aneuploid, and mosaic based on their genetic composition. Participants/materials, setting, methods The biopsy specimen area was measured using the Image J program. Mosaic blastocysts were categorized into either a chaotic mosaic group or another group, which included blastocysts with fewer than three chromosomal aneuploidies and segmental mosaicism. Further analysis was conducted by classifying blastocysts into high-level mosaic (>30% aneuploidy) and low-level mosaic (≤30% aneuploidy) groups. Group differences were analyzed using t-tests and chi-square, with a significance threshold set at p<0.05. Main results and the role of chance In the analysis of expanded mosaic blastocysts, the chaotic mosaic group had smaller biopsy specimens than those in the comparison group (p<0.05). The high-level mosaic group also had significantly smaller biopsy specimens than the low-level mosaic group (p<0.05). When categorizing the biopsied sample size into two groups—below 1350 µM2 (n = 98) and above 1500 µM2 (n = 97)—the rates of mosaic embryos were 37% (36/98) and 30% (29/97), respectively, showing a numerical difference without statistical significance. These findings suggest a correlation between the size of biopsy specimens and the chromosomal status of mosaic blastocysts, particularly indicating that a smaller sample size is associated with an elevated chance of mosaic embryos showing chaotic or high-level mosaic characteristics. Limitations, reasons for caution The biopsy specimens were photographed, and their areas were measured using the Image J program, lacking a comprehensive representation of the three-dimensional sample morphology. This constraint could potentially affect the accuracy and in-depth understanding of the spatial characteristics of the biopsy specimens. Wider implications of the findings The study emphasizes the crucial role of optimizing biopsy specimen size in PGT-A. The identified correlation with the chromosomal status in mosaic embryos, especially the association with chaotic or high-level mosaic embryo outcomes in smaller samples, highlights the need for careful size considerations. Trial registration number not applicable

P-515 The morphokinetic signature of high-level mosaic embryos in time-lapse ART analysis

Abstract Study question Do high-level mosaic embryos exhibit differences in developmental speed compared to euploid or low-level mosaic embryos? Summary answer High-level mosaic embryos exhibit slower development than euploid and low-level mosaic embryos during the blastulation and blastocyst formation stages. What is known already Time-lapse technology (TLT) has facilitated the real-time observation of human embryo development without disrupting culture conditions, generating a wealth of morphokinetic data linked to outcomes such as implantation potential, blastocyst development, and ploidy status. However, the developmental patterns of mosaic embryos based on their aneuploidy level using TLT remain not well understood. Consequently, we aimed to assess the clinical significance of morphokinetic parameters in human embryo development, with a specific focus on embryos categorized by their mosaic status. Study design, size, duration This retrospective study examined 385 blastocysts undergoing preimplantation genetic testing for aneuploidy (PGT-A) between January and December 2022. Out of the total, 191 expansion blastocysts underwent PGT-A biopsy on day 5. The PGT-A results, which categorized embryos into euploid, aneuploid, and mosaic based on their genetic composition, were determined through next-generation sequencing (NGS) by an analysis company. Mosaic embryos were further categorized into two groups: high-level mosaicism (>30% aneuploidy cells) and low-level mosaicism (≤30%). Participants/materials, setting, methods Differences in development rates were assessed through the examination of morphological and morphokinetic parameters. These parameters included the time to second polar body extrusion (tPB2), time to pronuclei appearance and fading (tPNa, tPNf), time to reach 2–8 cells (t2, t3, t4, t5, t6, t7, t8, t9), and time to the onset of blastulation and blastocyst formation (tSB, tB). Statistical analyses, incorporating ANOVA and post-hoc tests, were employed to compare developmental timings across different groups. Main results and the role of chance When categorizing mosaic embryos based on their level, high-level mosaic embryos showed a significantly slower developmental rate than low-level mosaic embryos, evident at both the blastulation stage (tsB, high-level vs. low-level: 101.80h vs. 97.3h, p=0.003) and blastocyst stage (tB, high-level vs. low-level: 109.98h vs. 105.92h, p=0.005). In comparison to euploid embryos, low-level mosaic embryos demonstrated comparable outcomes. Conversely, high-level embryos exhibited a significant difference in both blastulation (tsB, euploidy vs. high-level: 98.85h vs. 101.80h, p=0.026) and blastocyst (tB, euploidy vs. high-level: 106.73h vs. 109.98h, p=0.011) stages. These findings suggested that the mosaic level of embryos might influence developmental kinetics, proposing this approach as an additional tool for the selection of mosaic embryos in the context of embryo transfer. Limitations, reasons for caution This study focused on morphokinetic parameters captured using time-lapse imaging, which may not fully represent the complex cellular dynamics within embryos. Furthermore, the categorization of mosaic embryos into high-level and low-level groups based on the percentage of mosaicism (>30% and ≤30%, respectively) might not capture all embryo developmental potential. Wider implications of the findings The findings suggest valuable predictive potential in assessing the development of aneuploid and mosaic embryos. Further research into the development of euploid, aneuploid, and mosaic embryos, as well as high-level and low-level mosaic embryos, could provide valuable insights for ART practices. This could significantly impact ART decision-making and patient outcomes. Trial registration number not applicable

P-744 Reporting chromosomal mosaicism reduces the overall accuracy of preimplantation genetic testing for aneuploidies: results from the extended in vitro culture of 230 human embryos

Abstract Study question What is the accuracy of reporting chromosomal mosaicism in the context of next generation sequencing (NGS)-based preimplantation genetic testing for aneuploidies (PGT-A)? Summary answer Extended in vitro culture of human embryos demonstrated that reporting mosaicism increased the risk of false positives, reducing the overall accuracy of PGT-A to 70%. What is known already PGT-A has been introduced into IVF practice to improve clinical outcomes. However, diagnostic accuracy remains controversial, particularly regarding chromosomal mosaicism. While NGS allows the detection and reporting of mosaicism from a trophectoderm (TE) biopsy, the inability to discern biological variability from technical artefacts continues to limit the interpretation of PGT-A results. As reports of mosaicism often have a major impact on patient management, achieving an appropriate level of standardization will be vital to avoid overestimation. Extended in vitro culture of human embryos up to 12 days post fertilization (D12) delivers an important, clinically relevant platform for the validation of PGT-A. Study design, size, duration A total of 230 clinically unsuitable blastocysts were included in the study. Embryos were selected based on their original diagnosis following PGT-A as: uniformly abnormal (n = 158), aneuploid and mosaic (n = 21), mosaic only (n = 36) and euploid (n = 15) but carrying a monogenic disease mutation in homozygosis. Blastocysts were originally classified as mosaic if they contained 30-70% abnormal cells, with <50% mosaicism defined as low level and >50% considered high level mosaicism. Participants/materials, setting, methods Vitrified blastocysts were warmed and cultured to D12 using established protocols to generate embryo outgrowths. A total of 90 outgrowths were selected for NGS. Outgrowths were collected whole (n = 64) or separated into 2-4 portions (n = 26), which were then analyzed individually. We correlated the original PGT-A diagnoses to developmental outcomes and the chromosomal status of the embryo outgrowths. Fisher’s Exact test (two-sided) was used for evaluating associations. All p-values <0.05 were considered significant. Main results and the role of chance Following extended culture, 49.1% (113/230) of embryos remained viable and attached, while 50.9% (117/230) degenerated and detached. As previously observed in our model, euploid blastocysts and embryos diagnosed with trisomies, duplications or chromosomal mosaicism were significantly more likely to attach throughout the extended culture, while monosomies, deletions and complex chromosomal constitutions impaired in vitro development (total attached, 78.3% vs. 24.8%, p < 0.0001). When compared to the original biopsy, we established 100% concordance when reporting euploidy and uniform whole chromosome aneuploidies, demonstrating an overall high sensitivity of PGT-A. Of the blastocysts diagnosed as mosaic only, 80.6% (29/36) remained viable throughout the extended culture. These were sequenced either whole (n = 11) or in parts (n = 18). At D12, 69.0% (20/29) of these embryos were uniformly euploid, while 27.6% (8/29) were uniformly aneuploid across all outgrowth samples. Notably, the latter were all originally diagnosed as high level mosaics. One embryo (3.4%) revealed a true mosaic configuration, with evidence of a reciprocal event across the different outgrowth samples. Ultimately, the proportion of embryos accurately diagnosed as euploid was 25.0% (8/35), with an overall false positive error rate of 77.1%. False positives were largely attributed to the diagnosis of mosaicism, 85.2% (23/27) or segmental aberrations, 14.8% (4/27). Limitations, reasons for caution This study was performed using a single PGT-A assay and cannot be extrapolated to other platforms. To evaluate the predictive value of chromosomal mosaicism, we selected embryos based on their chromosomal profiles. This may underestimate the accuracy of PGT-A, as sampling of embryos was not performed randomly. Wider implications of the findings We confirm the high sensitivity of PGT-A, as euploidy and uniform whole chromosome abnormalities were detected with high accuracy. However, predictive value is considerably reduced when diagnosing mosaicism. Given the high false positive rate, reporting mosaicism remains difficult to justify, as potentially viable embryos continue to be excluded for transfer. Trial registration number NA

P-264 Correlations between the artificial intelligence scoring system (iDAScore v1.0) and live birth outcomes in preimplantation genetic testing for aneuploidy cycles

Abstract Study question Is the iDAScore v1.0 score correlated with pregnancy outcomes in single embryo transfer (SET) cycles following preimplantation genetic testing for aneuploidy (PGT-A)? Summary answer We demonstrated that elevated iDAScore v1.0 scores are positively correlated with the probabilities of pregnancy and live birth (LB) in SETs following PGT-A. What is known already To select the blastocysts with high development potential in advance, several groups have proposed the implementation of artificial intelligence (AI)-based software is capable of predicting implantation in women undergoing in vitro fertilization (IVF). It has also been known that using high resolution next generation sequencing preimplantation genetic screening (hr-NGS) for PGT-A can enable the exclusion of aneuploid embryos before embryo transfers and thus improve pregnancy outcomes of SETs. However, the capacity of AI-based assessments has remained unclear in IVF cycles with PGT-A. Study design, size, duration This retrospective study, approved by the Institutional Review Board of Chung Sun Medical University, was performed to assess the dataset of 317 SET cycles after PGT-A from January 2017 to September 2019. A single euploid or mosaic blastocyst with an AI score calculated using the iDAScore software (v1.0) was selected for frozen embryo transfer according to the given priority of blastocyst morphology. The iDAScore groups were categorized by quartiles of AI scores. Participants/materials, setting, methods Embryos were cultured in a time-lapse incubator and qualified blastocysts underwent next generation sequencing (NGS)-based PGT-A. The confounding factors associated with LB were evaluated using logistic regression analysis in generalized estimating equations (GEEs). The differences between iDAScore groups were assessed using the Mann–Whitney U test or Fisher’s exact test, as applicable. The receiver operating characteristic (ROC) curve analysis was used to estimate the predictive powers. A P value < 0.05 was considered statistically significant. Main results and the role of chance The results revealed the patient age, anti-Müllerian hormone, body mass index, oocyte sources (autologous or donor), PGT-A results (euploidy, low-level mosaicism, or high-level mosaicism), aberrant chromosome types (none, whole chromosome, segmental chromosome, or whole with segmental chromosome), and aberrant chromosome sites (0, 1, 2, or > 2) were not correlated with LB. However, embryo biopsy days (day 5 vs. day 6, OR = 2.271, 95% CI = 1.534–14.824), blastocyst morphology scores (OR = 1.185, 95% CI = 1.034–1.359), and AI scores (OR = 1.640, 95% CI = 1.231–2.183) were significantly correlated with LB. The AI scores were then divided into quartiles (group 1: 5.0–7.9; group 2: 8.0–8.5; group 3: 8.6–8.9; and group 4: 9.0–9.5). The ongoing pregnancy (29.1% vs. 54.2%–57.5%) and live birth (27.8% vs. 52.8%–56.3%) rates of group 1 were significantly lower than other groups. The blastocyst morphology score (5.3 ± 1.2), and KIDScore D5 scores (v1 = 3.0 ± 1.7; v3 = 4.6 ± 1.5) were also lowest in group 1. The ROC curve analysis confirmed a significant but limited LB prediction capability for iDAScore v1.0 (AUC = 0.6), which was similar to the KIDScore D5 (AUC = 0.61–0.62). Limitations, reasons for caution Because of the retrospective nature, the major limitation was the lack of randomization, which may present a risk of selection bias. According to the criteria of blastocyst selection for SETs, the embryos with low (< 5.0) or high (> 9.5) AI scores were not present in this dataset. Wider implications of the findings Although prediction capability of iDAScore remains perfectible, the AI score is still significantly associated with LB probability. Euploid or mosaic blastocysts with low AI scores (<8.0) processed a decreased LB rate, indicating the potential of annotation-free iDAScore system as a decision support tool for deselecting embryos with poor post-implantation development. Trial registration number not applicable

Targeted resequencing reveals high-level mosaicism for a novel frameshift variant in WDR45 associated with beta-propeller protein-associated neurodegeneration

Objectives Beta-propeller protein-associated neurodegeneration (BPAN) is a rare X-linked dominant neurodegenerative disease, which is characterized by iron accumulation in the basal ganglia. BPAN is associated with pathogenic variation in WDR45, which has been reported almost exclusively in females most probably due to male lethality in the hemizygous state. Methods Whole exome sequencing (WES) and targeted deep sequencing were performed for a male with a clinical diagnosis of BPAN at the age of 37. Results The novel frameshift variant in WDR45 detected by WES was further analyzed with targeted resequencing to detect a mosaic variant with a level of 85.5% in the blood sample of the proband. Discussion Although the main role of WDR45 remains elusive, recent studies show that WDR45 may contribute to neurodegeneration through defects in autophagy, iron storage and ferritin metabolism, mitochondria organization, and endoplasmic reticulum homeostasis. The extend of spatiotemporal haploinsufficiency of WDR45 frameshifting variants caused by mosaicism in males may lead to variable clinical severity, which may be hard to elaborate clinically. Promising genetic analysis strategies using targeted deep sequencing may help determine the clinical outcome of somatic mosaicism in neurological disorders including BPAN. Additionally, we suggest that deep sequencing should be conducted in cerebrospinal fluid samples to provide more reliable results in terms of reflecting the mosaicism level in the brain for future studies.

Mosaic results after preimplantation genetic testing for aneuploidy may be accompanied by changes in global gene expression.

Aneuploidy in preimplantation embryos is a major cause of human reproductive failure. Unlike uniformly aneuploid embryos, embryos diagnosed as diploid-aneuploid mosaics after preimplantation genetic testing for aneuploidy (PGT-A) can develop into healthy infants. However, the reason why these embryos achieve full reproductive competence needs further research. Current RNA sequencing techniques allow for the investigation of the human preimplantation transcriptome, providing new insights into the molecular mechanisms of embryo development. In this prospective study, using euploid embryo gene expression as a control, we compared the transcriptome profiles of inner cell mass and trophectoderm samples from blastocysts with different levels of chromosomal mosaicism. A total of 25 samples were analyzed from 14 blastocysts with previous PGT-A diagnosis, including five low-level mosaic embryos and four high-level mosaic embryos. Global gene expression profiles visualized in cluster heatmaps were correlated with the original PGT-A diagnosis. In addition, gene expression distance based on the number of differentially expressed genes increased with the mosaic level, compared to euploid controls. Pathways involving apoptosis, mitosis, protein degradation, metabolism, and mitochondrial energy production were among the most deregulated within mosaic embryos. Retrospective analysis of the duration of blastomere cell cycles in mosaic embryos revealed several mitotic delays compared to euploid controls, providing additional evidence of the mosaic status. Overall, these findings suggest that embryos with mosaic results are not simply a misdiagnosis by-product, but may also have a genuine molecular identity that is compatible with their reproductive potential.

The nature of embryonic mosaicism across female age spectrum: an analysis of 21,345 preimplantation genetic testing for aneuploidy cycles

To understand how mosaicism varies across patient-specific variables and clinics. Cross-sectional cohort. Genetic testing laboratory. A total of 86,208 embryos from 17,366 patients underwent preimplantation genetic testing for aneuploidy using next-generation sequencing. Mosaic embryos were classified as either low-level (20%-40%) or high-level (40%-80%) and by type of mosaic error: single segmental, complex segmental, single chromosome, or complex abnormal mosaic. The rate of mosaicism was stratified by the Society for Assisted Reproductive Technology age categories: <35 years, 35-37 years, 38-40 years, 41-42 years, and >42 years. Distribution of chromosomal findings and prevalence of mosaicism type by age. Probability of creating mosaic embryos in a subsequent cycle. Among all embryos, 44% were euploid, 40.2% were aneuploid, and 15.8% were mosaic. Both low-level and high-level mosaicism were more prevalent among younger patients. Of all mosaic embryos, the youngest age cohort <35 years had the highest proportions of single and complex segmental mosaicism (37.9% and 6.8%, respectively), whereas those aged >42 years had the highest single whole chromosome and complex abnormal mosaicism (37.1% and 34.0%, respectively). Although there was variability in mosaic rates across clinics, the median mosaic rate over 3 years ranged from 14.48% to 17.72%. A diagnosis of a mosaic embryo in a previous cycle did not increase a patient's odds for having a mosaic embryo in a subsequent cycle. Mosaicism is overall higher in younger patients, but the complexity of mosaic errors increases with age. A history of mosaicism was not associated with mosaicism in subsequent cycles. Additional research is needed to understand the etiologies of the various subtypes of mosaic embryos and clinical outcomes associated with their transfer.

The use of copy number loads to designate mosaicism in blastocyst stage PGT-A cycles: fewer is better.

The use of copy number loads to designate mosaicism in blastocyst stage PGT-A cycles: fewer is better.

Mosaic 45,X/46, XX at amniocentesis with high-level mosaicism for 45,X in a pregnancy with a favorable fetal outcome and postnatal decrease of the 45,X cell line

We present mosaic 45,X/46, XX at amniocentesis with high-level mosaicism for 45,X in a pregnancy with a favorable fetal outcome and postnatal decrease of the 45,X cell line. A 20-year-old, primigravid woman underwent amniocentesis at 17 weeks of gestation because of the non-invasive prenatal testing (NIPT) result of-4.82 Z score in sex chromosome at 12 weeks of gestation suggestive of Turner syndrome in the fetus. Amniocentesis revealed a karyotype of 45,X [18]/46,XX [15], and simultaneous multiplex ligation-dependent probe amplification (MLPA) on the DNA extracted from uncultured amniocytes showed mosaic Turner syndrome. Prenatal ultrasound and parental karyotypes were normal. She was referred for genetic counseling at 24 weeks of gestation, and continuing pregnancy was encouraged. At 39 weeks of gestation, a 2550-g phenotypically normal female baby was delivered. The karyotypes of cord blood, umbilical cord and placenta were 45,X [24]/46,XX [16], 45,X [23]/46,XX [17] and 45,X [28]/46,X,del(X) (q23)[12], respectively. When follow-up at age two months, the neonate was phenotypically normal in development. The peripheral blood had a karyotypes of 45,X [16]/46,XX [24]. Interphase fluorescence in situ hybridization (FISH) analysis on 103 buccal mucosal cells showed normal disomy X signals in all cells. High-level mosaicism for 45,X in 45,X/46, XX at amniocentesis can be associated with a favorable fetal outcome, cytogenetic discrepancy in various tissues, and postnatal decrease of the 45,X cell line.

Genomic testing for copy number and single nucleotide variants in spermatogenic failure.

To identify clinically significant genomic copy number (CNV) and single nucleotide variants (SNV) in males with unexplained spermatogenic failure (SPGF). Peripheral blood DNA from 97/102 study participants diagnosed with oligozoospermia, severe oligozoospermia, or non-obstructive azoospermia (NOA) was analyzed for CNVs via array comparative genomic hybridization (aCGH) and SNVs using whole-exome sequencing (WES). Of the 2544 CNVs identified in individuals with SPGF, > 90% were small, ranging from 0.6 to 75kb. Thirty, clinically relevant genomic aberrations, were detected in 28 patients (~ 29%). These included likely diagnostic CNVs in 3/41 NOA patients (~ 7%): 1 hemizygous, intragenicTEX11 deletion, 1 hemizygous DDX53 full genedeletion, and 1 homozygous, intragenic STK11 deletion. High-level mosaicism for X chromosome disomy (~ 10% 46,XY and ~ 90% 47,XXY) was also identified in 3 of 41 NOA patients who previously tested normal with conventional karyotyping. The remaining 24 CNVs detected were heterozygous, autosomal recessive carrier variants. Follow-up WES analysis confirmed 8 of 27 (30%) CNVs (X chromosome disomy excluded). WES analysis additionally identified 13 significant SNVs and/or indels in 9 patients (~ 9%) including X-linked AR, KAL1, and NR0B1 variants. Using a combined genome-wide aCGH/WES approach, we identified pathogenic and likely pathogenic SNVs and CNVs in 15 patients (15%) with unexplained SPGF. This value equals the detection rate of conventional testing for aneuploidies and is considerably higher than the prevalence of Y chromosome microdeletions. Our results underscore the importance of comprehensive genomic analysis in emerging diagnostic testing of complex conditions like male infertility.

O-076 Genomic and morphokinetic blueprint of mosaic embryos is evidence supporting their distinct clinical category

Abstract Study question Is there a correlation between blastocyst mosaicism, morphokinetic and morphologic characteristics and can it inform the clinical outcome of mosaic embryo transfers? Summary answer Mosaic (diploid/aneuploid) embryos have a distinct genetic, morphokinetic and morphologic signature that distinguishes them from euploid and abnormal embryos and supports their unique clinical identity. What is known already Advances in technologies for preimplantation aneuploidy testing (PGT-A) as the ultimate test for embryo selection has increased the resolution and sensitivity to detect intermediate chromosomal copy number as low as 20%. This has brought to light a biological phenomenon that has created clinical dilemma. Current recommendations support mosaic embryo transfer but with conflicting standpoints ranging from complete dismissal of mosaic embryos to arguing their existence. Time-lapse imaging technologies allow collection of morphokinetic data as a non-invasive assessment tool for embryo development. The aim of this study was to correlate genomic and morphokinetic data with clinical outcomes of mosaic embryos. Study design, size, duration This is a retrospective cohort study that took place in a single academic IVF centre, where we analysed the results from high resolution PGT-A, morphokinetic, morphologic and cell division features of 4113 embryos cultured in the time lapse imaging incubator (Embryoscope™) from 2016-2021. Associated patient data and transfer outcome of 770 euploid and 147 mosaic embryo transfers were analyzed. Participants/materials, setting, methods High resolution NGS PGT-A was performed using the Illumina platform and BluGnome and NxClinical for data analysis. Clinically relevant findings were aberrations &amp;gt;10Mb and mosaicism from 25%-75%. Time-lapse imaging was performed with an Embryoscope™. Comparison of clinical, morphological, morphokinetic characteristics of euploid, aneuploid, low-level mosaic (25%-&amp;lt;50% aneuploid cells in trophectoderm biopsy), and high-level mosaics (50% -75%) was done with R-statistical package, Mathlab and SSPS software were used, and p &amp;lt; 0.05 with CI 95% was considered significant. Main results and the role of chance Aneuploidy was detected in 26.2%, euploidy in 56.7% and diploid-aneuploid mosaicism in 17.1% of embryos included in this study. Compared with euploid embryos, mosaic embryos had significant delays in t2 (27.04±0.35vs.26.4±0.17 h-post-ICSI(hpi), p = 0.0029), which was not correlated with transfer outcome. Embryos with whole chromosome mosaicism (WCM) had longer t2 compared to those with segmental chromosome mosaicism (SCM) (27.29±0.59vs.26.4±0.3hpi,p=0.013). The time to first mitosis was also longer for mosaic vs. euploid embryos (4.57± 0.59vs.3.4±0.25hpi,p=0.000056) and for mosaic embryos with WCM vs SCM embryos (4.64± 0.9 vs 3.5 ±0.5hpi,p=0.04). Mosaic embryos had longer tB &amp;tSB than euploid embryos (110.12±0.69vs.109.13hpi±0.4,p=0.01). Embryos with WCM had longer tB &amp;tSB than those with SCM (111.07±1.09vs.108.7±0.8hpi,p=0.0006). Time to blast was longer for mosaic vs euploid embryos (13.69±0.52vs.12.7±0.4hpi,p=0.0035) and for mosaic WCM vs. SCM embryos (14.3±0.7vs.12.8±0.5hpi,p=0.00084). Both euploid and mosaic embryos that implanted had shorter time to tB&amp;tSB than mosaic embryos that didn’t (p = 0.029; p = 0.007 and p = 0.002; p = 0.006 and p = 0.029 respectively); and mosaic embryos that implanted had shorter time to first mitosis than euploid embryos that didn’t (p = 0.00228). High level mosaic embryos had significantly more multinucleation at 2-cell stage (MN2) vs. euploid (p = 0.04,OD1.58-95%CI[1.01-2.45]),aneuploid (p = 0.02,OD1.7-95%CI[1.08-2.8]), as well as MN at 4-cell stage (euploid-p=0.0007,OD2.6-95CI1.4-4.7] and aneuploid-p=0.039,OD1.8-95CI1.02-3.4]). Limitations, reasons for caution Although this is the largest study to date that evaluated genomic, morphokinetic and outcome data of mosaic transfers, the numbers are still low to analyze the impact of specific chromosomal aberrations on morphokinetics and pregnancy outcome. The retrospective nature and cohort design limited the generalizability of our results. Wider implications of the findings Mosaic embryos have significant developmental potential and result in birth of healthy babies. Our study reveals the genomic interconnection of genomic and morphokinetic features of mosaic embryos and provides further evidence that mosaic embryos are a distinct category that requires specific clinical attention. Trial registration number Not applicable

The inconsistency between two major aneuploidy-screening platforms\u2014single-nucleotide polymorphism array and next-generation sequencing\u2014in the detection of embryo mosaicism

BackgroundIn preimplantation genetic testing for aneuploidy (PGT-A), appropriate evaluation of mosaic embryos is important because of the adverse implications of transferring embryos with high-level mosaicism or discarding those with low-level mosaicism. Despite the availability of multiple reliable techniques for PGT-A, data comparing the detection of mosaicism using these techniques are scarce. To address this gap in the literature, we compared the detection ability of the two most commonly used PGT-A platforms, next-generation sequencing (NGS) and the single-nucleotide polymorphism (SNP) array, for mosaic embryos.ResultsWe retrospectively reviewed the data of PGT-A or preimplantation genetic testing for chromosomal structural rearrangements (PGT-SR) conducted at our center from January 2018 to October 2020, and selected blastocysts that underwent aneuploidy screening with both an SNP array and NGS. Trophectoderm biopsy, multiple displacement amplification (MDA), and aneuploidy screening with an SNP array were conducted on the enrolled blastocysts. When the SNP array indicated mosaicism, NGS was performed on the corresponding MDA product for verification. Among the 105 blastocysts diagnosed with mosaicism with the SNP array, 80 (76.19%) showed mosaicism in NGS, with complete and partial concordance rates of 47.62% (50/105) and 18.10% (19/105), respectively. The complete discordance rate of the two platforms was 34.29% (36/105). That is, 10.48% (11/105) of the blastocysts were diagnosed with completely different types of mosaicism with the two platforms, while 13.33% (14/105) and 10.48% (11/105) of the embryos diagnosed as showing mosaicism with SNP were detected as showing aneuploidy and euploidy with NGS, respectively.ConclusionsThe consistency of NGS and the SNP array in the diagnosis of embryo mosaicism is extremely low, indicating the need for larger and well-designed studies to determine which platform is more accurate in detecting mosaic embryos.

THE MORPHOLOGY OF MOSAICISM: CHARACTERIZING LOW- AND HIGH-LEVEL MOSAIC EMBRYOS COMPARED TO EUPLOID EMBRYOS

The developmental patterns of mosaic embryos have yet to be understood.1 The aim of this study is to determine whether low and high level mosaic embryos differ from euploid embryos in morphologic characteristics.

P–564 Analysis of efficiency and outcomes of assisted reproductive technology (ART) programs after embryo transfer with a low-level of mosaicism

Abstract Study question How does embryo transfer with a low-level of mosaicism affect the success of ART programs, pregnancy, and live birth in comparison with euploid embryo transfer? Summary answer The transfer of mosaic embryos results in the delivery of a healthy baby however significantly decreases the outcome of ART programs and live birth rate. What is known already Present methods of preimplantation genetic testing of aneuploidy (PGT-A) allow detecting a mixture of euploid and aneuploid cells at the blastocyst stage with high accuracy. Such embryos are classified as mosaics with varying levels according to the guidelines of the International Society for Preimplantation Genetic Diagnosis (PGDIS). Numerous sources describe that number of mosaic embryos can vary from 4 to 22%. Several publications report that mosaic embryos can lead to successful pregnancies and healthy childbirth, but with a lower frequency and higher rates of pregnancy loss compared to euploid embryos. Nevertheless, the effect of mosaicism on ART outcomes remains controversial. Study design, size, duration It has been analyzed 2506 embryos from 648 patients undergoing the ART program with PGT-A at the Institute of Reproductive Medicine for 2018 - 2019. Embryos after PGT-A were classified as euploid, aneuploid, and having mosaicism of less than 40% as low level and more than 40% as high level following PGDIS guidelines. Patients of (group A) were transferred 467 single euploid embryos, and 43 patients (group B) underwent single low-level mosaic embryo transfer. Participants/materials, setting, methods The embryos on day 5 or 6 were graded by Gardner Scoring System. Approximately 5–10 TE cells were biopsied from good quality blastocysts and subsequently vitrified. PGT-A was performed utilizing an array comparative genomic hybridization (aCGH) (Agilent). The transfer of mosaic embryos was performed in the absence of an alternative, only after medical genetic counseling with a risk explanation and the subsequent signing of an informed agreement. Statistical tests processed by Pearson’s chi-squared test. Main results and the role of chance Of all analyzed embryos, the proportion of euploid embryos was 48.6% (n = 1002), the total number of mosaics was 18.6% (n = 384) and aneuploid ones were 32.8% (n = 676). Depending on the level of mosaicism, the ratio between embryos with low-level mosaicism (≤40%) / high-level (≥40%) was 38.3% / 61.7%, respectively. According to the study, there was a significant decrease in the indicator of clinical pregnancy rate after embryo transfer with a low-level of mosaicism of 44.1% versus 63.2% transferred euploid embryo (р&amp;lt;0,01), however, despite an increase losses pregnancy in the group B (26.3%) there was no significant difference (p = 0.16) in comparison with the control group (15.4%). The live birth rate (LBR) significantly decreased (p &amp;lt; 0.001) after the transfer of the mosaic embryo by 32.5%, while in the control group the indicator was 53.9%. In all cases, after the transfer of the mosaic embryo, healthy babies were born. There were 2 cases of high-level mosaic embryo transfer as a result of which pregnancy did not occur. According to the survey, about 70% of patients agree to replant mosaic embryos, 20% are ready to go to the new program, and 10% cannot make a decision. Limitations, reasons for caution The number of patients in group B was significantly lower than in group A. Not enough cases of embryo transfer with a high-level of mosaicism. Wider implications of the findings: The current study might help to develop and to select a more appropriate strategy for transfer mosaic embryos. The next series of studies should focus on obstetric and neonatal outcome data from mosaic embryo transfer to gain a better understanding of the chromosomal and physiological health of children. Trial registration number Not applicable

THE IMPACT OF USING ARTIFICIAL INTELLIGENCE IN REPORTING HIGH RESOLUTION NGS RESULTS UPON PRIORITIZATION OF EMBRYOS SELECTED FOR TRANSFER

There are many factors that influence the interpretation of the plots of high resolution NGS (hr-NGS) in PGT A examination of the embryos, such as the Genetist experience, cut-off points, and the chromosomal number. The PGT A results differentiate between euploidy embryos that are given first priority for embryo transfer (ET), second priority for low mosaic, and aneuploidy or high level mosaic those who are not transferred . This study compares between manual calling of the results versus using artificial intelligence platform. This is a prospective ongoing study. A total of 720 biopsied sample from 280 patients processed according to Miseq Illumina protocol. Results are manually called subjective methodology (SM) using Bluefuse software and also uploaded up to PGTaiSM platform Coopersurgical. The mean age of the female partners is 34 years. There was a difference in 138 results out of 720 (19%) comparing between the “SM” and PGTaiSM platform interpretation. The following table shows the differences between four categories in both SM and PGTai results:Tabled 1categoriesSMPGTaiSMNo. of embryosEuploid3114 low level mosaic11 high level mosaic5 aneuploid1 QA failAneuploid271 low level mosaic15 high level mosaic1 euploid10 QA failHigh level mosaic292 low level mosaic14 euploid12 aneuploid1 QA failLow level mosaic4612 high level mosaic28 euploid5 aneuploid1 QA failThe PGTai platform changes some of the results in all reported categories. That would change the prioritization of embryos for transfer. There were 16 embryos that were reported euploidy by SM, while they are deemed “untransferable” by PGTai, being 5 aneuploidy and 11 being of high level mosaic. Again, in the same group 14 embryos were of low mosaic level which would have been given a second priority. In the aneuploidy group only 2 embryos (1 euploid and 1 low level mosaic) would have been possibly transferred according to PGTai.In the high level mosaic group 16 embryos would have been given priority for embryo transfer as 14 were found euploidy while 2 were of low mosaic level. In the low level mosaic group 28 embryos would have been given first priority as found to be euploid, while 17 embryos would have must been considered to be as 12 of high level mosaic and 5 were aneuploidy. Open table in a new tab The PGTai platform changes some of the results in all reported categories. That would change the prioritization of embryos for transfer. There were 16 embryos that were reported euploidy by SM, while they are deemed “untransferable” by PGTai, being 5 aneuploidy and 11 being of high level mosaic. Again, in the same group 14 embryos were of low mosaic level which would have been given a second priority. In the aneuploidy group only 2 embryos (1 euploid and 1 low level mosaic) would have been possibly transferred according to PGTai. In the high level mosaic group 16 embryos would have been given priority for embryo transfer as 14 were found euploidy while 2 were of low mosaic level. In the low level mosaic group 28 embryos would have been given first priority as found to be euploid, while 17 embryos would have must been considered to be as 12 of high level mosaic and 5 were aneuploidy. PGTai is thought to be more valid than manual subjective calling of results as based upon true data set of live births and sustained pregnancy outcomes. This study shows a strong difference of 19% in results that changes how embryos are prioritized for ET. Moreover, same embryos would have been not transferred as being aneuploidy as of high mosaic level. Further studies are needed to evaluate if using PGTai would improve live birth rate and decrease miscarriage by better selection of embryos.

Clinical Outcomes of Single Mosaic Embryo Transfer: High-Level or Low-Level Mosaic Embryo, Does it Matter?

Recently, reports showed that embryos identified as mosaic after preimplantation genetic testing for aneuploid (PGT-A) could result in live birth with lower pregnancy and higher pregnancy loss rates compared with euploid embryos. However, the effects of mosaicism level on reproductive outcomes remain controversial. This study aimed to examine the level of mosaicism on pregnancy outcomes. Single mosaic embryo transfer was offered to 108 women who only had mosaic embryos. Mosaic embryos were labeled by utilizing next generation sequencing (NGS) based PGT-A for day 5/6 trophectoderm (TE) biopsies. TE biopsies containing < 50% abnormal cells were classified as low-level mosaicism and ≥ 50% as high-level mosaicism. To further confirm the concordance of chromosome constitution between TE and inner cell mass (ICM), 41 remaining embryos designated as mosaic blastocysts donated for research were also analyzed. Comparable live birth rate (LBR) but higher miscarriage rate (MR) was found in the high-level group. (LBR: low vs. high: 44.5% vs. 36%; p = 0.45, MR: low vs. high: 5.1% vs. 30.7%; p = 0.012). Analyses of TE and ICM from the remaining mosaic blastocysts show a poor concordance. This preliminary study demonstrated that high-level mosaic embryos could result in comparable LBR but higher MR.

Frequency of low-level and high-level mosaicism in sporadic retinoblastoma: genotype-phenotype relationships.

Somatic mutational mosaicism is a common feature of monogenic genetic disorders, particularly in diseases such as retinoblastoma, with high rates of de novo mutations. The detection and quantification of mosaicism is particularly relevant in these diseases, since it has important implications for genetic counseling, patient management, and probably also on disease onset and progression. In order to assess the rate of somatic mosaicism (high- and low-level mosaicism) in sporadic retinoblastoma patients, we analyzed a cohort of 153 patients with sporadic retinoblastoma using ultra deep next-generation sequencing. High-level mosaicism was detected in 14 out of 100 (14%) bilateral patients and in 11 out of 29 (38%) unilateral patients in whom conventional Sanger sequencing identified a pathogenic mutation in blood DNA. In addition, low-level mosaicism was detected in 3 out of 16 (19%) unilateral patients in whom conventional screening was negative in blood DNA. Our results also reveal that mosaicism was associated to delayed retinoblastoma onset particularly in unilateral patients. Finally we compared the level of mosaicism in different tissues to identify the best DNA source to identify mosaicism in retinoblastoma patients. In light of these results we recommended analyzing the mosaic status in all retinoblastoma patients using accurate techniques such as next-generation sequencing, even in those cases in which conventional Sanger sequencing identified a pathogenic mutation in blood DNA. Our results suggest that a significant proportion of those cases are truly mosaics that could have been overlooked. This information should be taking into consideration in the management and genetic counseling of retinoblastoma patients and families.

Megalencephaly syndromes associated with mutations of core components of the PI3K-AKT-MTOR pathway: PIK3CA, PIK3R2, AKT3, and MTOR.

Megalencephaly (MEG) is a developmental abnormality of brain growth characterized by early onset, often progressive, brain overgrowth. Focal forms of megalencephaly associated with cortical dysplasia, such as hemimegalencephaly and focal cortical dysplasia, are common causes of focal intractable epilepsy in children. The increasing use of high throughput sequencing methods, including high depth sequencing to more accurately detect and quantify mosaic mutations, has allowed us to identify the molecular etiologies of many MEG syndromes, including most notably the PI3K-AKT-MTOR related MEG disorders. Thorough molecular and clinical characterization of affected individuals further allow us to derive preliminary genotype-phenotype correlations depending on the gene, mutation, level of mosaicism, and tissue distribution. Our review of published data on these disorders so far shows that mildly activating variants (that are typically constitutional or germline) are associated with diffuse megalencephaly with intellectual disability and/or autism spectrum disorder; moderately activating variants (that are typically high-level mosaic) are associated with megalencephaly with pigmentary abnormalities of the skin; and strongly activating variants (that are usually very low-level mosaic) are associated with focal brain malformations including hemimegalencephaly and focal cortical dysplasia. Accurate molecular diagnosis of these disorders is undoubtedly crucial to more optimally treat children with these disorders using PI3K-AKT-MTOR pathway inhibitors.

Embryo morphokinetics is potentially associated with clinical outcomes of single-embryo transfers in preimplantation genetic testing for aneuploidy cycles.

Are the morphokinetics of euploid blastocysts evaluated by a generally applicable algorithm associated with the clinical outcomes of single-embryo transfer (SET)? Time-lapse microscopy was used to compare morphokinetic variables between expanded blastocysts derived from preimplantation genetic testing for aneuploidy cycles using high-resolution next-generation sequencing (hr-NGS). The clinical efficacy of the morphokinetic algorithm KIDScore D5 was evaluated after euploid SET. Compared with euploid blastocysts, low-level mosaic blastocysts presented comparable morphokinetic and morphological features. However, high-level mosaic blastocysts exhibited significant delays in t5 (median 51.9 h post insemination (hpi), P = 0.034) (where t is the time for the embryo to reach the specific stage in hours after ICSI or conventional IVF) and t8 (median 58.6 hpi, P = 0.032) accompanied by a prolonged time period for the third cell cycle (median 14.7 h, P = 0.012). A significantly higher incidence (P = 0.011) of multinucleation indicated a susceptibility of high-level mosaic blastocysts to mitotic errors. Only a delay in the time for the embryo to reach the full blastocyst stage (median 106.0 hpi, P = 0.039) was revealed in aneuploid blastocysts, reflecting the reduced formation of good-quality blastocysts (42.6% versus 65.7%, P < 0.001). Euploid blastocysts with specific morphokinetic characteristics were graded using the KIDScore D5 algorithm. Grade C embryos achieved significantly lower rates of clinical pregnancy, implantation and ongoing pregnancy (25%, 25% and 10%, respectively) compared with the grade A (76.2%, 79.4% and 68.3%, respectively) or grade B (62.5%, 66.7% and 62.5%, respectively) embryos (P = 0.0171 to <0.0001). Although morphokinetic features appear dissimilar in embryos with different diploid-aneuploid mosaic levels, predicting chromosomal abnormalities using morphokinetics alone is still insufficient. When combined with hr-NGS, use of the generally applicable KIDScore D5 algorithm has the potential to discriminate euploid blastocysts with different developmental competence.

Reporting of Mosaics as High-level and Low level mosaics makes more number of embryos available as alternatives for transfer when no euploid embryos are available

Introduction It is relatively common for human preimplantation embryos produced during the course of IVF treatments to contain two or more cytogenetically distinct cell lines. This phenomenon, known as chromosomal mosaicism, can result in either a mixture of euploid and abnormal cells, or abnormal cells bearing distinct chromosomal gains and losses. NGS allows mosaicism to be detected with much greater sensitivity than earlier preimplantation genetic testing (PGT-A) methods. The application of NGS to trophectoderm biopsies, taken from embryos before transfer to the uterus, has provided insight into the clinical impact of mosaicism. However, because some mosaic embryos are have produced successful pregnancies, it may be appropriate to consider transfer of mosaic embryos in the absence of fully euploid embryos and following patient counseling. Materials and Methods Samples were amplified by SurePlex, sequenced with VeriSeq PGS assay (Illumina) on MiSeq (Illumina) and analyzed with BlueFuse Multi analysis (Illumina). Embryos with at least one fully aneuploid chromosome or one fully aneuploid chromosome and one mosaic chromosome were called aneuploid. Embryos with 3 chromosome abnormalities were designated complex abnormal. Mosaicism was reported when embryos had 20-80% abnormal sequencing profiles. Embryos with low degrees of mosaicism in sequencing profiles were designated as ‘Low –Level Mosaics’. When embryos had high degree of mosaicism, they were reported as ‘High- Level Mosaics’. Design Retrospective analysis of PGS procedures involving TE biopsy and NGS performed by laboratories serving over 250 fertility clinics over three years. A total of 3951 embryos resulting from 952 IVF cycles were analyzed. Results Gross aneuploidy (includes complex abnormals and polyploidies) rates in egg donors (n=402) was 25.62%. Euploid rate in egg donors was 51.00%. 20.90% embryos from egg donors were mosaic, with 11.94% being classified as High-level mosaic and 8.96% being classified as Low-level mosaic. In the non-egg donor group (n=3,545 embryos) aneuploidy (includes complex abnormals and polyploidies) ranged at 44.23%. Euploid rate in non-egg donors was about 36.47%. About 15.94% embryos were mosaic with no trend observed in advanced maternal age and mosaicism. About 8% of all embryos in the non-egg donor group were High-level mosaics while and equal percentage of embryos were Low- level mosaics. A total of 33% cycles (n=317 cycles) resulted in cohorts which had zero euploid embryos. Of these 317 cycles however, 112 cycles (35%), had at least one mosaic embryo to transfer. Of the zero-euploid group a total of 52 cycles (16%) resulted in a Low-level mosaic for patients who otherwise do not have any normal/euploid embryos to transfer. Only 7 cycles from the donor group resulted in cohorts with zero euploid embryos. Conclusion The ability of PGT-A by NGS to delineate between high-level and low-level mosaic embryos allows patients without euploid embryos for transfer the ability to identify mosaic embryos with the greatest likelihood of producing a successful pregnancy. Approximately 8% of all embryos tested by PGT-A are low-level mosaic, with 16% of cycles without identified euploid embryos producing at least one low-level mosaic embryo. The results herein can be used to counsel patients and guide expectations.