Chromosomal Status Research Articles

Comparative analysis of reduced toxicity conditioning regimens between fludarabine plus melphalan and fludarabine plus busulfex in adult patients with acute lymphoblastic leukemia.

Reduced-toxicity conditioning (RTC) regimens aim to mitigate regimen-related toxicity while maintaining anti-leukemic efficacy in allogeneic hematopoietic stem cell transplantation (allo-HSCT). We assessed outcomes of RTC regimens utilizing melphalan versus intravenous busulfan combined with fludarabine in adult acute lymphoblastic leukemia (ALL) patients. A retrospective analysis was conducted with 149 consecutive adult ALL patients (median age 51, range 18-60) in remission undergoing allo-HSCT. Patients received either fludarabine 150 mg/BSA plus 2 days of melphalan 70 mg/BSA (FM140, n = 76) from 2009 to 2015 or fludarabine plus 3 days of busulfan 3.2 mg/kg (FB9.6, n = 73) from 2016 to 2021. At 5 years post-HSCT, FM140 demonstrated superior disease-free survival (53.4% vs. 30.5%, p = 0.007) and lower cumulative relapse (27.4% vs. 46.8%, p = 0.026) than FB9.6. Five-year overall survival and non-relapse mortality did not significantly differ. FM140 exhibited a higher incidence of acute graft-versus-host disease (GVHD) grades II-IV (49.3% vs. 30.3%, p = 0.016), though rates of acute GVHD grades III-IV and chronic GVHD were similar. Multivariate analysis identified Philadelphia chromosome and minimal residual disease positive status, and FB9.6 conditioning as predictors of increased relapse and poorer disease-free survival. FM140 RTC regimen displayed significantly reduced relapse and superior disease-free survival compared to FB9.6 in ALL patients undergoing allo-HSCT, highlighting its current clinical utility.

Modeling X chromosome inactivation using t5iLA naive human pluripotent stem cells

BackgroundX chromosome inactivation (XCI) is a critical epigenetic event for dosage compensation of X-linked genes in female mammals, ensuring developmental stability. A robust in vitro model is required for mimicking XCI during the early stages of embryonic development. This methodology article introduces an advanced framework for the in-depth study of XCI using human pluripotent stem cells (hPSCs). By focusing on the transition between naive and primed pluripotent states, we highlight the role of long non-coding RNA X-inactive specific transcript (XIST) and epigenetic alterations in mediating XCI.ResultsOur methodology enables the distinction between naive and primed hESCs based on XIST expression and the activity of X-linked reporters, facilitating the investigation of XCI initiation and maintenance. Through detailed experimental procedures, we demonstrate the utility of our hESC lines in modeling the process of human XCI, including the establishment of conditions for random XCI induction and the analysis of X chromosome reactivation.MethodsThe study outlines a comprehensive approach for characterizing the X chromosome status in hPSCs, employing dual fluorescent reporter hESC lines. These reporter lines enable real-time tracking of XCI dynamics through differentiation processes. We detailed protocols for the induction of X chromosome reactivation and inactivation, as well as the X status characterization methods including cultivation of hESCs, flow cytometric analysis, RNA fluorescence in situ hybridization (FISH), and transcriptome sequencing, providing a step-by-step guide for researchers to investigate XCI mechanisms in vitro.ConclusionsThis article provides a detailed, reproducible methodology for studying XCI mechanisms in vitro, employing hPSCs as a model system. It presents a significant advance in our ability to investigate XCI, offering potential applications in developmental biology, disease modeling, and regenerative medicine. By facilitating the study of XCI dynamics, this methodological framework paves the way for deeper understanding and manipulation of this fundamental biological process.

Survival in Patients with Uveal Melanoma Is Linked to Genetic Variation at HERC2 Single Nucleotide Polymorphism rs12913832

Uveal melanoma (UM) is a rare disease, with the highest incidence in people with fair skin and light eyes. Eye color is largely genetically determined and is defined by a set of single nucleotide polymorphisms (SNPs). We set out to determine whether we could identify a SNP related to prognosis. We sequenced DNA from peripheral blood mononuclear cells of 392 patients with UM and obtained the genotype of 6 common eye color-related SNPs. Clinical and histopathologic tumor characteristics, tumor chromosome status, and patient survival were compared among patients with different genotypes. Three hundred ninety-two patients who underwent enucleation for UM at the Leiden University Medical Center, Leiden, The Netherlands. We isolated DNA from peripheral blood leukocytes of 392 patients with UM and performed sequencing, using 6 eye color SNPs from the HIrisPlex-S assay (Erasmus MC, Walsh lab). The genotypes extracted from the sequencing data were uploaded onto the HIrisPlexwebtool (https://hirisplex.erasmusmc.nl/) for eye color prediction. We tested the association of eye color SNPs with tumor characteristics and chromosome aberrations using Pearson's chi-square test and the Mann-Whitney U test and evaluated survival with Kaplan-Meier curves with the log-rank test and Cox regression. Uveal melanoma-related survival. Of 392 patients with analyzable genotype data, 307 patients (78%) were assigned blue eyes, 74 patients (19%) were assigned brown eyes, and 11 patients (3%) could not be assigned to either blue or brown. Patients with a genetically blue eye color showed worse survival (P= 0.04). This was related to 1 genotype: patients with the G/G genotype of rs12913832 (HERC2), which codes for blue eye color showed a worse prognosis (P= 0.017) and more often had high-risk tumors (monosomy of chromosome 3; P= 0.04) than in patients with an A/G or A/A genotype. The G/G genotype of rs12913832 (HERC2), which is related to blue eye color, not only is a genetic factor related to the risk of UM develop, but also is linked to a worse prognosis because of an association with a higher risk of a high-risk UM developing (carrying monosomy of chromosome 3). The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Preconception carrier screening and preimplantation genetic testing in the infertility management.

Genetic testing serves as a valuable element of reproductive care, applicable at various stages of the reproductive journey: (i) before pregnancy, when a couple's genetic reproductive risk can be evaluated; (ii) before embryo implantation, as part of in vitro fertilization (IVF) treatment, to ascertain several inherited or de novo genetic/chromosomal diseases ofthe embryo before transfer; (iii) during the prenatal period, to assess the genetic costitution of the fetus. Preconception carrier screening (CS) is a genetic test typically performed on couples planning a pregnancy. The primary purpose of CS is to identify couples at-risk of conceiving a child affected by a severe genetic disorder with autosomal recessive or X-linked inheritance. Detection of high reproductive risk through CS allows prospective parents to be informed of their predisposition and improve reproductive decision-making. These include undergoing IVF with preimplantation genetic testing (PGT) or donor gametes, prenatal diagnosis, adoption, remaining childless, taking no actions. Both the presence of the affected gene (PGT-M) and chromosomal status (PGT-A) of the embryo can be comprehensively assessed through modern approaches. We provide a review of CS and PGT applications to equip healthcare providers with up-to-date information regarding their opportunities and complexities. The use of CS and PGT is currently considered the most effective intervention for avoiding both an affected pregnancy whilst using autologous gametes in couples with known increased risk, and chromosomal abnormalities. As our understanding in the genetic component in pathological conditions increases, the number of tested disorders will expand, offering a more thorough assessment of one's genetic inheritance. Nevertheless, implementation and development in this field must be accompanied by scientific and ethical considerations to ensure this approach serves the best long-term interests of individuals and society, promoting justice and autonomy and preserving parenthood and the healthcare system. The combination of CS and PGT aligns with principles of personalized medicine by offering reproductive care tailored to the individual's genetic makeup.

Establishment and transcriptome analysis of single blastomere-derived cell lines from zebrafish

Maintaining chromosome euploidy in zebrafish embryonic cells is challenging because of the degradation of genomic integrity during cell passaging. In this study, we report the derivation of zebrafish cell lines from single blastomeres. These cell lines have a stable chromosome status attributed to BMP4 and exhibit continuous proliferation in vitro. Twenty zebrafish cell lines are successfully established from single blastomeres. Single-cell transcriptome sequencing analysis confirms the fidelity of gene expression profiles throughout long-term culturing of at least 45 passages. The long-term cultured cells are specialized into epithelial cells, exhibiting similar expression patterns validated by integrative transcriptomic analysis. Overall, this work provides a protocol for establishing zebrafish cell lines from single blastomeres, which can serve as valuable tools for in vitro investigations of epithelial cell dynamics in terms of life–death balance and cell fate determination during normal homeostasis.

O-272 Preimplantation genetic testing of the first polar body is useful for predicting embryo euploidy: data from 1,836 donor oocytes

Abstract Study question Can preimplantation genetic testing of the first polar body (PGT-1BP) accurately identify donor oocytes that will produce euploid embryos? Summary answer Oocyte chromosomal status determined through PGT-1BP had a strong association with embryo euploidy. What is known already Aberrant meiosis during oocyte development leads to aneuploidy, diminished embryo quality, and ultimately, poor reproductive outcomes. Preimplantation genetic testing for aneuploidies (PGT-A) is increasingly employed in donor oocyte cycles, detecting blastocyst euploidy rates ranging from 30% to 80%. However, this variability, together with the clinical and patient resources required to generate blastocysts, support the need for alternative approaches. Oocyte chromosomal status can safely be determined with PGT-1PB. This strategy has shown that 20–30% oocytes obtained from donors with proven fertility are aneuploid, allowing clinicians and patients to make informed reproductive decisions. Study design, size, duration In this prospective study, PGT-1BP was performed on 1,836 fresh donor oocytes collected between September 2017 and January 2022. Vitrified-thawed euploid oocytes were grouped according to donor age (n = 714, n = 554, and n = 568 from 20–24, 25–29, and 30–34-year-old donors, respectively). Oocyte survival and chromosomal status, fertilization through ICSI, blastocyst use and euploidy status were analyzed. Participants/materials, setting, methods Oocytes were derived from donors (n = 115) whose oocytes previously led to at least one live birth. First polar body biopsies were performed 2 hours after in vitro maturation, which corresponds to 38 hours post-hCG trigger. PGT-1BP and PGT-A analyses were performed on the Illumina platform. Oocyte and embryology data as well as the association between oocyte and embryo chromosomal status was evaluated using Mann-Whitney U test. Main results and the role of chance The post-thawing viability rates for the biopsied oocytes were 98.2%, 99.5%, and 96.5% for the three age groups, respectively. Donor oocyte euploidy significantly decreased with advancing age (81.9% vs. 76.9% vs. 70.0%, respectively; p ≤ 0.05). The rate of blastocyst use was comparable across age groups (68.1% vs. 71.7% vs. 68.7%, respectively; p > 0.05). Blastocysts derived from euploid oocytes maintained their euploid status in 88.7%, 90.1%, and 85.6% of cases, respectively. Euploid oocyte-euploid blastocyst matching rates were 89.2%, 90.1%, and 88.1%. Limitations, reasons for caution Although PGT-1BP may not guarantee euploid embryos, it could allow clinicians and couples to make informed decisions about proceeding with fertilization and embryo culture. The associations between euploidy rate of embryos derived from euploid donor oocytes should be confirmed in further studies with larger cohorts. Wider implications of the findings Selecting euploid oocytes with a high probability of becoming euploid embryos may increase the odds of achieving a successful pregnancy at the first attempt. This approach is cost-effective, could prevent unnecessary emotional distress for patients, and is an attractive option for couples with limited sperm available for IVF/ICSI. Trial registration number not applicable

O-314 Investigating the embryonic contribution to recurrent pregnancy loss: a retrospective analysis of embryo quality, chromosomal status and morphokinetics in 11,169 embryos

Abstract Study question What is the embryonic contribution, in terms of embryo quality, chromosomal status and morphokinetics, to the reproductive success of recurrent pregnancy loss (RPL) patients? Summary answer RPL patients undergoing PGT-A had significantly fewer embryos available for biopsy, higher aneuploidy rates and showed morphokinetic alterations around the time of blastulation. What is known already RPL is diagnosed following the loss of two or more pregnancies before the 24th week of gestation. RPL has a multifactorial etiology encompassing both maternal and embryonic factors, such as uterine malformations, the presence of anti-phospholipid antibodies, maternal obesity or embryo aneuploidy, among others. While some studies have shown increased rates of chromosomal abnormalities in blastocysts from idiopathic RPL patients, a comprehensive characterization of the embryonic contribution to the disorder remains limited, and the role of embryo morphokinetics is still underexplored. Study design, size, duration This retrospective cohort study analyzed 11,169 embryos from 3,005 PGT-A cycles performed across 9 European fertility clinics between January 2017 and July 2023. Differences in the number of biopsied embryos, aneuploidy rates and embryo quality were compared between RPL and non-RPL patients. Morphokinetic timings were evaluated in a subset of 704 RPL embryos from 2 fertility centers, which was then compared to a separate cohort of embryos (n = 952) derived from double gamete donation cycles (n = 134). Participants/materials, setting, methods Genetic and clinical outcomes were assessed by multivariate analyses adjusting for maternal age, paternal age, the presence of male factor and embryo grade, as appropriate. Kaplan-Meier survival curves were constructed for each morphokinetic parameter (tPNf, t2, t3, t4, t5, t8, tSB and tB) and analyzed by Cox regression using maternal age and male factor as adjusting variables. P-values <0.05 were considered statistically significant. Main results and the role of chance RPL patients undergoing PGT-A represented 15.47% (n = 465/3,005) of the cohort and were significantly younger than the rest of the patients (34,97±6.6 vs 36.38±6.6, p < 0.0001). RPL patients obtained significantly fewer blastocysts available for biopsy (coefficient -0.39, 95%CI: -0.703- -0.076, p = 0.01) and had significantly higher aneuploidy rates (OR = 1.19, 95%CI: 1.06-1.35, p = 0.004). However, embryo quality (excellent vs. rest, p > 0.05) was comparable between the two groups (n = 11,169 embryos). As anticipated, biochemical and clinical pregnancy rates were comparable between RPL patients and non-RPL patients (OR = 0.94, 95%CI:0.72-1.24, p = 0.68; OR = 0.95, 95%CI: 0.72-1.24, p = 0.71, respectively), however patients with RPL presented with significantly higher miscarriage rates (OR = 1.66, 95% CI:1.03-2.74, p = 0.04). Compared to double donation embryos, RPL embryos exhibited significant delays in all analyzed times (tPNf: 23.7±3.1 vs. 22.7±3.1; t2: 26.5±4.1 vs. 25.6±3.4; t3: 37.1±5.4 vs. 36.0±4.7; t4: 39.2±6.3 vs. 37.9±5.3; t5: 50.2±7.9 vs. 49.3±7.4; tsB: 99.4±10.3 vs. 97.9±9.7; tB: 110.3±11.5 vs. 106.4±11.8; p < 0.05). Notably, delays in blastocyst formation were also observed when adjusting for maternal age and male factor (Hazard Ratio=0.38, 95% CI: 0.18-0.79, p = 0.009). Significantly, morphokinetic alterations persisted even when exclusively comparing euploid RPL embryos to the double donation group (n = 41, t8: Hazard Ratio=1.78, 95% CI: 1.22-2.57, p = 0.002; tsB: Hazard Ratio=1.70, 95% CI: 1.17-2.48, p = 0.006). Limitations, reasons for caution The main limitation of the study is its retrospective design, where known RPL risk factors leading to the selection of a PGT-A cycle were not considered, potentially introducing biases. Wider implications of the findings These findings highlight the intricate interplay between embryo quality, aneuploidy, and morphokinetics in RPL, guiding personalized patient management. Higher embryo aneuploidy and morphokinetic alterations observed even in euploid RPL embryos suggest an embryonic role, underscoring the condition’s multifactorial nature and urging exploration of molecular mechanisms linking embryonic factors to RPL. Trial registration number not applicable

P-530 Precise quantification of DNA in spent culture media sheds light on associations between DNA quantity, embryonic ploidy status and morphological quality

Abstract Study question Is it possible to accurately measure cell-free DNA (cfDNA) quantity in embryo spent culture media, and are amounts correlated with embryo morphological and/or chromosomal status? Summary answer A sensitive DNA quantification method was created, demonstrating that cfDNA quantity is not associated with embryo ploidy, but increases when embryos are of poor morphology. What is known already Preimplantation genetic testing involves embryo biopsy, necessitating specialist equipment and highly trained staff, increasing costs. Additionally, there have been concerns over potential embryo damage to the embryo resulting from biopsy. The discovery of cfDNA in medium in which embryos were cultured, led to suggestions that non-invasive PGT (niPGT) might be possible. However, the amount of cfDNA in media samples, and potential correlations with other aspects of embryo biology, require clarification. We aimed to provide the first accurate measurement of the amount of DNA in spent culture medium, providing insights into whether niPGT is likely to provide acceptable accuracy rates. Study design, size, duration This prospective analysis involved media samples from 151 fresh embryos, which were produced at a single IVF clinic over the course of one year. Embryos were cultured individually, with a medium change on day-3 of development. Trophectoderm biopsy was undertaken on day-5/-6, followed by PGT-A. Embryo morphology was assessed according to ASEBIR criteria (ranked A-C). Participants/materials, setting, methods The culture and biopsy protocols used were standard for the clinic. Media was collected prior to biopsy and underwent a novel quantitative whole genome amplification method, allowing precise measurement of DNA with respect to a standard curve generated using samples containing known amounts of DNA. PGT-A involved the use of a highly validated method, based upon next generation sequencing. Correlation between DNA quantity and embryo morphological quality was also studied. Data were analyzed using ANOVA. Main results and the role of chance There was no difference in the quantity of DNA in media samples associated with euploid and aneuploid embryos (mean DNA quantity and standard deviations were 1.72 ± 3.23 pg and 1.54 ± 3.66 pg for aneuploid and euploid embryos, respectively; p = 0.75). Overall, 91% of media samples contained less than the equivalent of one cell (<5pg of DNA) and almost two-thirds of samples (65%) contained less than 1pg of DNA. Given such low quantities of embryonic DNA, achieving accurate genetic testing is likely to be challenging. Drops that held embryos until day-6 had significantly higher levels of DNA than those associated with culture to day-5 (P < 0.0001), suggesting cfDNA accumulates with time. Thus, longer culture periods are likely to yield more DNA for analysis. Comparing the amount of cfDNA with morphological status revealed a significant association. Embryos of fair quality (C) tended to have higher quantities of DNA in spent culture medium, whereas top-quality embryos (classified as A or B) typically had less (mean and standard deviation of DNA quantity: 1.23 ± 3.14 pg for class A and B versus 2.60 ± 3.85 pg for class C; p < 0.05). This may reflect increased cell death and DNA release in poorer quality embryos. Limitations, reasons for caution This project is limited by its single-centre nature and relatively small sample size. A large-scale, multicentre study would be necessary to confirm these results. Wider implications of the findings It has been hypothesised that cfDNA quantity in culture medium may be predictive of embryo chromosomal status. We found no evidence to support this. Although DNA quantities were higher after culture to day-6, levels remained low, calling into question the ability of niPGT to deliver high accuracy diagnosis of embryos. Trial registration number not applicable

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-273 Image-based oocyte model predictive of blastocyst development correlates with ploidy status across a broad spectrum of PGT-A indications, accounting for confounding variables

Abstract Study question Does MAGENTA, an AI assessment of oocyte quality, indicate ploidy potential of the developed blastocysts in the presence of confounding variables? Summary answer MAGENTA correlates to oocytes that develop into euploid embryos across diverse demographics, highlighting its robust ability to assess oocyte quality and genetic potential. What is known already MAGENTA is a non-invasive AI-image analysis tool that assesses images of mature oocytes and provides a score from 0-10, where higher scores indicate higher chances of developing into a blastocyst. MAGENTA has also displayed a correlation to blastocyst morphological quality. The ploidy status of blastocysts plays a significant role in cycle success and is often utilized for the selection of embryos to transfer. Despite the selection of embryos based on their genetic normality, many transfers are unsuccessful. With most chromosomal errors arising in the oocyte, assessment at the gamete level is critical to improving outcomes. Study design, size, duration A retrospective study using CRM Weill Cornell Medicine data included 3,342 mature oocytes that developed into blastocysts and underwent PGT-A in 2021. Blastocysts tested as mosaic (n = 753) or inconclusive (n = 13) were excluded. Images of the oocytes were obtained immediately post-ICSI using EmbryoScope time-lapse incubators (Vitrolife, Sweden). Blastocyst morphological grades were categorized by quality: highest (ICM+TE grade A), high (ICM+TE grade A/B), medium (ICM+TE grade B), or low quality (ICM or TE grade C/D). Participants/materials, setting, methods Data was obtained from 629 patients (females 26-45 years old) with an average of 5.3 blastocysts (range 1-20) with PGT-A results per patient. MAGENTA assessed each oocyte image and provided a score (0-10), with higher scores representing a greater potential to develop into a blastocyst embryo. In this dataset, 1827 (55%) blastocysts were tested as euploid and 1515 (45%) were tested as aneuploid. PGT-A was conducted for various specific and non-specific indications in these patients. Main results and the role of chance MAGENTA was positively associated with oocytes that developed into blastocysts of increasing quality, with significance between highest and high-quality (7.4 vs 7.0,p<0.05) and medium and low-quality (6.9 vs 6.1,p<0.01). Of the blastocysts, the MAGENTA score further displayed significant differences between oocytes that developed into euploid (7.0) compared to aneuploid blastocysts (6.6) (p < 0.001;Welch’s t-test). Although MAGENTA does not predict ploidy, a threshold of 8.8 MAGENTA score was determined to best distinguish between ploidy outcomes with 61% euploidy rate above this threshold compared to 51% below, a significantly different proportion (p < 0.001;Two-Sample Proportions z-test). Therefore, oocytes scored ≥8.8 display a 19.6% relative increase in chance of developing into a euploid blastocyst. To account for female age, subgroup analysis was conducted and found MAGENTA scores to be significantly different between oocytes that developed into euploid and aneuploid blastocysts for patients <35 years old (7.2 vs 6.6, p < 0.01;Welch’s t-test) and ≥35 years old (6.8 vs. 6.5, p < 0.05;Welch’s t-test). Blastocyst morphological grade was also investigated as a confounding factor. No significant differences existed between MAGENTA scores of euploid and aneuploid blastocysts among the low, high, or highest-quality. Significantly, differences were only found within medium quality blastocysts (7.0 vs 6.6,p<0.05), which had the greatest sample size (n = 1854). Limitations, reasons for caution MAGENTA was not trained to predict euploid embryo development from images of mature oocytes. Further data is required to discern the correlation of MAGENTA to chromosomal ploidy status, apart from blastocyst quality. Additionally, investigation into sperm DNA fragmentation as a potential contributor to ploidy determination is warranted. Wider implications of the findings MAGENTA can identify oocytes of better quality that not only have higher chances of blastocyst development and higher morphological grades, but also indicates oocytes that are more likely to become euploid embryos across patients of various PGT-A indications and a broad age demographic. Trial registration number not applicable

O-004 Oocyte image assessment by novel artificial intelligence (AI) model predicts blastocyst PGT-A results, outperforming age as a predictor

Abstract Study question Can a non-invasive, image analysis artificial intelligence (AI) model be built to predict the likelihood that a mature oocyte will develop into a euploid blastocyst? Summary answer A novel AI model was developed to assess images of mature oocytes, predicting chromosomal ploidy status of the developed blastocyst with an AUC of 0.71. What is known already The genetic status of blastocysts greatly impacts implantation and cycle success; therefore, PGT-A has become widely adopted as routine to aid in blastocyst selection for transfer and aims to decrease the time to pregnancy. Most chromosomal abnormalities arising in a blastocyst embryo are of maternal meiotic origin–indicating that the oocyte may have valuable information to be harnessed at the earliest stage in the process. Gaining insights into the potential genetic status of a blastocyst at the oocyte stage in a non-invasive manner would provide critical information to the overall fertility cycle. Study design, size, duration This study included a labeled dataset—9,260 images of mature denuded oocytes, immediately post-ICSI—from 3 clinics (Canada, United States (US), Spain) with euploid or aneuploid trophectoderm biopsy results (excluding mosaic/inconclusive embryos, n = 980). The labeled dataset was split into training (60%), validation (20%), and test (20%). An unlabeled dataset was also included—34,709 oocyte images (2 clinics; Canada, US)—and utilized in training. An external dataset (n = 473) from a separate US clinic was assessed for model generalization. Participants/materials, setting, methods Fully-supervised and semi-supervised machine learning models were experimented to predict blastocyst PGT-A from oocyte images. The labeled data were utilized in both methods. The semi-supervised method integrates unlabeled data using the well-known Mixmatch algorithm—learning important features from augmented images (with pseudo labels) that were then utilized in the supervised phase to predict outcomes. Age was incorporated into the models, therefore, a model only using age to predict ploidy was developed to act as a comparison. Main results and the role of chance Following various experiments, the fully-supervised model displayed an AUC 0.61, sensitivity 0.41, specificity 0.76, and accuracy 0.62 on the test set. The semi-supervised model displayed a performance of AUC 0.71, sensitivity 0.63, specificity 0.67, and accuracy 0.65–notably higher AUC and balanced performance on the positive/negative classes. The semi-supervised method benefits from both labeled and augmented unlabeled data for enhanced learning efficacy. The model utilizing age only to predict ploidy was extremely imbalanced in performance on the test set with AUC 0.63, sensitivity 1, specificity 0, and accuracy 0.48, confirming the importance of image features. Performance of the semi-supervised model was assessed by clinic. On Clinic 1 (n = 364; 40% euploid) it displayed AUC 0.61, sensitivity 0.60, specificity 0.56, and accuracy 0.58; on Clinic 2 (n = 675; 52% euploid) it displayed AUC 0.64, sensitivity 0.56, specificity 0.65, and accuracy of 0.60; on Clinic 3 (n = 466; 50% euploid) it displayed significantly better performance with AUC 0.85, sensitivity 0.76, specificity 0.79, and accuracy 0.77–warranting further investigation into model generalizability. On the external dataset (n = 473, 58% euploid), the semi-supervised model displayed improved performance (AUC 0.62, sensitivity 0.61, specificity 0.61, accuracy 0.60) in comparison to the age only model (AUC 0.59, sensitivity 1, specificity 0, accuracy 0.58). Limitations, reasons for caution This dataset includes oocytes that developed into blastocysts and underwent PGT-A. Datasets including oocytes that did not reach the blastocyst stage must also be assessed to validate the model’s clinical utility. Despite utilizing diverse data, advancing the generalizability of the model requires additional investigations to ensure balanced performance across clinics. Wider implications of the findings Utilizing images of mature oocytes, an AI model predicts the ploidy status of resulting blastocysts, providing a powerful tool for assessing oocyte quality and genetic integrity. These preliminary findings are promising for those undergoing oocyte cryopreservation by striving to elucidate genetic potential at the earliest phase of the IVF process. Trial registration number Not applicable.

P-559 Pregnancy rates in conventional and non-invasive pre-implantation genetic testing – a multicentric retrospective cohort study

Abstract Study question To determine the pregnancy rates with embryo diagnosed as euploid by non-invasive preimplantation genetic testing (niPGT-A) and trophectoderm based pre-implantation genetic testing (cPGT-A) Summary answer Biochemical pregnancy rates are comparable in couples undergoing transfer with embryos diagnosed as euploid as judged by niPGT-A or cPGT-A What is known already NiPGT-A utilizes cell-free DNA in culture media to assess the chromosomal status of embryos. Several studies have shown that the niPGT-A and cPGT-A have a reasonable concordance rate and niPGT-A can be considered as an alternative in predicting the ploidy status of the embryos. However, limited information exist on the clinical outcomes of embryos deemed euploid by niPGT-A. Study design, size, duration Data of 1209 participants between June 2022 and Dec 2022 was collected from 46 centers of Indira IVF Hospital. These were couples who had experienced recurrent implantation failures (>3 failed ET), recurrent miscarriages (>2 miscarriages) or had advanced maternal age (>35 years) and opting either for niPGT-A or cPGT-A at our centres for euploid embryo transfers. Women with anatomical uterine defects and Asherman syndrome were excluded. Participants/materials, setting, methods Trophectoderm biopsy was done in 1383 Day 5 grade A embryos and PGT-A was done using NGS. Culture media of 3083 Grade A embryos were collected on D5 or D6 and frozen. NGS was done at an external facility. Embryos deemed euploid in either of the groups were transferred. Biochemical pregnancy was determined by measurements of beta hCG. Main results and the role of chance There were 906 patients in the niPGT-A group and 303 in the cPGT-A group. The clinical characteristics of the participants were comparable in both groups. Biochemical pregnancy rates were 60% and 69%, respectively. This difference was not statistically significant (p < 0.6). The euploidy rates were comparable in the niPGT-A and cPGT-A groups (40% and 46%) respectively. The aneuploidy rates were lower in the niPGT-A group as compared to the cPGT-A group (15% vs 26%). The mosaicism rates were higher in the niPGT-A group as compared to the cPGT-A group. Limitations, reasons for caution This is a preliminary data analysis of only the biochemical pregnancy rates. Clinical pregnancy, miscarriage, and live birth outcomes are subject to further investigation. Wider implications of the findings The study will aid IVF specialist to consider niPGT-A as a possible alternative to cPGT-A in clinical practice. Trial registration number ’not applicable’

P-198 Time to maximum blastocyst expansion (tmBE) may represent a novel metric for assessing embryo competence

Abstract Study question Can the time to achieve maximum blastocyst expansion (tmBE) and the frequency of spontaneous collapses serve as useful indicators of embryo competence? Summary answer Compared to the frequency of spontaneous collapses, tmBE was a better indicator of embryo competence, potentially serving as a valuable metric for embryo selection. What is known already Identifying the most viable blastocyst for transfer is essential for success in assisted reproduction. In scenarios with good prognosis, such as oocyte donation, blastocyst selection may require a more nuanced approach, given the high number of available good quality embryos. Exploring indicators of embryo competence beyond traditional morphology-based criteria may be beneficial, however an in-depth understanding is still lacking. Blastocyst expansion and contraction dynamics have been suggested to play a role in developmental and chromosomal fitness. However, their predictive value has not been investigated across a wide range of patient populations. Study design, size, duration This is a single-center retrospective study of 287 blastocysts transferred in autologous (n = 88) and heterologous (n = 199) cycles, performed between September 2019 and September 2022. Only fresh single embryo transfers were considered. We compared blastocysts across two groups: advanced maternal age (AMA) patients (>35 years old, n = 88) and oocyte donors (<35 years old, n = 199). We evaluated well-established indicators of embryo quality, as well as novel metrics, including tmBE and the frequency of spontaneous collapses. Participants/materials, setting, methods Conventional indicators of embryo quality included blastocyst grade and morphokinetic timings (tPNf, t2, t3, t4, t5, t8, tSB and tB). tMBE was defined as the maximum time (hpi) required for the blastocyst to reach its largest diameter, extending beyond tB. Blastocyst contractions were measured as a decrease and subsequent recovery of the blastocoel area over time. ImageJ was employed for size measurements. p<0.05 was considered statistically significant after Two-tailed Student’s t-test or Chi-square test. Main results and the role of chance We observed a significant difference in live birth rates between oocyte donors and AMA patients (40% vs 18%, p=0.0004). Nevertheless, there were no differences across blastocyst expansion scores between oocyte donors and AMA patients (5= 58% vs 65%; 4= 38% vs 30% and 3= 4% vs 5%, p > 0.05), nor in their morphology score (Excellent= 57% vs 61%; Good= 24% vs 18%; Average= 15% vs 16% and Poor= 4% vs 5%). In terms of morphokinetics, when comparing oocyte donor and AMA embryos, the only significant difference was observed at tPNF (22.0±2.4 vs 22.6±2.3; p= 0.04). No differences were found at t2, t3, t4, t5, t8, tSB and tB. Additionally, the frequency of spontaneous collapses showed no significant differences between the oocyte donor and AMA group (1.4±1.2 vs 1.5±1.0, p=0.66). However, blastocysts from oocyte donors reached tmBE significantly faster than AMA embryos (115.0±4.8 vs 117.2±3.4; p = 0.0003). When these variables were further correlated to clinical outcomes, the frequency of spontaneous collapses did not appear to influence live birth in either group. Notably, however, tmBE was significantly shorter for embryos resulting in a live birth, across both the oocyte donor (113.5 vs 115.4, p = 0.04) and AMA group (115.6 vs 117.6, p = 0.04). Limitations, reasons for caution This is a single center retrospective study with a relatively small sample size, especially in the AMA group. Caution should be made when generalizing results to other patient populations, as confounding were not taken into consideration. No data on the chromosomal status of embryos was available. Wider implications of the findings Our results suggest that tMBE may serve as a reliable indicator of embryo potential, allowing a more nuanced embryo selection, particularly in cases with a high number of good quality embryos. This study opens avenues for further research into novel metrics for assessing embryo competence Trial registration number not applicable

O-273 A transcriptomic approach for embryo selection after preimplantation genetic diagnosis for aneuploidy

Abstract Study question Is it possible to predict an euploid chromosomal constitution from RNA-Seq data and identify a transcriptomic profile compatible with extended embryonic development? Summary answer It has been possible to obtain a karyotype comparable to PGT-A, in addition to acquiring a transcriptomic signature of embryos suggestive of improved implantation capacity What is known already Conventional assessment of embryo competence, based on morphology and morphokinetic, lacks knowledge of molecular aspects and faces controversy in predicting ploidy status. Understanding the embryonic transcriptome is crucial, as gene expression influences development and implantation. Preimplantation genetic testing (PGT) has improved pregnancy rates, but problems persist when high-quality euploid embryos do not reach term. In fact, around 50 to 60% implant, of which 10% miscarry. Comprehensive approaches, including RNA-Seq, offer potential to discover molecular markers of reproductive competence. Extended-embryo culture platforms up to day 14 can be utilized as a proxy to study embryo development at post-implantation stages Study design, size, duration Prospective pilot cohort study conducted from March 2023 to August 2023. A total of 30 vitrified human blastocysts with previous PGT-A diagnosis on day 5 (D5) or day 6 (D6) of development were analysed: n = 15 euploid and n = 15 aneuploid embryos. Finally, 21 embryo samples were included in the study (n = 10 euploid and n = 11 aneuploid), the rest (n = 9) were excluded due to poor quality after sequencing. All embryos were donated for research purposes Participants/materials, setting, methods Following warming and re-expansion, embryos underwent a second trophectoderm biopsy. The remaining embryo was cultured until day 11 to assess its development. After biopsy analysis by RNA-Seq, karyotypes were studied, as well as differential expression gene (DEG) of the comparison: unattached embryos (n = 12) vs embryos attached (n = 9) to the plate (padj<0.05 and FC > 1). In addition, we obtained a transcriptomic signature specific to embryos with a “theoretical” capacity for sustained implantation (raw p-value<0.05 and shrunk LFC>1.1) Main results and the role of chance Digital karyotype concordance was obtained with a sensitivity of 81%, a specificity of 83%, a Kappa index of 65.5% and an area under the ROC curve of 90%. At the gene level, 76 DEGs (56 upregulated and 20 downregulated) were found in the comparison of embryos unattached to the plate in extended culture versus those that were attached. To address the functional implications of these differences, significantly deregulated pathways according to GO and KEGG categories were identified. The mural trophectoderm of the analysed blastocysts showed 63 significantly deregulated terms (30 GO-Biological Process, 13 GO-Cellular Component, 12 GO-Molecular Function and 8 KEGG pathways), displaying upregulation of autophagy, apoptosis, protein kinase and ubiquitin-like protein ligase activity, and downregulation of ribosome, spliceosome, kinetochore, segregation, and chromosome condensation processes. The first described transcriptomic signature specific to embryos with theoretical enhanced implantation capacity consists of 501 genes, including: EMP2, AURKB, FOLR1, NOTCH3, LRP2, FZD5, MDH1, APOD, GPX8, COLEC12, HSPA1A, CMTM7, BEX3, which are related to implantation and embryonic development. These findings indicate that it would be possible to identify euploid embryos with a greater capacity for implantation and development, excluding those embryos that, although they could be implanted and viable, present chromosomal alterations Limitations, reasons for caution This study included a small sample size and a remarkable variability between samples. Structural chromosomal abnormalities were not included. It is not possible to diagnose mosaic embryos. Trophectoderm biopsy does not assure the chromosomal status of the whole embryo. Maximum number of days for in vitro development was 11 Wider implications of the findings Gene expression profiles of embryos with theoretical increased implantation capacity have been obtained. Clinical implementation of this technique, which assesses chromosomal content and transcriptomic profile, could be used to significantly increase clinical outcomes in assisted reproduction, allowing informed decisions to be made about future treatments and the prospects of success Trial registration number Not applicable

P-511 Euploidy potential of day 7 embryos and the relevance in Assisted Human Reproduction treatments

Abstract Study question Is it worth waiting embryos with late blastulation (Day 7) for preimplantation genetic analysis? Summary answer Embryo biopsy on Day 7 (D7) benefits cases of ovarian insufficiency and severe male factor with a euploidy rate of 15.1%. What is known already New technologies and improvements in IVF laboratories has allowed the extended cultivation of blastocysts to increase the pool of embryos per IVF cycle, resulting in greater chances, especially in cases of low blastulation rate, blastocysts with low quality or with few cells at Day 5(D5) and Day 6(D6). The variation in the time of the compaction stage depends on many factors, which may take more time, leading some embryos to reach the blastocyst stage only on D7 of development. These embryos have a lower euploidy rate, but there is embryonic competence and these embryos can be eligible for embryo transfer. Study design, size, duration Retrospective study that evaluated the chromosomal status (by Next Generation Screening - NGS) of 33 blastocysts biopsied on day 7 of development, between 2016 and 2023. Subsequently, it was compared the rate of euploidy and mosaicism with 295 blastocysts biopsied at D5 and D6, biopsied from January to October 2023. D7 embryos were originated from IVF cycles of couples with female age between 26 and 44 years, and males age between 29 and 56 years. Participants/materials, setting, methods D7 blastocysts from fresh and own oocytes fertilized by ICSI. The euploidy and mosaicism rates was compared, as well as the morphology of the ICM and trophectoderm (A-C; by Gardner Blastocyst Grade), the day which biopsy was performed and the respective infertility factors, in the groups: Group 1: D7 euploid and Group 2: D7 aneuploid. Subsequently, the rate of euploidy and mosaicism was compared between D7 embryos (Group 3) and D5/D6 embryos (Group 4). Main results and the role of chance There was no difference (p > 0.05) between maternal age for group D7 euploid (38.4 years) vs group D7 aneuploid (37.9 years) and group 3 (38.4 years) vs group 4 (38.2 years). Embryonic morphological evaluation of D7 embryos showed that ICM was classified as B (18.2%) or C (81.8%) and trophectoderm as A (6.1%), B (24.2%) or C (69.7%). Comparisons of ICM and trophectoderm classifications between euploid (group 1) and aneuploid (group 2) in D7 blastocysts showed no difference (p > 0.05). There was a predominance of 5CC embryos in both groups (Group 1: 40.0% vs Group 2: 42.4%), with no difference between them (p > 0.05). In most cases, the biopsy was performed on two days (D5+D7 – 39.3%; D6+D7 – 35.7%), while in 10.7% of cases only on Day 7, when the infertility factor was 100% ovarian insufficiency. In cases of biopsy within 3 days (D5+D6+D7), we observed that 100% of cases were related to severe male factor. When comparing the euploidy rate of D7 embryos – group 3 (15.1%) with that of D5/D6 – group 4 (34.2%), we observed a statistical difference (p < 0.05), while, for in mosaicism rates, D7 embryos represented 6.1% and D5/D6 embryos 9.1%, with no statistical difference (p > 0.05). Limitations, reasons for caution The retrospective nature of our study may lead to bias derived from clinical practices and variability in relation to patient age, embryonic quality and infertility factor. Wider implications of the findings Extended culture and biopsy on D7 benefits cases of ovarian insufficiency and severe male factor by optimizing the IVF cycle. Although the euploidy rate in D7 embryos is lower than D5/D6 embryos, 15% of D7 embryos are euploid, demonstrating that the slower development is not caused exclusively by aneuploidy Trial registration number NA

O-216 The impact of maternal age on cumulative life birth rates after single euploid embryo transfer: differences between autologous and donor oocyte cycles

Abstract Study question How much of an impact has the maternal age on cumulative life births after euploid Single Embryo Transfer (SET) in autologous and donor oocyte cycles? Summary answer A maternal age ≥ 42 years old affects negatively the cumulative Life Birth Rates (cLBRs) even after euploid SET, but only in the autologous cycles. What is known already Despite the improvements in assisted reproduction technologies (ART) in the last decades, the LBRs remain suboptimal, especially when advanced maternal age is considered. Although several factors could explain these outcomes (i.e. infertility history; uterine pathologies/surgery; advanced paternal age), maternal age-related aneuploidies remain the most limiting factor for a favorable clinical outcome. Thus, the Genetic Testing for Aneuploidies (PGT-A) has been integrated into ART as a strategy to evaluate chromosomal status before transfer. Interestingly, recent studies show that, even after the transfer of euploid blastocyst, the maternal age negatively influences the LBRs. Study design, size, duration This study, carried out in Villa Mafalda Reproductive Medicine Department (Italy), included 568 euploid SET: 542 from autologous cycles (divided in four subgroups according to maternal age: ≤ 34 y.o. (n = 207); 35-38 y.o. (n = 193); 39-41 y.o. (n = 106); ≥ 42 y.o. (n = 36)) and 26 from oocyte donation (OD) cycles (average recipient age is 44.2). All cycles were performed from May 2020 to February 2023. PGT for Monogenic disorders (PGT-M) cycles were excluded. Participants/materials, setting, methods All blastocysts included in this study were analyzed by Next Generation Sequencing (NGS) after a day 5/6 trophectoderm biopsy. For each subgroup was annotated the embryo morphology- divided into good (AA-AB-BA); average (BB-BC-CB) and poor (CC) according to Gardner/Schoolcraft blastocyst grading system- and the day of expanded blastocyst formation (grade 4 of Gardner/Schoolcraft blastocyst grading system). Primary analysis included the cumulative LBRs. Secondarily, blastocyst morphology and blastocyst expansion were examined for all embryo categories considered. Main results and the role of chance The pairwise comparison between groups was performed by Chi-square analysis and p < 0.05 was considered statistically significant. The cLBRs for each subgroup were respectively: ≤ 34 (63.09%); 35-38 (58.47%); 39-41 (56.84%); ≥ 42 (48.57%); OD (69.23%). Data showed that cLBRs decreased with the increase of maternal age, although statistical significance was observed only among women ≤ 34 y.o. and ≥ 42 y.o. (P=0.029). Notably, there was no statistical significance in cLBRs comparing women ≤ 34 y.o. and OD patients (P=0.086). The good-quality blastocyst rate was respectively: ≤ 34 y.o. (54.58%); 35-38 y.o. (47.66%); 39-41 y.o. (60.37%); ≥ 42 y.o. (50%); OD (59.37%). The pairwise comparison showed no significant difference between groups. Furthermore, there was not found significant difference about the day of expanded blastocyst formation: ≤ 34 y.o. (D5: 78.92%; D6 21.07%); 35-38 y.o. (D5: 78.49%; D6: 21.50%); 39-41 y.o. (D5:75.70%;D6: 24.29%); ≥ 42 y.o. (D5:69.44%; D6: 30.55%); OD (D5: 87.5%; D6: 12.5%). Data showed that maternal age ≥ 42 y.o. affects negatively cLBRs even after euploid SET, suggesting that other factors age-related, rather than aneuploidies, influence implantation. Interestingly, these factors do not affect OD recipients, with advanced maternal age, who, instead, reach successful LBRs like patients ≤ 34. Limitations, reasons for caution These results should be evaluated carefully because of the small sample size of the OD patients. A greater number of OD cycles are needed to verify the results obtained. Wider implications of the findings Our findings suggest that embryonic age-related factors - changes in gene expression, metabolism or epigenetics - could influence the embryo capability to, successfully, support implantation other than the ploidy status. This thesis was supported by the OD recipient’s behavior, who had cLBRs comparable to young autologous patients. Trial registration number Not Applicable

Correlation Analysis of Apparent Diffusion Coefficient Histogram Parameters and Clinicopathologic Features for Prognosis Prediction in Uveal Melanoma.

To investigate the correlation between apparent diffusion coefficient (ADC) histograms and high-risk clinicopathologic features related to uveal melanoma (UM) prognosis. This retrospective study included 53 patients with UM who underwent diffusion-weighted imaging (DWI) between August 2015 and March 2024. Axial DWI was performed with a single-shot spin-echo echo-planar imaging sequence. ADC histogram parameters of ADCmean, ADC50%, interquartile range (IQR), skewness, kurtosis, and entropy were obtained from DWI. The relationships between histogram parameters and high-risk clinicopathological characteristics including tumor size, preoperative retinal detachment, histological subtypes, Ki-67 index, and chromosome status, were analyzed by Spearman correlation analysis, Mann-Whitney U test, or Kruskal-Wallis test. A total of 53 patients (mean ± SD age, 55 ± 15 years; 22 men) were evaluated. The largest basal diameter (LBD) was correlated with kurtosis (r = 0.311, P = 0.024). Tumor prominence (TP) was correlated with entropy (r = 0.581, P < 0.001) and kurtosis (r = 0.273, P = 0.048). Additionally, significant correlations were identified between the Ki-67 index and ADCmean (r = -0.444, P = 0.005), ADC50% (r = -0.487, P = 0.002), and skewness (r = 0.394, P = 0.014). Finally, entropy was correlated with monosomy 3 (r = 0.541, P = 0.017). The ADC histograms provided valuable insights into high-risk clinicopathologic features of UM and hold promise in the early prediction of UM prognosis.

Loss of Chromosome Y in Neuroblastoma Is Associated With High-Risk Disease, 11q-Deletion, and Telomere Maintenance.

Neuroblastoma (NB) is a heterogeneous childhood cancer with a slightly higher incidence in boys than girls, with the reason for this gender disparity unknown. Given the growing evidence for the involvement of loss of the Y chromosome (LoY) in male diseases including cancer, we investigated Y chromosome status in NB. Male NB tumor samples from a Swedish cohort, analyzed using Cytoscan HD SNP-microarray, were selected. Seventy NB tumors were analyzed for aneuploidy of the Y chromosome, and these data were correlated with other genetic, biological, and clinical parameters. LoY was found in 21% of the male NB tumors and it was almost exclusively found in those with high-risk genomic profiles. Furthermore, LoY was associated with increased age at diagnosis and enriched in tumors with 11q-deletion and activated telomere maintenance mechanisms. In contrast, tumors with an MYCN-amplified genomic profile retained their Y chromosome. The understanding of LoY in cancer is limited, making it difficult to conclude whether LoY is a driving event in NB or function of increased genomic instability. Gene expression analysis of Y chromosome genes in male NB tumors showed low expression of certain genes correlating with worse overall survival. KDM5D, encoding a histone demethylase stands out as an interesting candidate for further studies. LoY has been shown to impact the epigenomic layer of autosomal loci in nonreproductive tissues, and KDM5D has been reported as downregulated and/or associated with poor survival in different malignancies. Further studies are needed to explore the mechanisms and functional consequences of LoY in NB.

Chromosomal copy number and mutational status are required to authenticate ovarian cancer cell lines as appropriate cell models

BackgroundThe mutational status of ovarian cancer cell line IGROV-1 is inconsistent across the literature, suggestive of multiple clonal populations of the cell line. IGROV-1 has previously been categorised as an inappropriate model for high-grade serous ovarian cancer.MethodsIGROV-1 cells were obtained from the Netherlands Cancer Institute (IGROV-1-NKI) and the MD Anderson Cancer Centre (IGROV-1-MDA). Cell lines were STR fingerprinted and had their chromosomal copy number analysed and BRCA1/2 genes sequenced. Mutation status of ovarian cancer-related genes were extracted from the literature.ResultsThe IGROV-1-NKI cell line has a tetraploid chromosomal profile. In contrast, the IGROV-1-MDA cell line has pseudo-normal chromosomes. The IGROV-1-NKI and IGROV-MDA are both STR matches (80.7% and 84.6%) to the original IGROV-1 cells isolated in 1985. However, IGROV-1-NKI and IGROV-1-MDA are not an STR match to each other (78.1%) indicating genetic drift. The BRCA1 and BRCA2 gene sequences are 100% identical between IGROV-1-MDA and IGROV-1-NKI, including a BRCA1 heterozygous deleterious mutation. The IGROV-1-MDA cells are more resistant to cisplatin and olaparib than IGROV-1-NKI. IGROV-1 has a mutational profile consistent with both Type I (PTEN, PIK3CA and ARID1A) and Type II ovarian cancer (BRCA1, TP53) and is likely to be a Type II high-grade serous carcinoma of the SET (Solid, pseudo-Endometroid and Transitional cell carcinoma-like morphology) subtype.ConclusionsRoutine testing of chromosomal copy number as well as the mutational status of ovarian cancer related genes should become the new standard alongside STR fingerprinting to ensure that ovarian cancer cell lines are appropriate models.

Exploring karyotype by developing novel oligonucleotide probes for Leucanthemum paludosum and other plant species

Leucanthemum paludosum is a small, popular, perennial herbaceous flower that is widely used as a potted plant and in flower beds. It is highly adaptable and fast-growing, blooms from early to late spring, and can be maintained for two–three months, thus making it a potential model plant for cytological studies. However, it lacks applicable markers for identifying its chromosome numbers, and no karyotyping or cytological study about L. paludosum has been reported. In the present study, we aimed to analyze the morphology and chromosomal status of L. paludosum to provide a reference for its systematic utilization. The results showed that L. paludosum is diploid (2n = 2x = 18). The bivalent number was substantially higher than the monovalent bodies. L. paludosum also conducted a loosely patterned karyotype, with no fluorescence in situ hybridization (FISH) signal when using a conventional oligonucleotide (oligo) probe library. To address this problem, we developed a novel repetitive oligo probe ((ATC)10) using transcriptome sequences. Furthermore, we explored its feasibility in different plants and provided effective repetitive oligoprobes for species such as Avena sativa, Vicia faba, Allium sativum, Lilium davidii var. unicolor, and Antirrhinum majus. Our study provides new cytological data for L. paludosum and developed a novel method for designing oligo probes for karyotype detection.