Chromosomal Status Research Articles

O-084 Computer vision can distinguish between euploid and aneuploid embryos. A novel artificial intelligence (AI) approach to measure cell division activity associated with chromosomal status

Abstract Study question Can we distinguish between top-grade euploid and aneuploid embryos by AI measurement of cell edges in time-lapse videos? Summary answer Aneuploid embryos can be distinguished from euploid embryos by AI determination of a longer time to blastulation and higher cell activity. What is known already Continuous monitoring of the embryo development has brought out morphokinetic parameters that are used to predict pre-implantation genetic testing (PGT) results. Previous publications showed that euploid embryos reach blastulation earlier than non-euploid embryos. However, time-lapse data are currently under-utilized in making predictions about embryo chromosomal content. AI and computer vision could take advantage of the massive amount of data embedded in the images of embryo development. This is the first attempt to distinguish between euploid and aneuploid embryos by computer vision in an objective and indirect way based on the measurement of cell edges as a proxy for cell activity. Study design, size, duration We performed a retrospective analysis of 1,314 time-lapse videos from embryos cultured to the blastocyst stage with PGT results. This single-center study involved two phases; a comparison of the start time of blastulation between euploid (n = 544) and aneuploid embryos (n = 797). In phase two, we designed a novel methodology to examine whether precise measurement of cell edges over time could reflect cell activity differences in blastulation. Participants/materials, setting, methods We assumed that the delay in blastulation is reflected by higher cell activity that could be determined accurately for the first time using computer vision and machine learning to measure the length of the edges (from t2 to t8). We compared computer vision based measurements of cell edges, reflecting cell number and size, in videos of 231 top-grade euploid (n = 111) and aneuploid (n = 120) embryos. Main results and the role of chance The mean and standard deviation of blastulation start time was 100.1±6.8 h for euploid embryos and 101.8±8.2 h for aneuploid embryos (p < 0.001). Regarding the measurement of cell activity, a computer vision algorithm identified the edges and provided a certainty score for each edge, higher when the algorithm is more certain that this is a cell edge (as opposed to noise in the images). A threshold was set to distinguish cell edges from noise using this score. The following results for top-grade embryos are shown as the sum of the edge lengths (µm) average of 160 pictures per embryo (frames between t2 and t8). The total length of the cell edges increased from two cells (420±85 µm) to eight cells (861±237 µm), in line with the mitosis events. Both the average total edge measured (450±162 µm for euploid embryos and 489±215 µm for aneuploid embryos, p < 0.01) and the average total of the difference between consecutive frames (135±47 µm for euploid embryos and 153±64 µm for aneuploid embryos, p < 0.01) were higher for aneuploid embryos than for euploid embryos. A regression model to differentiate between the two classes achieved 73% sensitivity and 73% specificity on this dataset. Limitations, reasons for caution The main limitation of this study is the difficulty to correlate our findings to other measure of cell activity. A more robust AI function (using not only cell edges lengths) would be required for future analysis to measure the cell activity in cell division up to the blastocyst stage. Wider implications of the findings Our results show for the first time that an AI based system can precisely measure microscopic cell edges in the dividing embryo. Using this novel method, we could distinguish between euploid and aneuploid embryos. This non-invasive method could further enhance our knowledge of the developing embryo. Trial registration number Not Applicable

O-004 Self-correction in human preimplantation development: What do we know?

Abstract text Recent advances in preimplantation genetic testing for aneuploidy (PGT-A) and time-lapse imaging have improved our understanding of the early human embryo confirming the variable patterns of development and chromosomal status. Aneuploidy is common and increased sensitivity in PGT-A allows the non-binary reporting of euploid-aneuploid mosaicism. The PGT-A result is the inference of the biopsied embryo’s ploidy status at a point in time, by assessment of a small percentage of cells, and, whilst concordance with the rest of the embryo is high; it is not absolute. Many reports have demonstrated that, with the transfer of embryos with increasing severity and complexity of mosaicism, comes compromised implantation, reduced ongoing pregnancy rates and increased miscarriage rates. Segmental mosaic embryos have been reported to have slightly reduced implantation potential compared with euploid counterparts. However, complex mosaic embryos are widely reported to result in severely reduced implantation success, if transferred. Outside of PGT-A treatment cycles, undoubtedly fertility clinics are unwittingly transferring mosaic and aneuploid embryos daily, with variable success. The transfer of embryos in which mosaicism has been detected, although associated with lower implantation and higher miscarriage rates than euploid embryos, can lead to normal pregnancies and healthy births. We know that the placenta can harbour chromosomal aberrations which are absent from the fetus, and there are few reports of births with demonstrably high levels of mosaicism through fetal development. This raises the question as to whether correction mechanisms exist. In other words, do conceptuses become chromosomally more normal as development progresses, and what are the mechanisms, if so? PGT-A, time lapse, novel live cell imaging and in vitro model techniques have enabled a more detailed study of early embryo development and consideration of the phenomenon of self-correction. This has provided insights and hypotheses surrounding the mechanisms of development and of self-correction. The relatively lower levels of chromosome abnormality in the blastocyst, compared with cleavage stage, are well documented and indicative of some form of correction. A recent investigation reported that a large proportion of embryos initially diagnosed as mosaic were later diagnosed as euploid when assessed at day 12 of development; providing evidence of the depletion of abnormal cells throughout the early post-implantation stages. There are many time-lapse reports of anomalous ‘direct’ or multichotomous blastomere divisions being associated with aneuploidy, and leading to developmental arrest or reduced implantation potential and of temporal delays in aneuploid embryos compared with their euploid counterparts. It is possible, therefore, that errors and even attempts to repair them, in individual cells in the rapidly developing embryo; which involve complex biochemical systems, could delay karyo- and cytokinesis, resulting in these detectable delays. The embryonic mortality model suggests that there is selection against embryos based on their degree of aneuploidy, such that aneuploid cell lines are lost during implantation. We know that irregularities in blastomere cleavage can generate chromosome segregation errors but these may sporadically be confined to cells excluded, or extruded, from the morula or from the blastocyst; a possible exhibition of the clonal depletion or embryo mortality model. The trisomic/monosomic rescue model suggests that aneuploid cells can give rise to diploid cells (and possibly uniparental disomy) through mitotic chromosome losses or gains. We know that an abnormal number of pronuclei does not always produce an aneuploid blastocyst and that early embryos exhibiting multinucleation can result in healthy live births. Finally, the preferential allocation of aneuploid cells to the trophectoderm model is based on the hypothesis that euploid cells are preferentially retained in the ICM in order to achieve viability. This presentation aims to consider what we know, and discuss the theories and available evidence for self-correction.

P–552 Delayed blastocyst development is associated with a higher risk of aneuploidy in patients of advanced maternal age

Abstract Study question Is delayed blastocyst development, assessed by the day of trophectoderm (TE) biopsy, associated with higher rates of aneuploidy? Summary answer Our findings show an association between delayed blastocyst development and poorer prognosis, in terms of euploidy rates, in patients of advanced maternal age. What is known already Extended culture of embryos past day 5 of development has become routine practice in all freeze-all cycles, including those applying preimplantation genetic testing for aneuploidies (PGT-A). As healthy live births have been obtained from day 6 and day 7 blastocysts, increasing the pool of embryos available for PGT-A is beneficial, particularly for patients of advanced maternal age who face higher cancellation rates. Nevertheless, the association between delayed blastocyst development and aneuploidy rates remains unclear. As current studies have reported opposing findings, detailed analysis of the chromosomal constitution of slowly developing embryos remains paramount. Study design, size, duration Retrospective, international, multicentre cohort study of 4211 patients undergoing preimplantation genetic testing for aneuploidy (PGT-A) from January 2016 to July 2020. We evaluated the chromosomal status of 14757 blastocysts tested using TE biopsy and next generation sequencing (NGS). Both autologous and donation cycles were included in the analysis. Cycles were excluded if they utilised preimplantation genetic testing for monogenic disorders (PGT-M) or preimplantation genetic testing for structural rearrangements (PGT-SR). Participants/materials, setting, methods We evaluated euploidy, aneuploidy and mosaicism rates reported in day 5 (n = 9560), day 6 (n = 4753) and day 7 (n = 262) blastocysts, stratified by SART-defined maternal age categories (<35, 35–37, 38–40, 41–42, >42). We further assessed the type and frequency of abnormalities reported in all blastocysts classified as clinically unsuitable, according to the day of biopsy. Finally, we examined the specific chromosomes affected in embryos diagnosed with a single uniform (n = 3882) or single mosaic (n = 518) abnormality. Main results and the role of chance The mean maternal age within our patient cohort was 39.9±3.7. Overall, slowly developing blastocysts were significantly more likely to be classified as clinically unsuitable (60.6%) compared to day 5 embryos (55.2%; p < 0.0001). This correlation was also observed when stratified by age, with the exception of the <35 age group (p = 0.25). Markedly, the risk of aneuploidy in slowly developing blastocysts became progressively higher with advancing maternal age (p < 0.0001). We did not observe any significant differences in the types of abnormalities diagnosed in slowly developing embryos compared to day 5 blastocysts. Nevertheless, abnormalities affecting all chromosomes were present at the blastocyst stage. Single trisomies and monosomies were the most frequent across all age groups, and were equally prevalent in day 5, 6 and 7 blastocysts. These most commonly affected chromosomes 16, 22, 21 and 15. We observed no significant differences in the incidence of segmental aneuploidies in relation to the day of biopsy, across all age groups. When considered separately, day 7 blastocysts presented with higher rates of structural aberrations, however low numbers limited statistical power. Finally, delayed blastocyst development was not associated with higher mosaicism rates (p = 0.79). Interestingly, single mosaic trisomies and monosomies were most frequently associated with chromosome 19. Limitations, reasons for caution Due to the retrospective nature of the study, full elucidation of all potential confounders may not be possible in all instances. The low number of day 7 blastocysts limited statistical power. As such, the results from day 6 and day 7 embryos were evaluated together. Wider implications of the findings: Our findings offer an important clinical resource for counselling patients of advanced maternal age. Maternal aging may be associated with a higher incidence of aneuploidy in slowly developing blastocysts. Nevertheless, extended culture increases the pool of biopsiable blastocysts, ultimately improving the chance of having a euploid embryo for transfer. Trial registration number NA

P–214 Effect of artificial activation of oocytes (AOA-ICSI) on the ploidy status of the resultant blastocysts. A sibling-oocytes pilot study

Abstract Study question Will artificial activation of oocytes alter the ploidy status of the resultant blastocysts? A sibling-oocytes pilot study Summary answer AOA-ICSI does not increase the risk of having aneuploidy blastocysts and can improve the fertilization rate in patients with sperm factor deficiency. What is known already Despite introducing ICSI as an aid to improve chances of fertilization, fertilization failure can still occur in 2–3% of ICSI cycles. Fertilization is a complex process triggered by a cascade of events following calcium (Ca2+) oscillations. Evidence suggests that the deficiency, localization or altered structure of the sperm-derived protein PLCζ in oocyte activation may be a reason for meiotic II arrest in the oocyte. Artificial oocyte activation has been proposed to compensate for the lack of calcium oscillation and resumes meiotic progression. There are however insufficient studies to determine its effect on the chromosomal status of the resultant blastocysts. Study design, size, duration This is a prospective, randomized study conducted at our Center from August-October 2020. A total of 20 couples intended for ICSI + Preimplatation Genetic Testing for Aneuploidy (PGT-A) cycles were recruited based on fulfilling one of the following criteria: 1) previous total fertilization failure (TFF), 2) history of low fertilization rate (<30%), 3) more than 2 cycles of failed IVF cycles (no implantation) 4) poor embryo development (no blastocysts formed) and 5) severe male factor. Participants/materials, setting, methods A total of 231 MII oocytes underwent randomization in a 1:1 ratio between AOA-ICSI and control group. All oocytes are subjected to ICSI treatment. Oocytes in the AOA-ICSI group are treated in 25μl droplets 10μM ready to use bicarbonate buffered calcium ionophore (Kitazato, Japan) for 15 minutes post-ICSI. The blastocysts were biopsied and subjected to PGT-A. Primary outcome was the aneuploidy rate and secondary outcomes were fertilization rate and blastocyst rate. Main results and the role of chance There were 11 out of 40 (27.5%) aneuploid blastocysts in the AOA-ICSI group and 7 out of 23 aneuploid blastocysts (30.4%) in the control group [odds ratio (OR) = 0.87; 95% confidence interval (CI) 0.28–2.68, p = 0.8040). There was no statistically significant difference between both groups. However, fertilization rate of the AOA- ICSI group was significantly higher than the fertilization rate in the control group (68.6% vs 49.6% respectively, OR = 2.22; 95% CI, 1.31–3.81, p = 0.0034). There were 40 blastocysts formed in the AOA-ICSI group and 23 blastocysts formed in the control group. It was found that the AOA-ICSI group yielded a higher blastocyst rate (49.4%) compared to the control group (41.1%) (OR = 1.40; 95% CI, 0.71 to 2.78, p = 0.3379) but the difference was not statistically significant. Limitations, reasons for caution The possibility of TE cells biopsied may not be representative of the whole blastocyst makes it possible to have false clinical data. The dosage and time were also not evaluated in this study as exposure time was found to be a critical factor of fertilization rate in a previous study. Wider implications of the findings: This study showed that AOA-ICSI does not increase the risk of having aneuploidy blastocysts and can improve the fertilization rate in patients with sperm factor deficiency. Additional studies involving a larger number of patients with more specific indication can further justify the benefits of AOA as a therapeutic application. Trial registration number NA

P–066 Does microfluidic sperm sorting (MSS) affect embryo euploidy rates in couples with high sperm DNA fragmentation (SDF)?

Abstract Study question Does MSS (microfluid chip-sorted spermatozoa selection) provide improvement on embryo quality and euploidy rates in couples with high SDF (sperm DNA fragmentation) and previous failed in vitro fertilization/ intracystoplasmic sperm injection (IVF/ICSI) cycles? Summary answer Use of MSS technique provides higher number of top quality blastocysts compared to density gradient centrifugation (DGC), however euploidy and live birth rates weren’t improved. What is known already Previously it has been reported that sperm DNA damage leads to poor embryo development and there is a significant association between SDF and high embryo aneuploidy rates. Recently this fact raised attention to sperm selection techniques such as MSS to enhance embryo quality, miscarriage rates and embryonic euploidy rates. Study design, size, duration This was a retrospective electronic medical record (EMR) analysis of a tertiary assisted reproduction center between 2016 and 2020. All EMR were reviewed to select eligible cases as; couples undergoing a new IVF/ICSI cycle with PGT-A (preimplantation genetic testing for aneuploidies). In total, data from 243 patients were obtained for the analysis that accounts for 688 embryos. Participants/materials, setting, methods Patients had at least 2 previous failed IVF cycles and males had at least 20% SDF. In their new cycles, MSS was offered, preceding ICSI and PGT-A. Couples who accepted the technique were assigned to MSS group (92 cycles with 310 embryos) and the rest were managed with DGC and assigned as controls (151 cycles with 378 embryos). Azoospermia cases and women with age>43, uterine abnormalities, trombophilia were excluded. Biopsies were performed at blastocyst stage. Main results and the role of chance Two groups were comparable in terms of demographic data including women and men age, SDF, sperm parameters and cycle characteristics. There was no difference between groups in terms of fertilization rates (MSS 85% vs DGC 79% p = 0.9), euploidy rates (MSS 53.2% vs DGC 50.7% p = 0.3), mean no of euploid embryo per patient (MSS 1.09 vs DGC 0.95 p = 0.3), positive pregnancy test (MSS 50% vs DGC 38.4% p = 0.06), clinical miscarriage (MSS 7.6% vs DGC 6.6% p = 0.7) and live birth rates (LBR)(MSS 42.4% vs DGC 31.7% p = 0.09). Total no of blastocysts and top quality blastocysts were significantly higher in MSS group than in DGC (3.9 vs 2.5 p < 0.01 and 1.6 vs 0.8 p < 0.001 respectively). Limitations, reasons for caution Retrospective design, small sample size, lack of proper randomization and power analysis are the main limitations. Wider implications of the findings: Offering PGT-A to couples with unexplained repeated IVF failures and high SDF seems feasible. MSS for such cases improves embryonic division process as improved blastulation rates were documented. However, euploidy rates were not improved in MSS group revealing that other factors influence comprehensive chromosomal status of an embryo. Trial registration number Not applicable

P–527 Embryonic cell-free DNA (cfDNA) in spent culture medium for aneuploidy screening and its concordance with trophoectoderm biopsy in PGT-A cycles

Abstract Study question Is embryonic cfDNA detectable in blastocyst spent culture medium; can its analysis via Next Generation Sequencing compare with trophoctoderm biopsy results of the respective embryos? Summary answer Embryonic cfDNA is detected in blastocyst spent culture medium (BCM) and gives comparable aneuploidy rates with trophoectoderm biopsy in PGT-A cycles. What is known already Currently, PGT- A involves the use of invasive techniques to obtain embryonic DNA, with significant technical and ethical limitations. Recently, spent culture medium (SCM) has been proposed as an alternative source of embryonic DNA. Studies have reported the detection of cfDNA in SCM and highlighted the diagnostic potential of non invasive PGT (niPGT) for assessing the genetic status of preimplantation embryos. Moreover, invasive PGT-A can lead to genetic misdiagnosis in case of mosaic embryos, while niPGT-A may be more represantative of the whole embryonic chromosome status. However, the reliability of this approach for clinical applications needs to be further determined. Study design, size, duration These are preliminary data from an observational study conducted in the period 2019–2020. 40 embryos from 13 patients, undergoing PGT-A, were analyzed. Trophoectoderm biopsies (TEB) and respective SCMs from individually cultured embryos were analyzed by Next Generation Sequencing NGS. The results from trophoectoderm biopsies and SCMs of the respective embryos were compared. Participants/materials, setting, methods The embryos were cultured individually in 10μl drops, from day 3 to the blastocyst stage (day5/6). On day 5/6, TEB was performed and the corresponding BCM was collected and stored at –80 °C, until analysed with NGS. Data were analysed with McNemar’s test and ROC analysis. The results were considered significant when P < 0.05. 95% Confidence intervals (95%CI) were calculated. Main results and the role of chance The amplification rate, for embryonic cfDNA from BCM samples collected from embryos cultured for 48–72 hours after day 3, was 100%. DNA concentration in each sample after whole genome amplification (WGA) ranged between 2500–30000 ng/ml for TEB and 2000–20400 ng/ml for BCM. Respective blank medium negative controls associated with each sample that underwent WGA showed no amplification in all cases. The trophoectoderm biopsy showed aneuploidy at a percentage of 61% (95% CI: 43–78%), vs. BCM aneuploidy at a percentage of 55% (95% CI: 37–72%). The overall agreement BCM vs. TE biopsy, from samples taken from the same embryo, was 27/33, 81.8% (95% CI: 68–96%). McNemar test: p = 0.687, non-significant. The aneuploidy agreement was 88.9% (sensitivity) and the euploidy agreement was 73.3% (specificity). In ROC analysis, AUC was 82.3% (95% CI 66.9–97.8). In 4 BCM samples detected euploidy, while TE biopsy showed embryo monosomes, possibly due to mosaicism. 7 samples were excluded due to low quality cfDNA. Of the 33 samples, 7 were male (XY), according to both TE biopsy and BCM analysis, a fact that confirms the safety of the method, as it shows no contamination by maternal DNA. Limitations, reasons for caution The study is limited by the small sample size. To become the niPGT reliable, several steps must be optimized: DNA collection methods, DNA amplification and downstream techniques for analysis. Also, the analysis of discarded whole blastocysts as a gold standard control may determine the method’s reliability. Wider implications of the findings: Non-invasive Preimplantation Genetic Testing (niPGT), is a promishing alternative that may give an accurate and reliable option of detecting euploid human embryos, also dealing with the problem of mosaicism in trophodectoderm biopsies. Further technical refinement is needed to perfect niPGT, so that it can be used in routine clinical practice. Trial registration number N/A

O-203 Application of machine learning to predict aneuploidy and mosaicism in embryos from in vitro fertilization (IVF) cycles

Abstract Study question Is it possible to predict the likelihood of an IVF embryo being aneuploid and/or mosaic using a machine learning algorithm? Summary answer There are paternal, maternal, embryonic and IVF-cycle factors that are associated with embryonic chromosomal status that can be used as predictors in machine learning models. What is known already The factors associated with embryonic aneuploidy have been extensively studied. Mostly maternal age and to a lesser extent male factor and ovarian stimulation have been related to the occurrence of chromosomal alterations in the embryo. On the other hand, the main factors that may increase the incidence of embryo mosaicism have not yet been established. The models obtained using classical statistical methods to predict embryonic aneuploidy and mosaicism are not of high reliability. As an alternative to traditional methods, different machine and deep learning algorithms are being used to generate predictive models in different areas of medicine, including human reproduction. Study design, size, duration The study design is observational and retrospective. A total of 4654 embryos from 1558 PGT-A cycles were included (January-2017 to December-2020). The trophoectoderm biopsies on D5, D6 or D7 blastocysts were analysed by NGS. Embryos with ≤25% aneuploid cells were considered euploid, between 25-50% were classified as mosaic and aneuploid with >50%. The variables of the PGT-A were recorded in a database from which predictive models of embryonic aneuploidy and mosaicism were developed. Participants/materials, setting, methods The main indications for PGT-A were advanced maternal age, abnormal sperm FISH and recurrent miscarriage or implantation failure. Embryo analysis were performed using Veriseq-NGS (Illumina). The software used to carry out all the analysis was R (RStudio). The library used to implement the different algorithms was caret. In the machine learning models, 22 predictor variables were introduced, which can be classified into 4 categories: maternal, paternal, embryonic and those specific to the IVF cycle. Main results and the role of chance The different couple, embryo and stimulation cycle variables were recorded in a database (22 predictor variables). Two different predictive models were performed, one for aneuploidy and the other for mosaicism. The predictor variable was of multi-class type since it included the segmental and whole chromosome alteration categories. The dataframe were first preprocessed and the different classes to be predicted were balanced. A 80% of the data were used for training the model and 20% were reserved for further testing. The classification algorithms applied include multinomial regression, neural networks, support vector machines, neighborhood-based methods, classification trees, gradient boosting, ensemble methods, Bayesian and discriminant analysis-based methods. The algorithms were optimized by minimizing the Log_Loss that measures accuracy but penalizing misclassifications. The best predictive models were achieved with the XG-Boost and random forest algorithms. The AUC of the predictive model for aneuploidy was 80.8% (Log_Loss 1.028) and for mosaicism 84.1% (Log_Loss: 0.929). The best predictor variables of the models were maternal age, embryo quality, day of biopsy and whether or not the couple had a history of pregnancies with chromosomopathies. The male factor only played a relevant role in the mosaicism model but not in the aneuploidy model. Limitations, reasons for caution Although the predictive models obtained can be very useful to know the probabilities of achieving euploid embryos in an IVF cycle, increasing the sample size and including additional variables could improve the models and thus increase their predictive capacity. Wider implications of the findings Machine learning can be a very useful tool in reproductive medicine since it can allow the determination of factors associated with embryonic aneuploidies and mosaicism in order to establish a predictive model for both. To identify couples at risk of embryo aneuploidy/mosaicism could benefit them of the use of PGT-A. Trial registration number Not Applicable

P–560 Comparative analysis of non-invasive preimplantation genetic testing of aneuploidies (niPGT-A), PGT-A and IVF cycles without aneuploidy testing: preliminary results

Abstract Study question Can non-invasive preimplantation genetic testing of aneuploidies (niPGT-A) improve the clinical outcome in IVF patients after proper validation? Summary answer We demonstrate the usefulness of the embryonic cell-free DNA (cfDNA) in the blastocyst culture medium to select more objectively the blastocysts with higher implantation potential. What is known already One of the greatest challenges in IVF is accurately selecting viable embryos that are more likely to achieve healthy livebirths following embryo transfer. Trophectoderm (TE) biopsy and PGT-A provide a direct assessment of chromosome status and improve implantation and clinical pregnancy rates per transfer. A non-invasive alternative is to analyse embryonic cfDNA in the blastocyst culture medium. Previous studies have shown that cfDNA testing in culture medium of blastocysts on day 6 of development allows aneuploidy detection with high concordance rates compared to TE biopsy and inner cell mass (Rubio et al., 2020). Study design, size, duration Observational study of the clinical application of niPGT-A (July 2020-December 2020). The clinical application consisted in a first validation phase, comparing TE biopsies with cfDNA in the media of 28 blastocysts. And, in a second phase, niPGT-A was applied and the outcome of 13 single embryo transfers (SETs) compared to 13 PGT-A SETs and 130 IVF/ICSI SETs performed in a period of six months. In the three groups, women and donors age was ≤38 years. Participants/materials, setting, methods Embryos were cultured in a Geri incubator (Merck) up to day 4, and then individually cultured in 10µl drops of CCSS (Fujifilm) until day 6 in a bench-top K-system. At day 6, blastocysts were vitrified, and media collected in sterile PCR tubes after at least 40 hours in culture. After collection, media were immediately frozen and analyzed by NGS analysis in our reference laboratory (Igenomix, Spain). Deferred transfer was performed according to media results. Main results and the role of chance Before the first clinical cases, a validation of the protocol comparing the results of cfDNA with the TE biopsies of the same day–6 blastocyst was performed, and ploidy concordance rates were 87.5%. Similar results were found for niPGT-A and PGT-A in terms of aneuploidy results and in clinical outcomes. The percentages of informative results were 95% and 97% and the aneuploidy rates were 44% and 46%, for niPGT-A and PGT-A, respectively. Clinical pregnancy rates were in both groups of aneuploidy testing, 69.2%, with 8 ongoing pregnancies (61.5%) and 4 tested by prenatal screaning NACE. For untested embryos clinical pregnancy (57.7%) and ongoing pregnancy rates (48.5%) were lower than in the two groups of tested embryos (niPGT-A and PGT-A). In the niPGT-A cycles embryo transfer was performed according to media results and morphology. We did a secondary analysis of which blastocyst we would transfer, if only morphology is considered. We observed that if we only select the embryos by morphology, in 61.5% of the cases we would choose the same embryo than with niPGT-A, and in 30.4% of the cases we would transfer a blastocyst with an aneuploid medium. Limitations, reasons for caution Our results are encouraging but should be interpreted with caution due to the small sample size. Larger and randomized controlled trials are needed to verify and extend our findings in each group. Wider implications of the findings: We observed consistent results for niPGT-A compared to TE biopsies in our internal validation. These results endorse the clinical application of niPGT-A in the routine of the laboratory and can avoid the embryo manipulation also reducing the subjectivity when embryos are selected only by morphology. Trial registration number Sa–16552/19-EC:428

Survival analysis following enucleation for uveal melanoma.

To determine survival outcomes following enucleation for uveal melanoma. To compare these outcomes with the 8th edition AJCC classification and determine the influence of cytogenetics, using Fluorescent in situ Hybridisation (FISH), on survival. To determine whether failure to gain sufficient sample for cytogenetics using Fine Needle Aspiration Biopsy (FNAB) correlates with survival. All patients undergoing primary enucleation for uveal melanoma at Moorfields Eye Hospital between 2012 and 2015 were included. Clinical, pathological, cytological and survival data were analysed for all patients. In total, 155 subjects were included. Mean age at enucleation was 65.9 years (SD 14.13). 88 (56.8%) patients died at a mean of three (SD 1.9) years following enucleation. Of these, 52 (33.5%) died from metastatic melanoma, 16 (10.3%) from other causes and 20 (12.9%) causes of death were unknown. Cumulative incidence analysis demonstrated AJCC grade, chromosome 8q gain and monosomy three all predict metastatic mortality. The greatest 5-year mortality rate (62%, SD10.1%) was in those with both chromosome abnormalities and AJCC stage III (Stage IV patients excluded due to low numbers). Largest basal diameter and chromosome status, both independently (p = 0.02 and p < 0.001) predicted metastatic mortality on multivariable regression analysis. Those who had an insufficient sample of cells gained during FNAB (n = 16) had no different prognosis. This study confirms, in this population, the poor survival of patients enucleated for uveal melanomas. It confirms the prognostic utility of adding AJCC grade to cytogenetic information. It demonstrates that the lack of sample in patients undergoing FNAB is not related to prognosis.

Evaluating prognostic value and relevant gene signatures of tumor microenvironment characterization in esophageal carcinoma.

Tumor microenvironment (TME) cells are an important part of tumor tissues. There is increasing evidence that the TME plays a vital role in tumor prognosis, and is associated with patient survival in various kinds of malignances. To date, very little research has been conducted on how to effectively use TME to better evaluate the prognosis of patients with esophageal carcinoma (EC). The concept of a "TME score" was introduced to better distinguish the prognosis of patients. We employed bioinformatic methods to investigate the TME infiltration patterns of 160 patients with EC from the Cancer Genome Atlas (TCGA) cohort. TME clusters were identified using k-means clustering methods with 1,000 resampling times. The significance of the survival difference among patients belonging to different TME clusters was assessed by the log-rank test and Kaplan-Meier survival curves. Correlations between immune cell types and survival were calculated by a Cox regression, and the Pearson correlation coefficient (PCC) was used to measure the relationship among different immune cell types. We classified patient into 2 subtypes based on the optimal breakpoint of TME score determined by R package maxstat. Two TME phenotypes were defined based on the immune cell type fractions, and patients with a high TME score phenotype had a better prognosis than those with a low TME score phenotype. Kaplan-Meier analysis for differentially expressed micro ribonucleic acids (RNAs) and messenger RNAs also showed that different TME score subtypes were significantly associated with the prognosis of EC. Just as tumor mutational burden can predict the efficacy of immunotherapy, the TME score can predict the efficacy of immune checkpoint inhibitors (ICIs). The genomic alterations of 2 TME score subtypes of EC further revealed that genomic instability is prevalent in TMEs, and patients with a low TME score subtype have a more unstable chromosome status than those with a high subtype. Thus, TME score is an emerging prognostic biomarker for predicting the efficacy of ICIs.

Chromosome status and yield characteristics of soybean (Glycine max L. (Merr)) in saline soil as affected by induced mutation

To fulfill the demand of soybeans, the expansion of the planting area can be one of the choices through the conversion of marginal land by utilizing suboptimal land, such as saline soil. One of the plant breeding techniques to obtain tolerant plants to salinity is the use of chemical mutations, such as colchicine. The study aimed to reveal the effect of salinity on the characters of soybean plants (mutated with colchicine) and the effect of colchicine on the number of chromosomes and ploidy level. This study was conducted from January to March 2020 and carried out in the plastic house of Faculty of Agriculture, Universitas Sumatera Utara. The experiment was arranged in a Randomized Block Design with salinity levels (0 dS.m-1, 2 dS.m-1, 4 dS.m-1, 6 dS.m-1) as treatments and consisted of ten replications in each treatment. The result showed that the soybean plants were not successfully induced by colchicine at a concentration of 0.04 % with soaking duration of 10 hours, as indicated by the number of chromosomes and analysis of ploidy level. The soybeans that had been induced were planted in saline soil. There were significant effects observed on the productive branch, number of filled pods, and the weight of seeds per plant. Colchicine treatment did not successfully affect the number of chromosomes of soybeans but had an impact on the chromosome pattern. In addition, the salinity treatment of 4 dS.m-1 significantly affected the number of filled pods compared to the treatment of 6 dS.m-1.

Blastomere movement correlates with ploidy and mosaicism in early-stage human embryos after in vitro fertilization.

Embryos undergo chaotic division and decrease in quality on day 3 with a reduction in the rates of subsequent blastocyst formation. Disordered cleavage causes a deterioration in embryonic quality, here we assessed the relationship between an cleavage model in first mitosis and the chromosomal status of human embryos, and discuss the potential biological and clinical implications for the cleavage model as a single parameter that can be used to assess embryonic quality. Thirty-two infertile couples, with normal karyotypes and who underwent their first IVF cycle were recruited to donate one normal two-cell-stage embryo each for this study between 2019 and 2020. Twenty-eight two-cell embryos underwent preimplantation genetic testing of each blastomere, and four chaotic-division embryos were stained with Hoechst and cultured in a confocal laser-scanning microscopy incubator system. This system showed high specificity and PPV but low sensitivity and NPV using the CM in the prediction of euploidy, indicating that CM could be considered a screening method for embryo selection; additional observational studies using the CM to select transferable embryos are needed before it can be used in clinical practice.

Abstract 2367: Role of miR-34a-MET axis in head and neck cancer squamous cell carcinoma

Abstract MicroRNAs (miR) are evolutionarily highly conserved non-coding RNAs that play roles in fundamental cellular functions by post-translational gene regulation. In cancer pathogenesis, miRs exert both anti-tumorigenic and pro-tumorigenic effects by virtue of miR-specific and context-dependent mechanisms. The role of chromosomal changes and mutation status on miR expression in cancer remains unknown. Here we provide evidence that chromosome arm 1p loss and P53 mutations are associated with lower levels of miR-34a, particularly in squamous cancers such as head and neck squamous cell carcinoma (HNSCC). miR-34a acts as a tumor suppressor and physically interacts with and functionally targets the proto-oncogene MET, and modulates cell proliferation. miR-34a expression is inversely correlated with MET in tumor tissue. We found that miR-34a suppresses HNSCC carcinogenesis, at least in part, by downregulating MET, consequently inhibiting HNSCC proliferation. Moreover, ectopic expression of miR-34a reduces HNSCC cell proliferation and tumor burden in vitro and in vivo, represses expression of genes and proteins involved in epithelial-mesenchymal transition, and negates the oncogenic effect of MET in mouse tumors. Our work demonstrates a role of miR-34a in maintaining tumor immunity, as overexpression of miR-34a resulted in a decease in percentage of anti-inflammatory (M2-like) TAMs and PDL1+TAMs, which are reported to have pro-tumorigenic activity. In HNSCC patient samples, higher levels of miR-34a are significantly associated with a higher frequency of Th1 cells, myeloid cells, and regulatory T cells. Our results demonstrate that miR-34a directly targets MET and maintains anti-tumor immune activity, and could potentially represent a new therapeutic approach for HNSCC. Citation Format: Xun Wu, Yi-Shing Lisa Cheng, Methew Matthen, Angela Yoon, Gary Schwartz, Shashi Bala, Alison M. Taylor, Fatemeh Momen-Heravi. Role of miR-34a-MET axis in head and neck cancer squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2367.

The Presence of Philadelphia Chromosome Does Not Confer Poor Prognosis in Adult Pre-B Acute Lymphoblastic Leukemia in the Tyrosine Kinase Inhibitor Era - a Surveillance, Epidemiology, and End Results Database Analysis

Translocation between chromosome 9 and 22 results in a BCR-ABL1 fusion gene identified in the form of an abnormal chromosome 22 (Philadelphia chromosome or Ph) in cytogenetics and/or other molecular studies. Ph+ acute lymphoblastic leukemia (ALL) constitutes about 25% of patients with pre-B ALL with increasing incidence with age. Prior to the introduction of tyrosine kinase inhibitors (TKI), presence of Ph chromosome conferred a poor prognosis in patients with ALL. Based on post-hoc comparisons between prospective clinical trials conducted before and after the introduction of this class of drugs, it appears that the use of TKIs inhibiting BCR-ABL have significantly improved the survival in Ph+ ALL. However, there has been no large-scale population based study comparing the survival of Ph+ ALL with Philadelphia negative (Ph-) ALL in the period when TKIs were widely used for treating Ph+ ALL. This information is highly relevant because survival of patients on clinical trials often exceeds results seen in real world. This could be due to study exclusion criteria, disparities in the number of elderly patients who receive TKI therapy, omission of patients with CNS involvement, etc. Thus the apparent improvement in outcomes has awaited confirmation at the population level. We compared the survival of Ph + and Ph-ALL during the period when TKIs were universally available in the US for Ph+ ALL, using a Surveillance, Epidemiology, and End Results (SEER) Database analysis. Patients with pre-B ALL ≥18 years in age, diagnosed between 2010 and 2014 were identified in SEER registries. 2694 patients with pre-B ALL were identified (206 Ph+ALL and 2488 Ph-ALL). The median OS was 32 months in Ph+ALL (95% CI 18 months-NE) and 27 months (95% CI 24-30 months) in Ph-ALL (Log-rank test p-value 0.34). To evaluate the progress in the survival of Ph+ ALL compared to Ph- ALL with time, the OS was estimated separately for patients diagnosed between 2010 - 2012 and 2013 - 2014 (Figure 3B and 3C). For 2010-2012, there was no significant difference (Log-rank p-value = 0.86) in the OS between Ph+ ALL (median OS = 24.5 months, 95% CI = 17.0-43.0) and Ph-ALL (median OS= 27, 95% CI = 23.0 - 30.0). For patients diagnosed between 2013 - 2014 period, there was a trend towards better survival for patients diagnosed with Ph+ ALL (1-year survival rate: 78.8%, 95% CI= 67.1%-86.7%) compared to Ph- (1-year survival rate: 69.2%, 95% CI= 65.5%-72.6%), although the difference was not statistically significant (Log-rank p-value = 0.094). Older age was associated with worse prognosis in both Ph+ALL and Ph-ALL. Age-adjusted OS was inferior in Hispanics and African-Americans compared to non-Hispanic whites. Survival of pre-B ALL shows continued improvement with time. Philadelphia chromosome status does not confer poor prognosis in pre-B ALL in the TKI era. Prognostic factors in pre-B ALL should be re-evaluated in the light of this finding. [Display omitted] DisclosuresKelly:Amgen: Honoraria; Abbvie: Honoraria; Jannsen: Honoraria; Pharmacyclics: Honoraria.

MDMX Is Widely Expressed in Adult Pre-Cursor B Cell B Acute Lymphoblastic Leukemia and Is a Potential Therapeutic Target.

MDMX is a human homolog of murine double minute 2 (HDM2), which like HDM2, is a negative regulator of p53. In pediatric precursor B-cell (pre-B) acute lymphoblastic leukemia (ALL), p53 expression is associated with poor prognosis. In this study, we assessed MDMX, HDM2, p53 and p21 levels in bone marrow samples from 55 adult patients with pre-B ALL diagnosis. A 110-core tissue microrray was subject to immunohistochemical analysis; in which 10% or more tumor cells stained were considered positive. Results were compared with Philadelphia chromosome (Ph) status (37 Ph-, 18 Ph+) and clinical data. MDMX was found to be expressed in 39 of 49 cases (80%), whereas HDM2 was expressed in 14 of 54 cases (26%). All 14 cases positive for HDM2 were also positive for MDMX (P < 0.05). Median survival duration was 138 weeks in the Ph- group and 48 weeks in the Ph+ group (P < 0.001). p53 was expressed more often in the Ph+ group than in Ph- group (10 of 17 versus 10 of 36, P < 0.05), as was p21 (11 of 17 versus 6 of 34, P < 0.003). In the Ph+ group, patients with tumor cells positive for p53, HDM2, and MDMX had a worse prognosis than other patients (35 weeks versus 61 weeks, P < 0.002). Western blot analysis of protein extracts of blasts isolated from patients with pre-B ALL confirmed the expression of MDMX or its variants. Furthermore, only 1 of 5 cases demonstrated missense mutation of p53 genes. Thus MDMX was expressed in most of these cases of pre-B ALL, suggesting that MDMX may be a potential therapeutic target. The higher incidence of MDMX expression seemed to be independent of p53. P21 expression by the blasts of a subset of ALL cases indicated that the p53 pathway was functional.

Consistency between chromosomal status analysis of biopsied human blastocyst trophectoderm cells and whole blastocyst cells.

PurposeThis study investigated the consistency between results of preimplantation genetic testing for aneuploidy performed on trophectoderm (TE) cells and remaining blastocyst cells.MethodsTE biopsy was performed on 29 surplus cryopreserved human blastocysts. Biopsy samples and remaining blastocysts were processed using the VeriSeq PGS kit, and chromosomal statuses were compared by next‐generation sequencing.ResultsDiscordance was observed in the chromosomal status of 11 out of 29 blastocysts between the biopsied TE and remaining blastocysts. Concordance was observed in 11 of 12 blastocysts classified as euploid by TE biopsy and in 7 of 17 blastocysts classified as aneuploid. There was 100% concordance (7/7) in cases diagnosed as aneuploid with no mosaicism by TE biopsy. However, discordance was observed in all 10 cases showing mosaicism or partial chromosomal abnormality.ConclusionChromosomal status analysis based on TE biopsy does not accurately reflect the chromosomal status of the whole blastocyst. The chromosomal status is usually the same between the TE and remaining blastocyst cells in cases diagnosed as euploid or aneuploid with no mosaicism. However, mosaic blastocysts and those with other types of structural rearrangements have a higher risk of inconsistency, warranting caution during embryo selection.

A Chinese multicenter retrospective study of isolated increased nuchal translucency associated chromosome anomaly and prenatal diagnostic suggestions

Extensive researches involving fetuses with multiple ultrasound anomalies have been conducted over the years, but only few were focused on the isolated increased nuchal translucency (NT). On top of that, these limited number of researches were all designed as single-arm studies and the control group was missing. In this study, we conducted a multicenter, retrospective study using amniotic fluid samples collected from 1197 pregnant women having fetuses with isolated increased NT (INT group) or normal NT values (NNT group). Copy number variation sequencing (CNV-seq) was performed to determine their chromosome status and pathogenic variations were validated using SNP array. Overall, 59 chromosome aneuploidies, 34 pathogenic CNVs and 23 copy number variants of unknown significance (VOUS CNVs) were discovered. the INT group had a significantly higher proportion of aneuploidy (19.44%) and pathogenic CNV (8.33%) than the control group (3.49% and 2.30% respectively), and 88.89% of the pathogenic CNVs were related to heart defects. Additionally, more male fetuses were presented in the INT group (68.51%), but they did not have a higher risk (Relative Risk = 1.03) of carrying pathogenic chromosome variations than female fetuses. Our results demonstrated that fetuses with isolated increased NT had a distinct pattern of chromosome abnormality and majority of detected pathogenic CNVs could be linked to the congenital heart disease. Furthermore, because a considerable proportion of pathogenic CNVs were detected, we strongly recommend to perform a joint test of karyotyping and CNV analysis in prenatal diagnosis for fetuses with isolated increased NT in order to decrease the incident of missed diagnosis.

Down-regulation of the tumor suppressor miR-34a contributes to head and neck cancer by up-regulating the MET oncogene and modulating tumor immune evasion

BackgroundMicroRNAs (miRs) have been shown to play an important role in tumorigenesis, including in head and neck squamous cell carcinoma (HNSCC). The miR-34 family is thought to play a role in tumor suppression, but the exact mechanism of their action in HNSCC is not well understood. Moreover, the impact of chromosomal changes and mutation status on miR-34a expression remains unknown.MethodsDifferential expression of miR-34a, MET, and genomic alterations were assessed in the Cancer Genome Atlas (TCGA) datasets as well as in primary HNSCC and adjacent normal tissue. The biological functions of miR-34a in HNSCC were investigated in samples derived from primary human tumors and HNSCC cell lines. The expression of MET was evaluated using immunohistochemistry, and the molecular interaction of miR-34a and MET were demonstrated by RNA pulldown, RNA immunoprecipitation, luciferase reporter assay, and rescue experiments. Lastly, locked nucleic acid (LNA) miRs in mouse xenograft models were used to evaluate the clinical relevance of miR-34a in HNSCC tumor growth and modulation of the tumor microenvironment in vivo.ResultsChromosome arm 1p loss and P53 mutations are both associated with lower levels of miR-34a. In HNSCC, miR-34a acts as a tumor suppressor and physically interacts with and functionally targets the proto-oncogene MET. Our studies found that miR-34a suppresses HNSCC carcinogenesis, at least in part, by downregulating MET, consequently inhibiting HNSCC proliferation. Consistent with these findings, administration of LNA-miR-34a in an in vivo model of HNSCC leads to diminished HNSCC cell proliferation and tumor burden in vitro and in vivo, represses expression of genes involved in epithelial-mesenchymal transition, and negates the oncogenic effect of MET in mouse tumors. Consistently, LNA-miR-34a induced a decreased number of immunosuppressive PDL1-expressing tumor-associated macrophages in the tumor microenvironment. In HNSCC patient samples, higher levels of miR-34a are significantly associated with a higher frequency of Th1 cells and CD8 naïve T cells.ConclusionsOur results demonstrate that miR-34a directly targets MET and maintains anti-tumor immune activity. We propose miR-34a as a potential new therapeutic approach for HNSCC.

Cell-free DNA in spent culture medium effectively reflects the chromosomal status of embryos following culturing beyond implantation compared to trophectoderm biopsy.

This prospective study evaluated the accuracy of non-invasive preimplantation genetic testing for aneuploidy (niPGT-A) using cell-free DNA in spent culture medium, as well as that of preimplantation genetic testing for aneuploidy (PGT-A) using trophectoderm (TE) biopsy after culturing beyond implantation. Twenty frozen blastocysts donated by 12 patients who underwent IVF at our institution were investigated. Of these, 10 were frozen on day 5 and 10 on day 6. Spent culture medium and TE cells were collected from each blastocyst after thawing, and the embryos were cultured in vitro for up to 10 days. The outgrowths after culturing beyond implantation were sampled and subjected to chromosome analysis using next-generation sequencing. Chromosomal concordance rate, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), false-positive rate (FPR), and false-negative rate (FNR) of niPGT-A and PGT-A against each outgrowth were analyzed. The concordance rate between the niPGT-A and outgrowth samples was 9/16 (56.3%), and the concordance rate between the PGT-A and outgrowth samples was 7/16 (43.8%). NiPGT-A exhibited 100% sensitivity, 87.5% specificity, 88.9% PPV, 100% NPV, 12.5% FPR, and 0% FNR. PGT-A exhibited 87.5% sensitivity, 77.8% specificity, 87.5% PPV, 75% NPV, 14.3% FPR, and 22.2% FNR. NiPGT-A may be more accurate than PGT-A in terms of ploidy diagnostic accuracy in outgrowths.

Outcomes after first relapse of childhood intracranial ependymoma.

Ependymoma is the third most common malignant CNS tumor in children. Despite multimodal therapy, prognosis of relapsed ependymoma remains poor. Approaches to therapy for relapsed ependymoma are varied. We present a single-institution retrospective review of the outcomes after first relapse of intracranial ependymoma in children. We performed a retrospective, IRB-approved chart review of patients with recurrent intracranial ependymoma treated at Dana-Farber/Boston Children's Cancer and Blood Disorders Center from 1990 to 2019. Thirty-four patients with relapsed intracranial ependymoma were identified. At initial diagnosis, 11 patients had supratentorial disease, 22 with posterior fossa disease and one with metastatic disease. Median time-to-first relapse was 14.9months from initial diagnosis (range 1.4-52.5). Seven patients had metastatic disease at first relapse. Gross total resection (GTR) was associated with improved 5-year progression-free survival (PFS) relative to subtotal resection (STR) and no surgery (p=.005). Localized disease at relapse was associated with improved 5-year overall survival (OS) when compared to metastatic disease (p=.02). Irradiation at first relapse seemed to delay progression but was not associated with statistically prolonged PFS or OS. Tumor location, histology, and chromosomal 1q status did not impact outcome at first relapse, although available molecular data were limited making definitive conclusions difficult. Median time-to-second relapse was 10months (range 0.7-124). Five-year PFS and OS after first relapse were 19.9% and 45.1%, respectively. Median PFS and OS were 10.0 and 52.5months after first relapse, respectively. Relapsed intracranial ependymoma has a poor prognosis despite multimodal therapy. Novel therapeutic strategies are desperately needed for this disease.