Ploidy Status Research Articles

Non Invasive Preimplantation Testing for Aneuploidies in Assisted Reproduction: A SWOT Analysis.

The implementation of non-invasive PGT-A offers a new strategy to genetically assess the preimplantation embryo and to enhance IVF results. The extraction of DNA from the embryo culture medium has been sufficiently demonstrated, and the ability to obtain chromosomal information as a result is particularly interesting. As morphological criteria have proven to have a weak correlation with embryo ploidy status, this technique emerges as a promising alternative for embryo selection. It also appears reasonable that avoiding biopsy may enhance further embryo development. However, there are growing concerns regarding several aspects of this technique, such as the origin of this cell free DNA, the degree of representativeness of the whole embryo, the need for extended culture or the absence of standardized protocols. Despite the published data on good prognosis couples are promising, niPGT-A is yet to be considered a substitute for trophectoderm biopsy. The current SWOT analysis aims to summarize both resolved and unresolved issues, as well as limiting aspects of niPGT-A.

Incidence of haploidy and triploidy in trophectoderm biopsies of blastocysts derived from normally and abnormally fertilized oocytes.

We aimed to identify the correlation between morphological pronuclear (PN) status and the genetically determined ploidy configuration in preimplantation embryos. A retrospective observational study was conducted on 1982 embryos displaying normal fertilization and 380 embryos showing an atypical PN pattern, tested for aneuploidies and ploidy status via preimplantation genetic testing (PGT) between May 2019 and May 2024. Ploidy prediction was performed using a validated targeted-NGS approach and a proprietary bioinformatic pipeline analyzing SNPs B-allele frequency information. Ploidy results were obtained in relation to the morphological PN pattern and further stratified by mode of PN observation, maternal age, and embryo quality parameters. Abnormal ploidy results in 2PN-derived embryos were 1% (n = 20/1982): 0.8% showed triploidy and 0.2% haploidy. Ploidy results in relation to PN number in atypical fertilization were as follows: 0PN (n = 150/380) associated with 87.3% of diploidy, 8.7% of haploidy, and 4.0% of triploidy; 1PN-derived blastocysts (n = 73/153) were haploid in 47.7% of cases, 6.5% were triploid, and 45.7% diploid; 2.1PN (n = 23/280) and 3PN patterns (n = 54/280) predicted a triploid result in 34.8% and 74.1% of cases, respectively. PN observation with time-lapse increased ploidy status predictivity from 28.3% to 80.4% (p < 0.01) and reduced expected diploid rates to 19.6% (p < 0.01). Diploidy rate was higher for maternal age ≤ 35years and for morphologically high-grade embryos. Morphological PN check can be improved by incorporating ploidy analysis within the conventional PGT workflow. Euploid 2PN-derived embryos can be further selected removing haploids and triploids, and some atypical PN pattern can be better classified.

Haploidy-linked cell proliferation defects limit larval growth in zebrafish.

Haploid larvae in non-mammalian vertebrates are lethal, with characteristic organ growth retardation collectively called 'haploid syndrome'. In contrast to mammals, whose haploid intolerance is attributed to imprinting misregulation, the cellular principle of haploidy-linked defects in non-mammalian vertebrates remains unknown. Here, we investigated cellular defects that disrupt the ontogeny of gynogenetic haploid zebrafish larvae. Unlike diploid control larvae, haploid larvae manifested unscheduled cell death at the organogenesis stage, attributed to haploidy-linked p53 upregulation. Moreover, we found that haploid larvae specifically suffered the gradual aggravation of mitotic spindle monopolarization during 1-3 days post-fertilization, causing spindle assembly checkpoint-mediated mitotic arrest throughout the entire body. High-resolution imaging revealed that this mitotic defect accompanied the haploidy-linked centrosome loss occurring concomitantly with the gradual decrease in larval cell size. Either resolution of mitotic arrest or depletion of p53 partially improved organ growth in haploid larvae. Based on these results, we propose that haploidy-linked mitotic defects and cell death are parts of critical cellular causes shared among vertebrates that limit the larval growth in the haploid state, contributing to an evolutionary constraint on allowable ploidy status in the vertebrate life cycle.

Do sperm factors influence embryonic aneuploidy? Long live the oocyte.

What is the impact of male age- and sperm-related factors on embryonic aneuploidy? Using a 3-fold analysis framework encompassing patient-level, embryo-level, and matching analysis, we found no clinically significant interactions between male age and sperm quality with embryo ploidy. While the effect of maternal age on embryo chromosomal aneuploidy is well-established, the impact of male age and sperm quality on ploidy is less well-defined. This retrospective cohort study analyzed autologous preimplantation genetic testing for aneuploidy (PGT-A) and frozen embryo transfer cycles from December 2014 to June 2021. The study involved 11087 cycles from 8484 patients, with a total of 35797 embryos. The aneuploidy rate, calculated as the ratio of aneuploid blastocysts to the total number of blastocysts biopsied in a single treatment cycle, was evaluated. In the embryo-level analysis, the main outcome measure was the ploidy state of the embryos. The study employed a multifaceted analytical approach that included a patient-level analysis using generalized linear mixed models, an embryo-level analysis focusing on chromosomal ploidy, and a propensity score matching analysis contrasting groups with distinct ploidy rates (0% and 100%). There were no interventions as this was an observational study of PGT-A cycles. No clinically relevant factors influencing ploidy rate related to male and sperm quality were revealed. In contrast, female age (coefficient = -0.053), BMI (coefficient = 0.003), prior ART cycle (coefficient = -0.066), and number of oocytes retrieved (coefficient = -0.018) were identified at the patient level. Embryo analysis identified age (coefficient = -0.1244) and ICSI usage (coefficient = -0.0129) as significant factors. Despite these, no significant interactions between male and female assessed factors on the ploidy rate emerged. Propensity score matching between maximal (100% vs 0%) euploid rates did not reveal significant differences of influence by male age and sperm quality. The focus on patients having blastocyst biopsy for PGT-A may not reflect the broader IVF population. Other semen quality parameters like DNA fragmentation were not included. Exclusion of embryo mosaicism from the analysis could affect aneuploidy rate interpretations. There may also be unmeasured influences like lifestyle or environmental factors. Male age and sperm quality parameters were consistent across both maximal and minimal ploidy rate comparisons. No significant clinical characteristics related to the factors assessed for the male-influenced blastocyst ploidy status, confirming the dominancy of the oocyte and female age. The study was not funded. There are no competing interests. N/A.

Effect of gamma irradiation on proliferation and growth of friable embryogenic callus and in vitro nodal cuttings of ugandan cassava genotypes.

Cassava (Manihot esculenta Crantz) production and productivity in Africa is affected by two viral diseases; cassava mosaic disease (CMD) and cassava brown streak disease (CBSD). Induced mutagenesis of totipotent/embryogenic tissues or in vitro plant material can lead to the generation of CMD and/or CBSD tolerant mutants. To massively produce non-chimeric plants timely and with less labor, totipotent cells or tissues are a pre-requisite. This study aimed to determine the effect of gamma radiation on the proliferation and growth of friable embryogenic callus (FEC) and in vitro nodal cuttings respectively. To obtain FEC, 2-6 mm sized leaf lobes of nine cassava genotypes were plated on Murashige and Skoog (MS) basal media supplemented with varying levels (37, 50, 70, 100) μM of picloram for production of organized embryogenic structures (OES). The OES of five cassava genotypes (Alado, CV-60444, NASE 3, NASE 13 and TME 204) were crushed and plated in Gresshoff and Doy (GD) basal media in combination with the amino acid tyrosine in varying concentrations for FEC production. FEC from five cassava genotypes and in vitro nodal cuttings of nine genotypes were irradiated using five different gamma doses (0, 5, 10, 15, 20 and 25 Gy) at a dose rate of 81Gy/hr. The lethal dose (LD)50 was determined using the number of roots produced and flow cytometry was done to determine the ploidy status of plants. The highest production of OES was noted in Alado across varying picloram concentrations, while TME 204 obtained the highest amount of FEC. The irradiated FEC gradually died and by 28 days post irradiation, FEC from all five cassava genotypes were lost. Conversely, the irradiated in vitro nodal cuttings survived and some produced roots, while others produced callus. The LD50 based on number of roots varied from genotype to genotype, but plants remained diploid post-irradiation. Accordingly, the effect of gamma irradiation on Ugandan cassava genotypes (UCGs) was genotype-dependent. This information is foundational for the use of in vitro tissues as target material for cassava mutation breeding.

Automatic ploidy prediction and quality assessment of human blastocysts using time-lapse imaging

Assessing fertilized human embryos is crucial for in vitro fertilization, a task being revolutionized by artificial intelligence. Existing models used for embryo quality assessment and ploidy detection could be significantly improved by effectively utilizing time-lapse imaging to identify critical developmental time points for maximizing prediction accuracy. Addressing this, we develop and compare various embryo ploidy status prediction models across distinct embryo development stages. We present BELA, a state-of-the-art ploidy prediction model that surpasses previous image- and video-based models without necessitating input from embryologists. BELA uses multitask learning to predict quality scores that are thereafter used to predict ploidy status. By achieving an area under the receiver operating characteristic curve of 0.76 for discriminating between euploidy and aneuploidy embryos on the Weill Cornell dataset, BELA matches the performance of models trained on embryologists’ manual scores. While not a replacement for preimplantation genetic testing for aneuploidy, BELA exemplifies how such models can streamline the embryo evaluation process.

Chromosome-scale genome assembly and annotation of the tetraploid potato cultivar Diacol Capiro adapted to the Andean region.

Potato (Solanum tuberosum) is an essential crop for food security and is ranked as the third most important crop worldwide for human consumption. The Diacol Capiro cultivar holds the dominant position in Colombian cultivation, primarily catering to the food processing industry. This highly heterozygous, autotetraploid cultivar belongs to the Andigenum group and it stands out for its adaptation to a wide variety of environments spanning altitudes from 1,800 to 3,200 meters above sea level. Here, a chromosome-scale assembly, referred to as DC, is presented for this cultivar. The assembly was generated by combining circular consensus sequencing with proximity ligation Hi-C for the scaffolding and represents 2.369 Gb with 48 pseudochromosomes covering 2,091 Gb and an anchor rate of 88.26%. The reference genome metrics, including an N50 of 50.5 Mb, a BUSCO (Benchmarking Universal Single-Copy Orthologue) score of 99.38%, and an Long Terminal Repeat Assembly Index score of 13.53, collectively signal the achieved high assembly quality. A comprehensive annotation yielded a total of 154,114 genes, and the associated BUSCO score of 95.78% for the annotated sequences attests to their completeness. The number of predicted NLR (Nucleotide-Binding and Leucine-Rich-Repeat genes) was 2107 with a large representation of NBARC (for nucleotide binding domain shared by Apaf-1, certain R gene products, and CED-4) containing domains (99.85%). Further comparative analysis of the proposed annotation-based assembly with high-quality known potato genomes, showed a similar genome metrics with differences in total gene numbers related to the ploidy status. The genome assembly and annotation of DC presented in this study represent a valuable asset for comprehending potato genetics. This resource aids in targeted breeding initiatives and contributes to the creation of enhanced, resilient, and more productive potato varieties, particularly beneficial for countries in Latin America.

Beyond black-box models: explainable AI for embryo ploidy prediction and patient-centric consultation.

To determine if an explainable artificial intelligence (XAI) model enhances the accuracy and transparency of predicting embryo ploidy status based on embryonic characteristics and clinical data. This retrospective study utilized a dataset of 1908 blastocyst embryos. The dataset includes ploidy status, morphokinetic features, morphology grades, and 11 clinical variables. Six machine learning (ML) models including Random Forest (RF), Linear Discriminant Analysis (LDA), Logistic Regression (LR), Support Vector Machine (SVM), AdaBoost (ADA), and Light Gradient-Boosting Machine (LGBM) were trained to predict ploidy status probabilities across three distinct datasets: high-grade embryos (HGE, n = 1107), low-grade embryos (LGE, n = 364), and all-grade embryos (AGE, n = 1471). The model's performance was interpreted using XAI, including SHapley Additive exPlanations (SHAP) and Local Interpretable Model-agnostic Explanations (LIME) techniques. The mean maternal age was 38.5 ± 3.85years. The Random Forest (RF) model exhibited superior performance compared to the other five ML models, achieving an accuracy of 0.749 and an AUC of 0.808 for AGE. In the external test set, the RF model achieved an accuracy of 0.714 and an AUC of 0.750 (95% CI, 0.702-0.796). SHAP's feature impact analysis highlighted that maternal age, paternal age, time to blastocyst (tB), and day 5 morphology grade significantly impacted the predictive model. In addition, LIME offered specific case-ploidy prediction probabilities, revealing the model's assigned values for each variable within a finite range. The model highlights the potential of using XAI algorithms to enhance ploidy prediction, optimize embryo selection as patient-centric consultation, and provides reliability and transparent insights into the decision-making process.

Live Motile Sperm Sorting Device Improves Embryo Aneuploidy: A Retrospective Cohort Study

Background: Conventional sperm selection methods, involving centrifugation, exert a detrimental effect on sperm DNA integrity due to mechanical stress. The recent noninvasive sperm selection device, CA0, based on live sperm sorting technology, facilitates the retrieval of highly motile sperm and minimizes sperm DNA fragmentation (SDF). This study was to investigate the impact of various sperm separation methods, with and without centrifugation, on embryo ploidy status. Methods: The retrospective study comprised 82 intracytoplasmic sperm injection (ICSI) cycles involving preimplantation genetic testing for aneuploidy (PGT-A) cases, with a focus on recruiting egg donation cycles to thoroughly investigate the impact of male factors. Two populations are classified based on semen quality: normozoospermic ([Formula: see text] = 33) and non-normozoospermic ([Formula: see text] = 49). Subjects were allocated to either swim-up (SU) or CA0. Preimplantation genetic testing results were recorded. Results: When comparing male characteristics between subgroups, no significant differences were observed except for a lower normal morphology rate in the CA0 group compared to SU (SU: 3 [3–4] vs. CA0: 2 [2–2.8], [Formula: see text] &lt; 0.0001) in the non-normozoospermic cohort. There were no differences in female factors such as age and mature oocyte count (MII) number between subgroups, indicating that this model is ideal for assessing the impact of male factors on clinical outcomes. In the normozoospermic cohort, euploidy rates were similar between SU and CA0 (SU: 71.9% vs. CA0: 64.2%). However, in the non-normozoospermic cohort, CA0 showed a significantly higher euploidy rate compared to SU (SU: 53.6% vs. CA0: 74.2%) and a lower aneuploidy rate (SU: 37.1% vs. CA0: 25.8%). Additionally, CA0 minimized the incidence of mosaic embryos, whereas a mosaicism rate of 9.3% was observed with SU. This trend highlights CA0’s distinct advantage in optimizing outcomes for non-normozoospermic cases. Conclusions: CA0 is a reliable intervention to optimize paternal genetic quality before assisted insemination, thereafter effectively reducing the incidence of embryo aneuploidy associated with male factors.

Species of the Sections Hedysarum and Multicaulia of the Genus Hedysarum (Fabaceae): Taxonomy, Distribution, Chromosomes, Genomes, and Phylogeny.

The genus Hedysarum L. (Fabaceae) includes about 200 species of annual and perennial herbs distributed in Asia, Europe, North Africa, and North America. Many species of this genus are valuable medicinal, melliferous, and forage resources. In this review, we consider the taxonomic history of the genus Hedysarum, the chromosomal organization of the species from the sections Hedysarum and Multicaulia, as well as phylogenetic relationships between these sections. According to morphological, genetic, and phylogenetic data, the genus Hedysarum is divided into three main sections: Hedysarum (= syn. Gamotion), Multicaulia, and Stracheya. In species of this genus, two basic chromosome numbers, x = 7 (section Hedysarum) and x = 8 (sections Multicaulia and Stracheya), were determined. The systematic positions of some species within the sections are still uncertain due to their morphological similarities. The patterns of distribution of molecular chromosomal markers (45S rDNA, 5S rDNA, and different satellite DNAs) in karyotypes of various Hedysarum species made it possible to determine their ploidy status and also specify genomic relationships within the sections Hedysarum and Multicaulia. Recent molecular phylogenetic studies clarified significantly the taxonomy and evolutionary development of the genus Hedysarum.

Morphological quality on Day 3 affects the pregnancy outcomes of low-quality euploid blastocysts: a retrospective cohort study.

Does the morphological quality on Day 3 influence the pregnancy outcomes of euploid blastocysts? The morphological quality on Day 3 affects the clinical pregnancy rate (CPR) and live birth rate (LBR) of low-quality euploid blastocysts. The morphological grading of Day 3 embryos affects the pregnancy outcome of cleavage-stage embryos and is an excellent indicator to predict embryo development potential. However, it is still unclear whether morphological quality on Day 3 is associated with pregnancy outcomes of the euploid blastocyst. This retrospective cohort study comprised 1275 patients who received single euploid blastocyst transfer between January 2016 and August 2021 at a tertiary teaching hospital. Patients were grouped into two groups according to the morphological grading on Day 3 of transferred blastocysts: high-quality (HQ, including Grades I and II) Day 3 embryos and low-quality (LQ, Grade III) Day 3 embryos. The primary outcomes were CPR and LBR. Interactions of development days (Day 5 and Day 6) and morphological quality (high- and low-quality) of blastocysts with morphological quality of Day 3 embryos on pregnancy outcomes were tested in the stratified analysis and logistic regression models. The multivariate logistic regression analysis was conducted to investigate the independent effect of the morphological quality of Day 3 embryos on pregnancy outcomes after adjusting for potentially confounding factors. The CPR and LBR of the HQ Day 3 embryos group were statistically higher than those of the LQ Day 3 embryos group (CPR: 59.73% versus 49.70%, respectively, P = 0.015; LBR: 49.73% versus 41.21%, respectively, P = 0.041). The development days of blastocysts did not exhibit a multiplicative interaction with the morphological quality of Day 3 embryos on the CPR (P for interaction = 0.648) and LBR (P for interaction = 0.925). The morphological quality of blastocysts exhibits a multiplicative interaction with the morphological quality of Day 3 embryos on the CPR (P for interaction = 0.020) and LBR (P for interaction = 0.012). After adjusting for potential confounders, the HQ Day 3 embryo group was positively associated with the CPR (adjusted odds ratio (aOR): 2.10, 95% CI: 1.31-3.36, P = 0.002) and LBR (aOR: 1.97, 95% CI: 1.20-3.25, P = 0.008) of LQ blastocysts. However, the morphological quality on Day 3 was not significantly associated with the CPR (aOR: 0.95, 95% CI: 0.58-1.55, P = 0.835) and LBR (aOR: 0.86, 95% CI: 0.53-1.40, P = 0.550) of HQ blastocysts. Selection and confounding bias introduced by the retrospective design cannot be completely eliminated in this study, although multivariable logistic analysis was conducted to adjust for potential confounders. Also, some subgroups had small sample sizes, which may reduce statistical power. Moreover, participants in our study only received single euploid blastocyst transfer, and whether the results could apply to blastocysts with unknown ploidy status is unclear. This study found that the morphological quality on Day 3 was significantly associated with the CPR and LBR of LQ blastocysts; Therefore, when only LQ euploid blastocysts are available for transfer, blastocysts derived from HQ Day 3 embryos are recommended. No external funding was obtained. The authors have no conflicts of interest to declare. N/A.

The impact of implementing a non-invasive preimplantation genetic testing for aneuploidies (niPGT-A) embryo culture protocol on embryo viability and clinical outcomes.

Are modifications in the embryo culture protocol needed to perform non-invasive preimplantation genetic testing for aneuploidies (niPGT-A) affecting clinical reproductive outcomes, including blastocyst development and pregnancy outcomes? The implementation of an embryo culture protocol to accommodate niPGT-A has no impact on blastocyst viability or pregnancy outcomes. The recent identification of embryo cell-free (cf) DNA in spent blastocyst media has created the possibility of simplifying PGT-A. Concerns, however, have arisen at two levels. First, the representativeness of that cfDNA to the real ploidy status of the embryo. Second, the logistical changes that need to be implemented by the IVF laboratory when performing niPGT-A and their effect on reproductive outcomes. Concordance rates of niPGT-A to invasive PGT-A have gradually improved; however, the impact of culture protocol changes is not as well understood. As part of a trial examining concordance rates of niPGT-A versus invasive PGT-A, the IVF clinics implemented a specific niPGT-A embryo culture protocol. Briefly, this involved initial culture of fertilized oocytes following each laboratory standard routine up to Day 4. On Day 4, embryos were washed and cultured individually in 10 μl of fresh media. On Day 6 or 7, blastocysts were then biopsied, vitrified, and media collected for the niPGT-A analysis. Six IVF clinics from the previously mentioned trial were enrolled in this analysis. In the concordance trial, Clinic A cultured all embryos (97 cycles and 355 embryos) up to Day 6 or 7, whereas in the remaining clinics (B-F) (379 cycles), nearly a quarter of all the blastocysts (231/985: 23.5%) were biopsied on Day 5, with the remaining blastocysts following the niPGT-A protocol (754/985: 76.5%). During the same period (April 2018-December 2020), the IVF clinics also performed standard invasive PGT-A, which involved culture of embryos up to Days 5, 6, or 7 when blastocysts were biopsied and vitrified. In total, 428 (476 cycles) patients were in the niPGT-A study group. Embryos from 1392 patients underwent the standard PGT-A culture protocol and formed the control group. Clinical information was obtained and analyzed from all the patients. Statistical comparisons were performed between the study and the control groups according to the day of biopsy. The mean age, number of oocytes, fertilization rates, and number of blastocysts biopsied were not significantly different for the study and the control group. Regarding the overall pregnancy outcomes, no significant effect was observed on clinical pregnancy rate, miscarriage rate, or ongoing pregnancy rate (≥12 weeks) in the study group compared to the control group when stratified by day of biopsy. The limitations are intrinsic to the retrospective nature of the study, and to the fact that the study was conducted in invasive PGT-A patients and not specifically using niPGT-A cases. This study shows that modifying current IVF laboratory protocols to adopt niPGT-A has no impact on the number of blastocysts available for transfer and overall clinical outcomes of transferred embryos. Whether removal of the invasive biopsy step leads to further improvements in pregnancy rates awaits further studies. This study was funded by Igenomix. C.R., L.N.-S., and D.V. are employees of Igenomix. D.S. was on the Scientific Advisory Board of Igenomix during the study. ClinicalTrials.gov (NCT03520933).

An expert opinion on rescuing atypically pronucleated human zygotes by molecular genetic fertilization checks in IVF.

IVF laboratories routinely adopt morphological pronuclear assessment at the zygote stage to identify abnormally fertilized embryos deemed unsuitable for clinical use. In essence, this is a pseudo-genetic test for ploidy motivated by the notion that biparental diploidy is required for normal human life and abnormal ploidy will lead to either failed implantation, miscarriage, or significant pregnancy complications, including molar pregnancy and chorionic carcinoma. Here, we review the literature associated with ploidy assessment of human embryos derived from zygotes displaying a pronuclear configuration other than the canonical two, and the related pregnancy outcome following transfer. We highlight that pronuclear assessment, although associated with aberrant ploidy outcomes, has a low specificity in the prediction of abnormal ploidy status in the developing embryo, while embryos deemed abnormally fertilized can yield healthy pregnancies. Therefore, this universal strategy of pronuclear assessment invariably leads to incorrect classification of over 50% of blastocysts derived from atypically pronucleated zygotes, and the systematic disposal of potentially viable embryos in IVF. To overcome this limitation of current practice, we discuss the new preimplantation genetic testing technologies that enable accurate identification of the ploidy status of preimplantation embryos and suggest a progress from morphology-based checks to molecular fertilization check as the new gold standard. This alternative molecular fertilization checking represents a possible non-incremental and controversy-free improvement to live birth rates in IVF as it adds to the pool of viable embryos available for transfer. This is especially important for the purposes of 'family building' or for poor-prognosis IVF patients where embryo numbers are often limited.

P-568 Correlation of ploidy analysis in two types of zygotes with 3 pronuclei (3PN) observed: same size 3PNs and one micronucleus 3PNs

Abstract Study question Do all zygotes with three observed pronuclei have an actual triploid chromosomal complement? Summary answer Not all zygotes with 3 pronuclei observed are triploid, most of embryos with two even pronuclei and one smaller micronucleus are diploid. What is known already In the in vitro fertilization (IVF) setting, normal oocyte fertilisation is commonly confirmed 16–19 hours after conventional insemination or intracytoplasmic sperm injection (ICSI) with the appearance of two pronuclei (2PN). The presence three pronuclei (3PN) shows an abnormal oocyte fertilization. The incidence of 3PN zygotes in IVF is between 5.0%- 8.1% in conventional insemination and between 2.0%- 6.2% in ICSI. 3PN fertilized embryos are commonly discarded due to the suspected increased risk of a triploid chromosomal complement. However, no definitive genetic evidence has been obtained to show that all 3PN zygotes are chromosomally abnormal. Study design, size, duration An Ethics Committee for Investigation accredited this prospective observational study performed in a private centre between June 2023 and January 2024. 120 3PN zygotes from 113 cycles were cultivated until blastocyst stage. 46 were biopsied to clarify ploidy and euploidy status. 3PNs were divided into 2 groups based on the size of their pronuclei: Micro Triploid (MT) (smallest pronucleus &amp;lt;70% of the diameter of the largest pronucleus) and Pure Triploid (PT) (smallest &amp;gt;70% of the largest). Participants/materials, setting, methods Patients with 1 or more 3PN embryo were offered to participate, culturing and assessing their 3PNs. If 3PNs reached blastocyst stage, they were biopsied and analysed using Next-Generation Sequencing strategy with Single Nucleotide Polymorphisms technology without the need for parental samples, to assess ploidy and aneuploidy status. Measures of the pronuclei size were taken to sort the 3PNs in both groups. Comparisons between these groups regarding blastocyst rates, ploidy status and aneuploidy rates were assessed. Main results and the role of chance 120 3PNs zygotes were identified in 1054 cycles, representing 1.9% of all fertilized embryos. The blastocyst rate (BR) for overall 3PNs was 38.3%, while the BR for 2PNs on those 113 cycles was 58.3%, showing significant differences (p &amp;lt; 0.001). 3PN zygotes were classified into the MT and PT groups described above. The MT group had 40.4% BR, and the PT group had 37.0% BR. No significant differences were found. Of the 46 biopsied embryos, 37 were informative (80.4%), 17 for the MT group and 20 for the PT group. The diploidy rate for the overall 3PNs was 37.8% (n = 14). The MT group had 82.4% (n = 14) of diploidy rate, while the PT group had 0% diploid rate (n = 0), showing significant differences between them. (p &amp;lt; 0.001). The overall euploidy rate for 3PN zygotes was 28.6% (n = 12) (36.2 mean age), which was similar to the euploidy rate (30.3%) for our overall 2PNs biopsied during 2023 (38.2 mean age). No differences found. Out of the 14 diploid 3PNs, 28.6% (n = 4) were euploid, showing no differences with our 2023 euploidy rate. Two diploid 3PNs were mosaic, resulting in a total of 6 embryos that could potentially be transferred. Limitations, reasons for caution This study is limited by the number of triploid embryos recruited. As blastocyst rate for 3PNs population is lower than observed in the 2PN population the number of biopsied embryos is reduced. Euploidy rates for 3PNs and 2PNs are very similar, but the mean age differs by 2 years. Wider implications of the findings It is very important to distinguish between both types of 3PNs observed: PT are not worthy to keep and analyse, while MT embryos are very likely to be diploid, and it is worthwhile to culture and analyse them. Patients with few embryos available in particular could benefit from this study. Trial registration number not applicable

P-252 Non-invasive artificial intelligence (AI) prediction of blastocyst development from mature oocytes unveils correlation with blastocyst ploidy for enhanced reproductive insight at earliest stage of development

Abstract Study question Can MAGENTA identify mature oocytes more likely to develop into a blastocyst of euploid status? Summary answer Although built to predict blastocyst development, MAGENTA analysis of mature oocytes additionally displays the ability to highlight those with higher potential to become euploid embryos. What is known already MAGENTA (AI image-analysis tool) has been trained to assess and predict blastocyst stage embryo development from mature, denuded oocytes. MAGENTA scores consistently identify oocytes of better quality – more likely to develop into a blastocyst, including blastocysts of higher morphological quality. However, in addition to achieving a blastocyst, the ploidy status of that embryo is crucial in dictating overall chances of implantation and cycle success. Although MAGENTA was not trained to predict blastocyst ploidy from oocytes, most chromosomal errors are of maternal meiotic origin. A correlation between MAGENTA and ploidy status would ignite research possibilities and provide valuable insights. Study design, size, duration A retrospective dataset of 1,324 blastocyst embryos that underwent PGT-A via NGS sequencing, between the years 2021-2023, was assessed. All embryos that were tested for ploidy from a patient cohort were included. Embryos reported as mosaic or ‘no diagnosis’ were excluded from analysis (N = 169). All embryos were cultured in a time-lapse incubator immediately post-ICSI. Of the biopsied blastocysts, 81% were of good morphological quality (expansion grade 1-6, ICM and TE grade A/B). Participants/materials, setting, methods The data includes 292 patients with a mean age of 38.5 (range 19-46) and an average number of tested blastocysts of 4 (range 1-14). Of the 1173 blastocysts, 847 were aneuploid (72.2%) and 326 were euploid (27.8%). 83% of the blastocysts were from patients of advanced maternal age (AMA, ≥38years old). The first time-lapse image, capturing the mature oocyte stage, was extracted, and scored by MAGENTA (scale 0-10). MAGENTA scores were compared by Welch’s t-tests. Main results and the role of chance Impressively, oocytes that developed into euploid blastocysts (n = 326) were identified by MAGENTA with a higher average score of 6.8 compared to those that developed into aneuploid blastocysts (n = 847) with a score of 6.4; a statistically significant differentiation (p &amp;lt; 0.05). 80% of the dataset were high quality blastocysts (expansion 4-6, ICM and TE grade A/B) with an imbalance of euploid (n = 295; mean score 6.8) and aneuploid (n = 643; mean score 6.6) blastocysts—likely a result of AMA. Therefore, subgroup analysis was focused on assessing the ploidy status relationships amongst several patient age groups within these AMA cases. Due to our clinic’s indication for PGT-A, the smallest subset of oocytes was from patients &amp;lt;38 years of age (N = 373, 48% euploid). In this age group there was no significant difference in MAGENTA scores between the oocytes of euploid and aneuploid embryo status (6.9 vs 6.7; p = 0.522). However, in oocytes from patients aged ≥38 (N = 800, 19% euploid), the distinction between those that became euploid and aneuploid blastocysts is evident with a statistically significant difference in MAGENTA scores (6.8 vs 6.3; p &amp;lt; 0.05). This relationship with ploidy status additionally remains with further stratification into the ≥41 age group (N = 467 oocytes, 13% euploid; 6.7 vs 5.9; p &amp;lt; 0.01). Limitations, reasons for caution Majority of the patients in this dataset underwent PGT-A due to AMA, further research is required to include a wider age demographic, particularly in patients &amp;lt;35 years old. Greater representation of varying blastocyst qualities is required to further assess MAGENTA correlation to ploidy outcomes amongst quality groups. Wider implications of the findings This study presents the first non-invasive assessment of mature oocyte quality that indicates oocytes with greater potential for euploid blastocyst development. This application of MAGENTA provides a novel method of gaining ploidy status potential at the mature oocyte stage, which is invaluable to both IVF and cryopreservation cycles. Trial registration number NOT APPLICABLE

P-216 Whole genome amplification (WGA) of spent culture media may not be a reliable marker of blastocyst prioritization for transfer

Abstract Study question Does the success or failure of DNA amplification from spent culture media of good-quality blastocysts correlate with their ploidy status and clinical pregnancy outcomes? Summary answer Samples with successful DNA amplification exhibited similar rates of euploidy and clinical pregnancy rates compared to spent culture samples with failed DNA amplification. What is known already Studies have detected DNA in spent culture media/blastocoelic fluid (BF) from fully developed blastocysts. After amplification, this DNA is sequenced to provide information on their ploidy status. Some authors reported simply detection of amplified DNA as an indicator of blastocyst viability, so that spent culture media/BF with DNA amplification derive from abnormal embryos, and failed DNA amplification from euploid blastocysts. Along with this line of rationale, some authors associated DNA amplification failure with higher clinical pregnancy rates. Reproducibility of such findings is warranted which may stem from various factors, such as culture conditions, amplification methods and statuses of blastocysts analysed. Study design, size, duration This preliminary set of results is part of a cohort (nonselective) ongoing study, from which 62 patients participated so far from two tertiary IVF centers during the period of January 2022 until October 2023. Only ICSI patients aged &amp;lt;43-year-old with &amp;gt;6 mature oocytes were included. Single frozen blastocyst transfers were performed blinded to the results of the whole genome amplification (WGA) and/or DNA sequencing and pregnancy outcomes recorded. Participants/materials, setting, methods Embryos were singly cultured into 20µl GTL-media in Embryoscope+ incubator (Vitrolife). On day 3, embryos were washed repeated times and placed into new culture dish. Spent media was collected from ≥BL4BB blastocysts (Gardner) on days 5/6 after laser collapse prior vitrification. DNA from culture medium were amplified using MALBAC (Yikon-Genomics). After WGA, DNA was quantified using Qubit 3.0 fluorometer (cut-off of 0.6 ng/µl) and then subjected to next-generation sequencing (NGS) with Illumina MiSeq platform. Main results and the role of chance A total of 104 spent culture media samples were analysed from individual blastocysts. About 70% of samples of spent media had amplified DNA and were sequenced further. From those sequenced samples, 55% resulted in euploidy and 45% in abnormal ploidy status. Embryo transfers took place from blastocysts cultured in 82/104 samples. A total of 36 (44%) clinical pregnancies were obtained from 82 transfers/samples from which, 78% derived from samples with DNA amplification and 22% from samples with failed DNA amplification. Outcomes of clinical pregnancies were verified for both conditions, samples with DNA amplification or failed DNA amplification. A total of 28/55 (51%) samples with DNA amplification resulted in clinical pregnancies, while only 30% (8/27) of failed DNA amplification samples did so (P = 0.06). Positive predictive values (PPV) and negative predictive values (NPV) of ploidy status of sequenced samples in relation to ongoing pregnancy and live birth rates are ongoing. Limitations, reasons for caution The limited sample size. Wider implications of the findings The presence of DNA in the spent culture media may offer an attractive alternative as a non-invasive test for aneuploidy. Research is still ongoing and challenging. Nevertheless, truncating the test at DNA amplification phase seems not a reproducible methodology to serve as a criterion to prioritize embryos for transfer. Trial registration number This research project is financially supported by Theramex Birth Grant program, a competitive grant supporting IVF research in Europe.

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&amp;lt;0.05) and medium and low-quality (6.9 vs 6.1,p&amp;lt;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 &amp;lt; 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 &amp;lt; 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 &amp;lt;35 years old (7.2 vs 6.6, p &amp;lt; 0.01;Welch’s t-test) and ≥35 years old (6.8 vs. 6.5, p &amp;lt; 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&amp;lt;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

P-583 The ploidy status in blastocysts generated from progestin primed ovarian stimulation and GnRH antagonist protocols: A comparison of 14.000 trophectoderm biopsies

Abstract Study question Is there any difference in the euploidy rate in a large cohort of blastocysts when produced after a progestin-primed or GnRH antagonist protocol? Summary answer Euploidy rates in trophectoderm biopsies when tested with next-generation sequencing (NGS) were similar after progestin-primed or GnRH antagonist protocol. What is known already Preimplantation genetic testing (PGT) might be employed in patients with advanced female age to improve efficiency of embryo transfer. Given the fact that there is no fresh embryo transfer in that policy, progestin primed ovarian stimulation (PPOS) protocol might be preferred over GnRH analogs due to its lower cost and advantage of oral administration instead of s.c injections. However, due to small sample sizes within the limited number of studies, there is lack of consensus whether ploidy status might be affected when an embryo is generated after a PPOS or GnRH analogs protocol. Study design, size, duration A retrospective cohort study was designed among patients that has undergone in-vitro fertilization (IVF) treatment between January-2018 and June-2023 in Bahceci Health Group. For the aim of PGT, only patients treated with PPOS or GnRH antagonists were included. PPOS protocol referred to utilization of either medroxyprogesterone acetate (10 mg/day) or dydrogesterone (20-30 mg/day) to avoid premature ovulation during ovarian stimulation. In GnRH antagonist protocol, flexible or fixed approach was preferred for pituitary suppression. Participants/materials, setting, methods During the study period, 11.616 and 2.480 trophectoderm biopsies had been performed from blastocysts (D5/6) generated after GnRH antagonist or PPOS protocol, respectively. Of them, 9.390 and 1.801 of them were analyzed with NGS. A total of 303 (3.2%) and 17 (0.9%) biopsies were excluded due to failure or non-informative results after testing. For the final analysis, 9.087 and 1.784 biopsies were included in the antagonist and PPOS arms for the current study. Main results and the role of chance The mean age of women treated with GnRH antagonist and PPOS was comparable (36.5±5.3 vs. 36.7±5.4 years, p = 0.17). The respective numbers of euploid embryos were 3.912 and 1.784. Per biopsied embryos, the rate of euploidy was 33.7% and 34.8% (p = 0.30). The rate of euploidy per metaphase II oocyte and 2-PN were 10.6% and 13.5% in the GnRH antagonist arm and they were 11.3% and 14.4% in the PPOS arm (p = 0.08 and p = 0.07) without any statistical significance. When only patients included in whom all available blastocysts were biopsied, the linear regression model revealed only female age (RR: -0.32 CI%95: -0.38 to – 0.26, p &amp;lt; 0.001) as the independent factor for the prediction of ploidy rate (euploid/euploid + aneuploid embryos) when body mass index, duration of infertility, AMH, number of oocytes, number of metaphase-II oocytes, and the type of ovarian stimulation protocol were entered into the model. This model represented female age as the sole independent factor (RR: -0.14 CI%95: -0.18 to – 0.11, p &amp;lt; 0.001) when euploidy rate per 2PN was tested with the same variables. Limitations, reasons for caution The inherent risks cannot be excluded due to the retrospective nature of the current study. Wider implications of the findings The current study includes the largest number of trophectoderm biopsies generated from PPOS and GnRH antagonist protocol, giving the opportunity to compare them with each other. Due to lack of any superiority of one protocol regarding ploidy status, financial cost and easiness might favor PPOS when PGT is planned. Trial registration number None

DNA ploidy and PTEN as biomarkers for predicting aggressive disease in prostate cancer patients under active surveillance

BackgroundCurrent risk stratification tools for prostate cancer patients under active surveillance (AS) may inadequately identify those needing treatment. We investigated DNA ploidy and PTEN as potential biomarkers to predict aggressive disease in AS patients.MethodsWe assessed DNA ploidy by image cytometry and PTEN protein expression by immunohistochemistry in 3197 tumour-containing tissue blocks from 558 patients followed in AS at a Norwegian local hospital. The primary endpoint was treatment, with treatment failure (biochemical recurrence or initiation of salvage therapy) as the secondary endpoint.ResultsThe combined DNA ploidy and PTEN (DPP) status at diagnosis was associated with treatment-free survival in univariable- and multivariable analysis, with a HR for DPP-aberrant vs. DPP-normal tumours of 2.12 (p < 0.0001) and 1.94 (p < 0.0001), respectively. Integration of DNA ploidy and PTEN status with the Cancer of the Prostate Risk Assessment (CAPRA) score improved risk stratification (c-index difference = 0.025; p = 0.0033). Among the treated patients, those with DPP-aberrant tumours exhibited a significantly higher likelihood of treatment failure (HR 2.01; p = 0.027).ConclusionsDNA ploidy and PTEN could serve as additional biomarkers to identify AS patients at increased risk of developing aggressive disease, enabling earlier intervention for nearly 50% of the patients that will eventually receive treatment with current protocol.

O-248 Novel early cleavage parameters improve embryo selection and reach ploidy predictive values comparable to artificial intelligence

Abstract Study question Can we improve embryo selection and ploidy prediction using novel morphokinetic parameters in blastocyst development, and obtain results comparable to artificial intelligence? Summary answer Time and pattern of novel parameter st2 are related to ploidy. Including them in embryo evaluation improves ploidy prediction, reaching predictive values comparable to AI. What is known already Time-lapse has allowed us to detect more morphokinetic parameters and improve embryo grading systems. However, ploidy prediction still relies on invasive techniques to obtain a reliable result. Artificial intelligence is promising and can already predict blastulation or implantation with good results, but not ploidy yet. Moreover, AI presents some other limitations now, including the black box behind its decisions, which doesn’t allow clinicians to know what parameters are being considered the most. Improving the manual evaluation tools used by embryologists can help improve clinical outcomes until AI is fully introduced in IVF. Study design, size, duration A single center retrospective study was performed. Data included a total of 608 blastocyst from 106 treatments between January 2018 and July 2022. Participants/materials, setting, methods 160 treatments of patients for preimplantation genetic testing cycles were studied. Embryos were cultured in time-lapse incubators and were studied for both morphologic and kinetic parameters. 608 of the embryos developed to good quality blastocyst and were able to be biopsied, and therefore analyzed by PGT for aneuploidies on Day5 o Day6. Annotation of several morphokinetic characteristics was performed on all analyzed embryos. The study was blinded for ploidy and all clinical outcomes. Main results and the role of chance Improved embryo grading systems used by embryologist can reach high predictive values comparable to artificial intelligence. We present a new parameter, “st2, start of t2”, which refers to the first cytoplasmic movements detected at the beginning of the first cell cleavage, as highly implicated in ploidy status. Earlier st2 is associated to better euploidy rates (p &amp;lt; 0.001). Not only the time, but also the phenotype of the movements appears as related to ploidy. Embryos showing none or un-patterned random cytoplasmic movements show a marked tendency towards aneuploidy. Contrary, circular waves prior to cell division are highly associated with euploidy (90% of the cases) (p &amp;lt; 0.0001). The implication of the described parameters was also confirmed by logistic regression, with a receiver operating curve (ROC) value of 0.69 for ploidy prediction (95% confidence interval (CI), 0.62 to 0.76). We processed raw time-lapse images with an automatic annotation software using artificial intelligence and obtained a ploidy predictive value of 0.64. This means that using the optimal parameters in manual embryo evaluation, we are able to improve our grading systems and reach ploidy predictive values comparable than with AI. Limitations, reasons for caution This is a retrospective study. The ploidy predictive potential is now being tested on a prospective study. Wider implications of the findings Optimizing the indicators to select the most suitable blastocyst, like including st2, can help reducing the time until the pregnancy of an euploid baby, avoiding invasive and expensive methods. Moreover, by combining AI with the novel parameter st2 we could improve the potential of the ploidy predictions. Trial registration number not applicable