Preimplantation Genetic Testing For Aneuploidy Research Articles

P-529 Optical Genomic Mapping (OGM) enable cryptic chromosomal rearrangements in a couple with recurrent miscarriages undergoing preimplantation genetic diagnosis

Abstract Study question Could Optical Genomic Mapping (OGM) detect chromosomal structural variations (SVs) below the detection limit of conventional cytogenetic diagnostics in couples with recurrent miscarriages? Summary answer The Next-generation cytogenetics of Optical Genomic Mapping confirmed a cryptic chromosomal translocation in a couple with recurrent miscarriages undergoing preimplantation genetic testing for aneuploidies (PGT-A). What is known already Chromosomal structural variants (SVs) significantly contribute to infertility, recurrent pregnancy losses and congenital defects... Approximately 2-4% of couples facing recurrent miscarriages carry chromosomal abnormalities. Currently, their detection is based on convencional cytogenetic methods (karyotyping, FISH or array-CGH), however cryptic chromosomal rearrangements are difficult to identify. Next-generation cytogenetics such as OGM is able to detect SVs with a resolution of < 500 kb, allowing a better understanding of the cause of patient’s infertility. In our study, we utilized OGM to confirm a cryptic translocation in a couple experiencing recurrent miscarriages, aiming to enhance our understanding of the underlying causes of their infertility. Study design, size, duration Our study focuses on a non-consanguineous couple diagnosed with recurrent pregnancy loss that attended to our private fertility clinic to undergo assisted reproductive treatment (ART) with their autologous semen, oocytes and PGT-A. Conventional cytogenetic technique was performed on both partners by G-banding karyotyping. PGT-A was performed by NGS (Veriseq, Illumina). Optical Genomic Mapping (OGM) served as a diagnostic tool, complemented by lymphocyte FISH analysis to confirm chromosomal structural variations in these infertile patients. Participants/materials, setting, methods Ultra High Molecular Weight genomic DNA (UHMW gDNA) was extracted from frozen human peripheral blood of both partners. UHMW gDNA was quantified using Qubit dsDNA assay BR kit and Qubit 4 Fluorometer (Thermo Fisher Scientific). We used Saphyr system (Bionano Genomics) for cytogenetic diagnosis. The consensus genomic map was constructed by de novo assembly in the Bionano Solve data analysis software. SVs were identified by aligning Optical Genomic Map with the reference genome GRCh38/hg38. Main results and the role of chance The couple had suffered four prior miscarriages and undergoing three PGT-A cycles. Both partners provided a normal karyotype. Among 13 biopsied embryos, four showed CNVs on chromosomes 2(p24.3è25.3, 12Mb) and 6(p24.1è25.3, 11Mb). Following this result we suspected that the couple might has a cryptic chromosomal structural abnormalities. To test our hypothesis, we performed OGM. We analysed UHMW gDNA samples from both partners. OGM generated an average of 823.67Gb. The average N50 molecule length (≥150kb) was 318kb. The average mapping rate was 90.75% and the average labeling rate was 15.30 labels per 100kb. The average effective coverage depth was 242X. OGM successfully identified a cryptic translocation in the male partner ogm[GRch38]t(2;6)(p24.3;24.1), while the female partner showed no abnormalities. Translocation breakpoints identified by OGM were Chr2:12.560.508 and Chr6:11.622.593. The translocated segments were near to the detection limit of conventional cytogenetic analysis and with similar pattern that could be the reason for missed detection by karyotyping. Importantly, we identified one gene disrupted by OGM rearrangement breakpoint: MIR3681HG. A previous study considered that CNV loci of 2p24.3, including MIR3681HG gene, could be associated with spermatogenesis. The lymphocyte FISH analysis finding corroborated the OGM result. Limitations, reasons for caution OGM is a very powerful method for detecting chromosomal structural variants in human genomes and is able to detect cryptic structural variants. However, this is a very new diagnostic application, so there is limited experience with OGM for correlating structural variations results to infertility diagnosis in couples with recurrent miscarriages. Wider implications of the findings A balanced cryptic translocation has been identified in a couple with recurrent miscarriages. Our case report demonstrates the high diagnostic capability of Optical Genomic Mapping for cryptic SVs. OGM technology allows a more accurate cytogenetic diagnosis by detecting cryptic structural variants with high resolution in infertile patients with recurrent miscarriages. Trial registration number not applicable

P-531 Embryo grading in PGT-A testing: does it really matter?

Abstract Study question Can embryo morphology grading be a good parameter to predict the embryo transfer’s outcome when euploid embryos are used? Summary answer Gardner’s grading is a useful predictor of LBR per euploid embryo transferred. Morphologic grading should be used for the embryo selection among euploid blastocysts. What is known already When preimplantation genetic testing for aneuploidies (PGT-A) is not performed, the selection of the embryo(s) to be transferred is based on embryo morphology. In PGT-A cycle, some studies have minimised the importance of embryo morphology, when euploid embryos are present. However, others still consider it as good predictor. Understanding the role of morphology in biopsied euploid embryos may help in optimizing frozen embryo transfer cycles. Study design, size, duration We retrospectively analysed PGT-A frozen embryo transfers with reported outcome, performed in our clinics between 2018 and 2022. The cycles included were divided into two groups based on Gardner’s grading of euploid embryos transferred: “good” embryos (AA, AB, BA and BB; n = 263) versus “poor” embryos (including AC, CA, BC, CB, and CC; n =112). Participants/materials, setting, methods We included in the study only single euploid embryo transfers, double embryos transfers and mosaic embryo transfers excluded. Body mass index (BMI), oocyte age and outcomes (pregnancy and live birth rates) were compared between the two groups. Data was statistically analysed using MedCalc Software, where P < 0.05 was considered as significant. Main results and the role of chance A total of 375 embryo transfer cycles were included in the study. Maternal ages were not significantly different between groups (good vs poor embryos) (37.5 vs 38.2). Similarly, oocyte age (37.7 vs 38.1, respectively), and BMI (22 vs 22.7, respectively) did not differ between good and poor embryos. On the contrary, clinical pregnancy (56.6% vs 40.1%) and live birth rates (27.7% vs 13.4%) were significantly higher when morphologically good grade embryos were transferred (p < 0.05). In fact, both rates were significantly higher after the transfer of blastocysts that exhibited A-grade or B-grade in inner cell mass and/or trophectoderm, as opposed to the transfers of blastocysts that exhibited a C-grade trophectoderm and/or inner cell mass. Limitations, reasons for caution The findings apply to the participating centres and may not represent worldwide trends. Data was analysed retrospectively, but details of the follow-up on PGT pregnancies and babies born were limited. The degree of blastocyst expansion and the day of biopsy were not taken into consideration in this study. Wider implications of the findings The Gardner embryo quality assessment improves the clinics’ ability to prioritize embryos after PGT. Preferential transfer based on morphologic criteria increases the chances of a positive outcome in terms of live birth rates. Trial registration number not applicable

P-535 Effect of advanced female age (AFA) on the ratio of patients receiving mosaic embryo transfers

Abstract Study question How does female age affect ratio of patients with at least one euploid embryo (≥1EE) and at least one mosaic embryo without any euploid ones(≥1ME)? Summary answer Percentage of patients receiving mosaic embryo transfers increases with female age. What is known already Introduction of Next Generation Sequencing (NGS) to the area of Preimplantation Genetic Testing for Aneuploidy (PGT-A) has enabled detection of mosaicism in human embryos. Studies reporting the birth of healthy babies after the transfer of embryos with a chromosomal mosaic result on PGT-A have been published (Greco et al., 2015; Kahraman et al., 2020; Viotti et al., 2021 and 2023). Data suggested lower implantation rates and higher miscarriage rates when mosaics were compared with euploid embryo transfers. The percentage of mosaic embryos in overall tested embryos decreases with increased maternal age, similar to euploid embryos’ decline (Sanders et al., 2023). Study design, size, duration This retrospective study includes 7287 cycles with PGT-A performed between January 2017 and December 2023. Number of biopsied blastocysts was 21,277 and all were tested with NGS. All PGT-A testing was conducted on Ion Torrent S5 (Thermo Fisher Scientific) according to the manufacturer guidelines. The mosaic embryo reporting thresholds were determined as 20 to 80%. Participants/materials, setting, methods Euploidy ratio (ER) was defined as (euploid embryos (n)/ all tested embryos(n)). Mosaicism ratio (MR) was defined as (mosaic embryos (n)/all tested embryos(n)). Alternative mosaicism ratio (AMR) was defined as the number of mosaic embryos divided by the sum of euploid embryos and mosaic embryos. In other words, AMR can be defined as the number of mosaic embryos divided by non-aneuploid embryos. Female ages were divided into five categories: FAG1:<35, FAG2:35-37, FAG3:38-40, FAG4:41-42, FAG5:>42. Main results and the role of chance The number of cycles, total number and average biopsied embryos with standard deviations were highest in FA1 and declined with AFA: FAG1:2537, 9298, 3.7±2.2; FAG2:1348, 4140, 3.1±1.7; FAG3:1701, 4298, 2.5±1.6; FAG4: 958,2116, 2.2±1.5; FAG5:738, 2407, 1.9±1.2. ER and MR were observed to decrease with AFA: FAG1:%48.4, %15.2; FAG2:%39.0, %12.7; FAG3:%26.5, %10.5; FAG4:%14.9, %6.0; FAG5:%5.0, %2.6. This decrease was statistically significant (p < 0.0001, Chi-Square Test). However, when AMR was calculated among age groups, AMR was increasing with AFA: FAG1:%23.9; FAG2:%24.6; FAG3:%28.5;FAG4:%28.7; FAG5:%34.5 and this was also statistically significant (p = 0.0002, Chi-Square Test). Correlation of MR and AMR with female age groups were tested with Pearson correlation coefficient and MR was found to be negatively correlated with AFA whereas AMR was positively correlated (r=-0.19, p < 0.0001; r = 0.07, p < 0.0001, respectively). The ratio of patients with ≥1EE and ≥1ME was found to be decreasing with AFA when compared to overall tested cycles: FAG1: %83%, %7; FAG2: %69, %9; FAG3: %47, %11; FAG4: %28, %9; FAG5: %9, %4. However, the ratio of ≥ 1ME in cycles with transferrable embryos significantly increased with AFA consisting of more than one fourth of embryo transfer cycles above 38 years of age: FAG1: %7; FAG2: %12; FAG3: %19; FAG4: %24; FAG5: 31%. Limitations, reasons for caution These are the results of a single ART and Reproductive Genetics Center with its strictly defined standard operating procedures for PGT-A testing and ovarian stimulation protocols. Although not expected, different PGT-A testing methodologies or stimulation protocols may influence the above-mentioned outcomes. Wider implications of the findings The alternative mosaicism ratio defined here is generally overlooked but impacts the treatment strategies of ART patients, specifically for the advanced maternal age groups. Trial registration number not applicable

O-089 Association of pituitary suppression method with euploid blastocyst yield in preimplantation genetic testing for aneuploidy (PGT-A) cycles

Abstract Study question Do progestin primed ovarian stimulation (PPOS) cycles have lower euploidy yield compared to pituitary suppression with gonadotropin releasing hormone (GnRH) antagonists? Summary answer The choice of pituitary suppression method has no bearing on euploid retrieval per blastocyst tested. What is known already Progestin primed ovarian stimulation cycles are an alternative to GnRH antagonists when fresh embryo transfer is not a priority. While the two approaches have been shown to be similar in terms of conventional stimulation outcomes such as mature oocyte retrieval, and blastulation rates, there is still some lingering controversy regarding cumulative pregnancy rates and the effects of progestins on embryo genetic constitution. Study design, size, duration This was a multicenter retrospective cohort study including data from two tertiary-level treatment centers. Participants/materials, setting, methods Infertile women undergoing ovarian stimulation with either progestin primed or GnRH antagonist pituitary suppression with the intention of transfer after PGT-A embryo testing with next-generation sequencing. The main outcome parameter was euploid embryo count per blastocyst tested and was modeled with mixed-effects log-binomial regression models. Results were adjusted for patient age and laboratory performing the PGT-A tests while they were not adjusted for embryo quality to avoid collider bias. Main results and the role of chance A total of 1425 PGT-A tested blastocysts from 383 cycles of 365 patients were included in the analysis. 243 cycles (63.4%) received GnRH antagonists for pituitary suppression while 140 (36.5%) received progestins. The overall euploidy rates per blastocyst tested in the GnRH antagonist and PPOS groups were 38.8% vs. 31.1%, 30.2% vs. 29.4%, 27.0% vs. 25.2%, and 13.8% vs. 14.5% for the age categories <35, 35-37, 38-40, and >40, respectively. After adjusting for the effect of female age at testing and the laboratory performing the PGT-A test, the type of pituitary suppression was not significantly associated with euploid retrieval per blastocyst tested (adjusted risk ratio: 0.94, 95% CI: 0.70 - 1.28, P = 0.701). After testing, 191 women received 233 embryo transfers. Human chorionic gonadotropin (hCG) positivity per transferred embryo was not significantly different between progestin primed and GnRH antagonist cycles (RR: 0.89, 95% CI: 0.71 – 1.08, P = 0.268). Limitations, reasons for caution This is a retrospective study with data from multiple centers with a non-standardized selection of blastocysts to be tested. In addition, the sample size and retrospective nature of the data may have biased the estimates. Wider implications of the findings The choice of pituitary suppression method does not appear to have a significant effect on euploid embryo retrieval rates or hCG positivity per transfer. The choice of medication can be based on the needs of the individual case without concern for suboptimal PGT-A outcomes. Trial registration number Not Applicable

P-553 Embryo collection improves clinical pregnancy outcomes in next-generation sequencing(NGS)-based preimplantation genetic testing for aneuploidy (PGT-A) euploid embryo transfers

Abstract Study question Is there still a significant impact of embryo-related characteristics on pregnancy rates in NGS based PGT-A euploid embryo transfers for advanced-age patients? Summary answer In single frozen euploid embryo transfers, priority of embryo transfer and grade significantly influence clinical pregnancy rates in women over 35 years old with PGT-A. What is known already It is widely recognized that better embryo grades and a shorter duration of blastulation are associated with favorable reproductive outcomes. Recent meta-analyses have affirmed these findings even in the context of transfer with euploid embryos. However, the impact of prioritizing the highest-ranked embryo among multiple vitrified euploid embryos on pregnancy rates remains unclear. The lack of research in this area implies a gap in understanding whether selecting embryos for transfer among multiple viable euploid embryos could enhance reproductive success. Study design, size, duration Throughout the year 2022, a retrospective review was conducted on 428 cycles of single frozen-thawed euploid embryo transfers performed at a single institution, targeting patients aged 35 and older. The study aimed to explore potential differences in clinical pregnancy rates based on embryo grade, embryo freezing date, and embryo selection priority. Participants/materials, setting, methods Women ≥ 35 years old with single frozen euploid blastocyst transfer cycles was categorized into three groups: (a) with one blastocyst (1/1), (b) with ≥ two blastocysts having the top-priority(½ or higher), and (c) lower-priority blastocysts (lower than ½) to examine potential differences in clinical pregnancy rates among groups based on embryo characteristics, including Gardner grading and day of embryo freezing. Results highlight the impact of both selection criteria and freezing conditions on pregnancy outcomes. Main results and the role of chance This study analyzed a total of 428 frozen embryo transfer (FET) cycles. In the context of frozen-thawed single embryo transfer, the clinical pregnancy rate was 52.2% (128/245) when only one euploid embryo was frozen and subsequently thawed for transfer (a). For cases where two or more euploid embryos were frozen, and embryos with a higher priority (½ or higher) were selected for transfer (b), the clinical pregnancy rate significantly increased to 71.3% (62/87). Conversely, when embryos with a lower priority (lower than ½ in order) were chosen for transfer (c), the clinical pregnancy rate was lower at 38.5% (15/38), indicating a statistically significant difference between the groups (p = 0.001). Categorizing embryos based on Gardner grading, “good” embryos (>3BB) demonstrated a clinical pregnancy rate of 66.8% (141/211), while embryos with lower quality (3BB or below) showed a significantly lower clinical pregnancy rate of 40.6% (88/217) (p < 0.001). The analysis of clinical pregnancy rates based on the embryo freezing day revealed significant differences among embryos freezing on 5 days (71.9%, 133/185), 6 days (39.9%, 71/178), 7 days (25%, 2/8), and thawing PGT-A embryos (38.2%, 21/55) (P < 0.001). Limitations, reasons for caution One limitation of this study is its retrospective nature, and the potential confounding effect of the close association between embryo selection and embryo grading. Further analysis will be conducted to confirm correlations and address this potential confounding effect. Wider implications of the findings In single euploid frozen embryo transfers, selecting the highest-quality embryo through careful embryo selection remains relevant. Morphological indicators of embryos significantly impact early pregnancy outcomes, highlighting the continued importance of strategic embryo accumulation for enhanced procedural success. Trial registration number not applicable

P-563 Improved diagnosis in PGT-A samples with intermediate sex chromosomes copy number changes by adding ploidy and contamination analysis

Abstract Study question Does triple analysis of aneuploidy, ploidy and contamination improve the diagnosis in samples with intermediate copy number changes for the X and the Y chromosomes? Summary answer PGT-A with additional ploidy and contamination analysis improves the diagnosis of samples with intermediate sex chromosomes copy number changes, reducing non-informativity and the re-biopsy need. What is known already Next Generation Sequencing (NGS) in Preimplantation Genetic Testing for Aneuploidies (PGT-A) identifies chromosomal aneuploidies in trophectoderm (TE) biopsies. However, diagnosis limitations exist when the TE biopsy displays intermediate copy number changes for the X and the Y chromosomes, observed in ∼0.3% of biopsies. Few explanations are plausible, including mosaicism for the sex chromosomes, presence of exogenous DNA contamination or triploidy. A second TE biopsy (re-biopsy) could be recommended to obtain a conclusive result. However, additional Single Nucleotide Polymorphism (SNP) analysis to determine the ploidy status of the embryo and the presence of contamination in the biopsy can be an alternative. Study design, size, duration In this retrospective study, the impact on the diagnosis of SNPs analysis with a target panel was evaluated in PGT-A samples showing intermediate copy number changes for the X and the Y chromosomes. A total of 21 TE biopsies from January to December 2023 were included in the study. Participants/materials, setting, methods The TE biopsies were analysed using NGS with a proprietary protocol for library preparation, Ion Chef and Ion S5 System instruments for sequencing and a proprietary bioinformatics pipeline (v2.0) for 24-chromosomes aneuploidy testing. A re-biopsy was analysed in 8 embryos using the same protocol. Eighteen original biopsies were retrospectively evaluated for ploidy and contamination using an Ion AmpliSeq Custom Panel including SNPs (Thermo Fisher Scientific, USA) and a proprietary algorithm (v1.0) for data analysis. Main results and the role of chance The 21 TE biopsies displaying intermediate copy number changes for the X and the Y chromosomes were initially diagnosed as non-informative and candidates for re-biopsy to rescue diagnosis. A total of 8 embryos were re-biopsied (38.1%): 6 re-biopsies (75.0%) showed the same intermediate sex chromosomes copy number changes suggesting a triploid status of the embryo, 1 re-biopsy (12.5%) showed trisomy 22 suggesting DNA contamination in the original biopsy and, 1 re-biopsy (12.5%) displayed a mosaic monosomy X/XY suggesting a diploid-mosaic status of the embryo. In 18 original TE biopsies (85.7%), an additional ploidy and contamination analysis was done: 10 samples were triploid (55.6%), 6 samples showed DNA contamination (33.3%) and 2 samples were diploid (11.1%). In 5 embryos, the re-biopsy with the aneuploidy testing had been done: (i) in 4 triploid embryos the re-biopsies showed the same intermediate copy number changes for the sex chromosomes than the original biopsy, confirming the triploid status of the embryo; (ii) in 1 diploid embryo the re-biopsy showed a mosaic monosomy X/XY, confirming the diploid status and the sex chromosome mosaicism of the embryo. From the 21 embryos initially non-informative, only the 6 contaminated ones remained non-informative and candidates for re-biopsy (33.3%). Limitations, reasons for caution The limited number of samples with both analysis, ploidy/contamination by SNPs in the original sample and aneuploidy by NGS in the re-biopsy precluded to drawn stronger conclusions. This double-check would reinforce the benefit of doing additional ploidy/contamination analysis in these specific cases. Wider implications of the findings A high incidence of samples with intermediate sex chromosomes copy number are triploid, some have exogenous DNA contamination, and few are real mosaics for the sex chromosomes. Additional ploidy and contamination analysis could improve the PGT-A diagnosis and decrease mosaicism and non-informativity rates, reducing the embryos that would need re-biopsy. Trial registration number Not applicable

P-244 Which embryo selection model should be prioritized for transfer of a single euploid blastocyst?

Abstract Study question Which embryo selection model would be given high priority for single euploid blastocyst transfer, iDAScore® v2.0, KIDScoreTM D5 v3.2 or conventional morphological grading system? Summary answer Prioritize morphological grading over embryo score for single euploid blastocyst transfers, and reverse the priority for single non-PGT-A blastocyst transfers. What is known already The use of effective tools, such as AI, morphokinetic data and the Gardner criteria, for embryo selection within in vitro fertilization (IVF) has the potential to increase the possibility of pregnancy. Nevertheless, previous studies indicated that the best embryo these tools prioritize may be inconsistent and controversial. Therefore, this study demonstrates integrated reference tables of FHB+ rates for selecting of single embryo transfer (SET) clinically, and assess the association between iDAScore®, KIDScoreTM, traditional morphological grading and embryonic ploidy status. Study design, size, duration Retrospective study from November 2022 to October 2023 with morphokinetic parameters of time-lapse monitoring analysis involving embryos from 264 patients who underwent day-5 single vitrified-warmed blastocyst transfer with or without preimplantation genetic testing for aneuploidies (PGT-A) (total SET=264; euploid=158; non-biopsied=106) using Embryocope® Plus incubators. After SET, the rates of FHB+ of each group were analyzed. Mean female age was 35.2 years. Participants/materials, setting, methods Blastocyst morphology was scored in 3 groups: good (AA, AB, BA), average (BB), poor-quality embryos (BC, CB, CC). Blastocysts were ranked established on the scores caused by iDAScore or KIDScore. All cycles were stratified into 4 score groups (8.1-9.9, 6.1-8.0, 3.7-6.0, 1.0-3.6). Assisted hatching was conducted on day 3. Trophectoderm biopsies were performed on day 5, and examine the embryonic ploidy status using a next-generation sequencing-based PGT-A platform (Thermo/Ion GeneStudio™ S5 Prime). Main results and the role of chance In D5 non-PGT-A SET group, the FHB+ rates of good-quality embryos were higher than those of average and poor-quality embryos (49% vs. 40% vs. 0%). According to the embryo score group, the group with higher scores showed better FHB+ rates (KIDScore: 56.9% vs. 47.2% vs. 16.7% vs. 0%; iDAScore: 59.1% vs. 49.6% vs. 14.9% vs. 0%). The non-PGT-A results indicated that embryo score should be given high priority. In D5 euploidy SET group, the FHB+ rates of good-quality embryos were higher than those of average-quality embryos (78.12% vs. 56.00% vs. 44.44%). According to the embryo score group, the group with higher scores did not show distinct better FHB+ rates (KIDScore: 65% vs. 69.51% vs. 47.6% vs. 0%; iDAScore: 64.1% vs. 67.7% vs. 38.8% vs. 0%). The PGT-A results indicated that morphological grading could be given high priority. Based on the data, we hypothesized that assisted hatching might lead to distortion in embryo scores. Limitations, reasons for caution The study is limited by its retrospective single center study with small sample size of single embryo transferred even some of the groups have no embryo transfer. Therefore, a prospective multicenter validation is necessary. Wider implications of the findings This study provides distinct tables for clinical embryo selection with or without PGT-A. Our findings support the potential application of the integration of AI, morphokinetic data and embryo quality prediction systems, and indicate the possibility of making appropriate decisions in different age groups clinically. Trial registration number Not Applicable

P-539 Chaotic blastocysts in preimplantation genetic testing for aneuploidies: prevalence, characterization and re-biopsy results

Abstract Study question Are we excluding blastocysts with favorable reproductive potential when embryos are designated as chaotic by Preimplantation Genetic Testing for Aneuploidies (PGT-A)? Summary answer The re-biopsy of chaotic blastocysts can rescue embryos with favorable reproductive potential only when there are ≥5 chromosomes displaying intermediate copy number values (CNV). What is known already An embryo is deemed as chaotic when multiple aneuploidies (5, 6 or more) are detected in the biopsied cells of the trophectoderm. Such embryos are classified as aneuploid, therefore their transfer is not recommended. There is evidence that chaotic embryos may result from the biopsy of apoptotic cells, cellular fragments and, more broadly, DNA degraded by multiple factors. Studies involving the re-biopsy and re-testing of a limited number of chaotic embryos have found a relevant percentage of euploid results. Moreover, there has been published a case report of a healthy live birth after the transfer of a chaotic embryo. Study design, size, duration This is a retrospective study of 1,442 cycles of PGT-A performed in twenty-two in vitro fertilization (IVF) clinics from November 2017 to December 2023. A total of 5,801 blastocysts were biopsied and analyzed in a single reference laboratory using Next-Generation Sequencing. Ninety-three chaotic blastocysts donated from patients who provided informed consent were thawed for re-biopsy in two IVF clinics. The subsequent re-analysis was performed in the same PGT laboratory using the same methodology. Participants/materials, setting, methods This study assessed the prevalence of chaotic embryos in all included cycles. Chaotic embryos were considered those with ≥5 chromosomes showing CNV below 1.75 or above 2.25. We sub-classified these embryos into pure chaotic (≥5 uniform aneuploidies), mixed chaotic (≥5 alterations considering uniform aneuploidies and chromosomes with intermediate CNV) and mosaic chaotic (≥5 chromosomes with intermediate CNV). Re-biopsies of 69 chaotic blastocysts were compared to the initial PGT-A results. Main results and the role of chance The prevalence of chaotic embryos was 4.3% (251/5,801); 13.5% (n = 34) pure, 53.0% (n = 133) mixed and 33.5% (n = 84) mosaic. After thawing 93 embryos, the survival rate was 74.2%. The 69 embryos that survived underwent re-biopsy yielding the following PGT-A results: 24.6% (n = 17) euploid, 37.7% (n = 26) chaotic, 26.1% (n = 18) aneuploid non-chaotic, 7.2% (n = 5) mosaic and 4.4% (n = 3) non informative. Nevertheless, outcomes varied substantially when the analysis was conducted based on the sub-category of chaotic embryos. In the pure category (n = 11), 72.7% (n = 8) showed identical results to the initial biopsy while 27.3% (n = 3) were aneuploid non-chaotic confirming at least one aneuploidy identified at the initial biopsy. Among the mixed category (n = 24), 54.2% (n = 13) were also mixed chaotic, and 45.8% (n = 11) were aneuploidy non-chaotic. All re-biopsies in this category also manifested at least one common aneuploidy with the initial biopsy or its opposite (trisomy/monosomy). In the mosaic category (n = 31), 54.8% (n = 17) were euploid, 16.1% (n = 5) were mosaic chaotic, 16.1% (n = 5) were mosaic non-chaotic and 12.9% (n = 4) were aneuploid non-chaotic. The concordance with the initial biopsy in terms of aneuploid result was 100% in the pure and mixed chaotic categories. However, over half of the embryos initially categorized as mosaic chaotic were, in fact, euploid. Limitations, reasons for caution Implantation, miscarriage and live birth rates are not explored. These outcomes are crucial for understanding the clinical significance of euploid embryos initially deemed as chaotic. The survival rate of chaotic embryos should be considered as it is lower than the survival rate of vitrified blastocysts reported in most IVF laboratories. Wider implications of the findings Previous research has suggested that re-biopsy can rescue embryos from PGT-A cycles given that a chaotic result has reduced predictive value. However, our current study reveals that not all chaotic embryos are suitable for re-biopsy, as 100% of pure and mixed chaotic blastocysts were aneuploid when re-tested. Trial registration number Not applicable

P-557 The contribution of PGT-A to PGT-M and PGT-SR clinical outcomes – A single centre retrospective analysis

Abstract Study question To determine if the introduction of concurrent testing with PGT-A has improved clinical outcomes for PGT-M/SR patients. Summary answer This study demonstrates that the introduction of concurrent testing may have improved clinical outcomes for this patient demographic irrespective of age. What is known already Preimplantation Genetic Testing (PGT) is utilised to investigate the genetic constitution of embryos. PGT-M (monogenic) and PGT-SR (structural rearrangements) are screening techniques considered in treatment for couples at risk of conceiving offspring with known genetic or structural chromosomal abnormalities respectively. Preimplantation Genetic testing for Aneuploidy (PGT-A) was developed in order to screen embryos for numerical chromosomal abnormalities, a leading cause of failed implantation. Nevertheless, routine use of PGT-A is discouraged due to lack of evidence regarding its efficacy. However, PGT-A is routinely used alongside PGT-M and PGT-SR despite the lack of evidence of its effectiveness in improving patient outcomes. Study design, size, duration The retrospective analysis of 496 PGT-M/SR single embryo transfer patient cycles between December 2013 and December 2021. Participants/materials, setting, methods The retrospective analysis of 496 PGT-M/SR single embryo transfer cycles at CRGH Portland was undertaken (single testing n = 77; concurrent testing n = 419). The clinical outcomes (positive urine pregnancy test, clinical pregnancy, live birth and miscarriage rates) were compared between groups using IBM SPSS Statistics (premium 27). Binary logistic regression was performed for statistical analysis, results with p < 0.05 were determined statistically significant. Main results and the role of chance Overall, concurrent testing was associated with statistically significant improved clinical outcomes compared to the reference group. Statistical significance was seen in positive urine pregnancy test (49% vs 68%, p = 0.012), clinical pregnancy (32.5% vs 63%; p = 0.000013), live birth rate (32.5% vs 59%; p = <0.00016) and miscarriage rates (34.2% vs 13%; p = 0.001). The results imply that the introduction of concurrent testing has improved the probability of obtaining a positive clinical outcome and has reduced the chance of miscarriage in this patient demographic. Further analysis showed concurrent testing improved the chances of obtaining a positive clinical outcome irrespective of the patients age or quality of the embryo. Limitations, reasons for caution This study has produced promising results. However, there are several limitations such as the small sample size, embryo biopsy techniques used, and the different molecular diagnostic techniques and providers utilised. Wider implications of the findings The results imply a justification for the use of PGT-A alongside PGT-M/SR screening techniques to improve embryo selection and clinical outcomes for this cohort of patients. Additionally, they demonstrate the efficacy of PGT-A in a previously underrepresented demographic. However, further research is required to confirm the clinical value of PGT-A. Trial registration number not applicable

O-065 PGT-A reveals triploid embryos among normally fertilized oocytes: Frequency and blastocyst features

Abstract Study question Is there any embryo parameter that could differentiate 2 pronucleus (PN) triploid from 2 PN euploid embryos? Summary answer Accuracy in the fertilization check is still necessary as 2 PN but triploid embryos are due mostly to retention of the second polar body (PB). What is known already Since blastocyst culture has evolved and the development of genetic technologies enables to know the karyotipe of the embryos, the concept of normal fertilization is being revised. Commonly, correct fertilization is denoted by the appearance of 2 PN 16-18h after insemination. Deviation from 2PN is considered evidence of abnormal fertilization and ploidy anomalies. However, not all oocytes exhibiting 2 PN are euploids. After performing NGS in trophectoderm biopsies for preimplantation genetic testing for aneuploidy (PGT-A), a percentage of embryos are categorized as triploids. Study design, size, duration Retrospective study including 1862 cycles from 1605 patients aged 18-45 years old who underwent PGT-A with NGS of trophectoderm biopsies in our IVF center from 2019 to 2023. From 6526 analyzed blastocysts in an independent Genetics laboratory, 59 were categorized as triploids. These 59 blastocysts were obtained from 57 PGT-A cycles and belonged to 54 women. In total, these patients had 266 biopsied blastocysts. From the remaining 207 embryos, 42 were called as euploids. Participants/materials, setting, methods In embryos cultured in time-lapse, fertilization was re-evaluated to check the appearance of 2PN and the second PB extrusion. Genetic triploid embryos were analysed in relation to the day of biopsy (day 5 vs day 6), the trophectoderm quality and the timing of the first cell divisions (time of cleavage from 2 to 6 cells: t2, t3, t4, t5, t6). Categorical variables are shown as rate. T-student tests were performed. P-value<0.05 was considered statistically significant. Main results and the role of chance The re-evaluation of the fertilization of the 59 triploids categorized as 2 PN revealed 50 oocytes 2 PN, 8 embryos 3 PN and 1 embryo 2 PN + 1 micronuclei configuration. Therefore, the prevalence of real triploidy in 2PN embryos was 0,8% (n = 50/6526; CI95%:0,6-1,0%). These data showed that 2PN zygotes may give rise to embryos with altered ploidy. From 27 triploid 2PN embryos was possible to access to the whole development time-lapse images. Just 33,3% of triploid embryos exhibited the extrusion of the second PB, while 100% of euploid sibling embryos showed this extrusion (N = 9/27 vs N = 24/24,;p<0,005). Comparing 2 PN triploid and the rest of the embryos from the same cohorts, there were no differences regarding the day of biopsy (N = 31/176 day 5 biopsy, 17.6% vs N = 19/81 day 6 biopsy, 23.5%; p > 0.05). On the contrary, a worse blastocyst quality showed a significant relation with triploidy (N = 23/161,14,2% of good quality embryos are triploid vs N = 27/96, 38,6% of fair quality embryos are triploids; p:0.006).Embryos cleavage time points up to 6-cell stage did not show any significative difference between triploid and euploid embryos (t2: 27.36h vs 25.8h; t3: 38.5h vs 35.6h; t4 39.4h vs 37.2h; t5 52.8h vs 47.0h; t6 56.2h vs 52.1h; all comparisons p > 0.05). Limitations, reasons for caution The incidence of triplody from 2PN zygotes may vary among IVF laboratory settings. The small sample size may be responsible of no difference in morphokinetics. Wider implications of the findings Fertilization check should persist in IVF procedures. Special consideration should be given to the second PB extrusion as triploidy is the most common form of aneuploidy observed in the first trimester miscarriages. The role of diploid spermatozoa, the size of the pronucleus and location should be explored in further studies. Trial registration number NOT APPLICABLE

P-545 Early pregnancy outcomes associated with age and AMH following Next-Generation Sequencing(NGS)-based Preimplantation Genetic Testing for Aneuploidy(PGT-A) in single euploid embryo transfers for advanced age patients

Abstract Study question Does age and AMH impact early pregnancy outcomes in euploid single embryo transfers using NGS-based PGT-A for individuals aged 35 and above? Summary answer In this NGS-based PGT-A study, no significant differences in early pregnancy rates were observed based on age or AMH among individuals aged 35 and above. What is known already A recent meta-analysis suggest a decline in pregnancy rates with advancing age in the context of euploid embryo transfer following PGT-A. However, it’s crucial to acknowledge variations in PGT-A techniques across studies. While individuals with low AMH levels may encounter challenges in obtaining euploid embryos, the precise impact of AMH on pregnancy rates post-embryo acquisition remains unclear. Study design, size, duration A retrospective cohort study was conducted on 428 cycles of single euploid embryo transfers in patients aged 35 and above, performed at the single infertility center from January to December 2022. Participants/materials, setting, methods The study examined frozen-thawed embryo transfer cycles in patients aged 35 and above, analyzing early pregnancy outcomes in single euploid embryo transfers. Participants were grouped by age(35-38, 38-42, 42 and above) and AMH levels(<1, 1-3, >3 ng/ml). NGS-based PGT-A conducted embryo biopsy. The analysis aimed to detect differences in initial pregnancy rates among age and AMH subgroups. The differences between groups were compared using Pearson’s chi-squared test. Main results and the role of chance The average age at transfer was 39.05 ± 2.53 years, with a mean AMH of 2.60 ± 1.86(ng/ml). The mean number of prior frozen embryo transfer (FET) cycles was 1.9 ± 2.04, with 119 patients (27.8%) having undergone three or more transfer cycles. Transfer cycles comprised 363 (84.8%) artificial cycles and 65 (15.2%) ovulatory cycles, with an average endometrial thickness of 8.95 ± 1.73 mm at the time of transfer. The β-hCG positive rate was 57.7% (247/428), and the clinical pregnancy rate was 53.5% (229/428). When analyzing positive b-hCG results by age, rates were 59.9% (82/137) for ages 35-38, 55.5% (121/218) for ages 38-42, and 60.3% (44/73) for ages 42 and above (p = 0.641). In terms of AMH, β-hCG positive rates were 52.1% (37/71) for <1, 59.1% (120/203) for 1-3, and 58.0% (83/143) for ≥3, with no statistically significant differences (p = 0.584). Clinical pregnancy rates were 54.0% for ages 35-38, 51.8% for ages 38-42, and 57.5% for ages 42 and above. For AMH, rates were 50.7% for <1, 53.2% for 1-3, and 54.5% for ≥3, with no statistically significant differences (p = 0.869). Limitations, reasons for caution Being a retrospective study analyzing patients undergoing embryo transfer, our study overlooks variables related to oocyte retrieval. Given the challenges of obtaining euploid embryos in advanced age, further investigation is warranted to explore the probability and duration of embryo acquisition in this population. Wider implications of the findings In this study, utilizing NGS-based PGT-A after generating euploid embryos, no significant differences in early pregnancy outcomes were observed for women aged 35 and older, considering age and AMH levels. The study suggests further research is needed to explore if NGS-based PGT-A can address limitations of traditional chromosomal testing methods. Trial registration number not applicable

P-146 The importance of cytoplasmic strings between inner cell mass and trophectoderm after blastulation on embryonic development and pregnancy outcome

Abstract Study question Whether the cytoplasmic strings between inner cell mass and trophectoderm during blastocyst expansion have a significant impact on embryo development and pregnancy outcomes? Summary answer The presence of cytoplasmic strings is related to morphological quality, but it has no impact on embryo ploidy or clinical outcome after euploid embryo transfer. What is known already Cytoplasmic strings are a dynamic cellular structure, and their width and length vary with the degree of embryonic expansion. The presence of cytoplasmic strings is considered a characteristic of embryos with poor vitality. In recent years, researchers have found the presence of cytoplasmic strings may indicate a higher embryonic developmental potential. However, the specific function of cytoplasmic strings remains unclear, and their impact on embryonic development is still controversial. Study design, size, duration A retrospective cohort analysis was conducted on clinical data of 530 patients who underwent single blastocyst transfer after pre-implantation genetic testing for aneuploidy (PGT-A) in our center from June 2019 to December 2021. Two embryologists annotated the presence of cytoplasmic strings, the morphological quality of blastocyst. Participants/materials, setting, methods A total of 2132 biopsied blastocysts were obtained from the 530 patients, with 534 blastocysts having cytoplasmic strings (+) and 1598 blastocysts having cytoplasmic strings (−). The quality and ploidy differences between the two groups were compared. After PGT-A, single euploid blastocysts were transferred, and the difference in pregnancy outcomes between cytoplasmic strings (+) (n = 115) and cytoplasmic strings (−) (n = 415) cycles was compared. Main results and the role of chance There was no significant difference in the euploidy rate between the two groups (44.6% vs. 45.3%). As the morphological quality of the embryos decreased, the rate of cytoplasmic strings (+) significantly decreased (Good-quality vs. Average-quality: 30.19% vs. 24.62%, P = 0.008; Average-quality vs. Low-quality: 24.62 vs. 12.63, P = 0.000). After adjusting for patient age, anti-Müllerian hormone, and gonadotropin dose, multivariate regression analysis showed that cytoplasmic strings were associated with embryo quality, but not with embryo ploidy. There were no significant differences in implantation rate (72.17% vs. 66.02%, P = 0.213), miscarriage rate (10.43% vs. 8.43%, P = 0.504), and live birth rate (61.74% vs. 57.59%, P = 0.424) between blastocysts with cytoplasmic strings (+) and without cytoplasmic strings (−). Limitations, reasons for caution Larger datasets are required to confirm putative associations between cytoplasmic strings and blastocyst ploidy, and the pregnancy outcome. Wider implications of the findings The presence of cytoplasmic strings is related to the embryological quality of the blastocyst, but it has no impact on embryo ploidy or clinical outcome after euploid embryo transfer. Trial registration number not applicable

P-717 Add-ons availability and quality of online information provided by Brazilian IVF clinics

Abstract Study question How do Brazilian fertility clinic websites advertise four IVF add-ons: assistedhatching (AH), preimplantation genetic testing for aneuploidies (PGT-A), ERA and sperm DNA fragmentation test. Summary answer Available information, about ivf add-ons in Brazil, on researched websites is heterogeneous and is not evidence-based according to the last published recommendations. What is known already Over 1 billion people worldwide use online resources to obtain health-related information. People struggling with infertility are increasingly turning to online resources for infertility-related content and social support. “Add-on” procedures are actively promoted on some fertility clinic websites as means to improve IVF success rates. However, the actual contribution of these interventions to IVF success rates remains debatable. ESHRE has recently issued 42 recommendations were formulated on the use of add-ons in IVF. At present, no study has evaluated the type and quality of the information provided on the Brazilian IVF clinics’ websites regarding the merits of “add-ons. Study design, size, duration Prospective evaluation of the availability and quality of information on four common add-ons provided by IVF clinics in the southeast of Brazil between October-November 2023. Clinics were identified using the national registry (SisEmbrio) as well as through manual online search. As only public online available information was used no ethical approval was necessary. Statistical analyses were restricted to descriptive of the frequency of each add-on and frequency of claims made in relation to their use. Participants/materials, setting, methods One reviewer took screenshots of the included clinic pages and claims and a second reviewer double-checked everything. The folowing information was colletced: availability of of the four add-ons; information on the effectiveness on: implanation rate(IR), pregnancy rate (PR) or live-birth rate (LBR); information on the scientifc uncertainty of the use of add-on and possible negative impact; for PGT-A if the clinic informed that the data obtained should be confirmed by prenatal diagnostic testing. Main results and the role of chance The websites of 122 IVF clinics were included. Nine clinics had no website or were offline. PGT-A was the most frequent available add-on (n = 77; 68%) but only two of them (2,5%) informed on PR. There was no information on IR nor LBR. Thirteen clinics (16,8%) acknowledged it could have adeverse effects but only one mentioned the scientifc uncertainty related to its use. Forty (35%) websites displayed AH with 7 reporting on possible negative effects but none disclosed success rates (PR, IR or LBR). Eight clinics admitted AH had uncertain benefits and seven noted possible negative impact. The sperm DNA fragmentation test was presented in 37 (33%) websites but none reported on potential adverse effects nor success rates in terms of PR, IR nor LBR. No website provided information on the uncertainty of the benefit of the test. The ERA-test was advertised in 28 (25%) websites with no information on its benefits on sucess rates. Three clinics advised it could have a negative impact and six agreed its effectiveness was unclear. The cost was never pesented for any of the add-ons researched in accordance to national regulations. Of note, 36 websites advertised other add-ond such as MSOME, ZYMOT and endometrial scrathcing. Limitations, reasons for caution The study was restricted to 122 clinics websites in the southeast of Brazil. Evalaution was restricted to four commons add-ons and the quality of the information provided could not be assessed as the clinics follow no specific regulation on how to advertise scientifc information to their patiens and general public. Wider implications of the findings The information provided by Brazilian IVF clinics could be misleading. The use of add-ons is surrounded by scientific uncertainly related to their benefits and cost-effectiveness therfeore it is of utmost importance that clinics reviewt heir websites and offer sound scientific data to patients and the general public as well. Trial registration number Not applicable

P-612 Euploidy rates between cycles triggered with gonadotrophin-releasing hormone agonist (GnRHa), human chorionic gonadotrophin (hCG) and dual trigger

Abstract Study question Is there any difference in euploidy rates between in vitro fertilization (IVF) cycles triggered with GnRHa, hCG or dual trigger? Summary answer There is no difference in the euploidy rate in preimplantation genetic testing for aneuploidy (PGT-A) cycles according to the trigger method used. What is known already The use of PGT-A has shown that the success of meiosis in the IVF laboratory is inversely correlated with maternal age. However, other uninvestigated factors involved in the IVF process may interfere with normal oocyte physiology. Since the final maturation obtained after ovarian stimulation depends on the use of drugs to promote the resumption of meiosis, it is worth investigating whether the triggering method could impact on oocyte and embryo euploidy. This study aims is to evaluate differences in embryo euploidy rates between PGT-A cycles triggered with GnRHa, hCG or dual trigger (GnRHa + hCG). Study design, size, duration This is a single-center retrospective study of 934 patients who underwent 1085 PGT-A cycles by Next-Generation Sequencing (NGS) from January 2017 to December 2023, using one of three possible trigger methods to induce final oocyte maturation: GnRHa, hCG, or dual trigger. The couples included in the study had normal karyotypes and no severe male factor. Participants/materials, setting, methods Data on the number of oocytes retrieved, ratio of metaphase II (MII) oocytes and fertilization rate, blastocyst development and embryo euploidy were assessed and compared between the GnRHa, hCG and dual trigger treatments. A secondary analysis was performed to evaluate data stratified by patient age. Main results and the role of chance A total of 1085 PGT-A cycles involving 934 patients (38.07 ± 3.01 years) and 2803 biopsied blastocysts were included in the analysis. Dual trigger was used in 726 (77.73%) cycles, GnRHa in 287 (26,4%) cycles and hCG in 72 (7.71%) cycles. The number of oocytes retrieved was significantly higher in the GnRHa group (GnRHa: 18.72 ± 8.59; hCG: 9.88 ± 5.64; dual trigger: 10.58 ± 5.65; p < 0.001) with similar maturation rate (GnRHa: 77.46%; hCG: 74.58%; dual trigger: 76.03%; p = 0.789), fertilization rate (GnRHa: 78.12%; hCG: 76.37%; dual trigger: 79.12%; p = 0.168), blastulation rate (GnRHa: 61.03%; hCG: 58.32%; dual trigger: 58.73%; p = 0,629), high-quality blastocyst rate (GnRHa: 27,88%; hCG: 33,67%; dual trigger: 27,88%; p = 0.543) and embryo euploidy per biopsied embryo (GnRHa: 42.48%; hCG:45.49%; dual trigger:46.98%; p = 0.112). Analysis of the data stratified by age showed similar rates of euploidy rates with the three trigger methods in women younger than 41 years (<35 years: GnRHa 66.50%; hCG 66.67%; dual trigger 56.97% p = 0.290; 35-37 years: GnRHa 49.45% hCG 51.04%; dual trigger 50.23%; p = 0.988; 38-40 years: GnRHa 40.93%; hCG 41.27%; dual trigger 39.13% p = 0.257). However, in patients ≥41 years old the euploidy rate was statistically higher in the GnRHa group (GnRH 31.51%; hCG 18.75%; dual trigger 16.89%; p = 0.032). Limitations, reasons for caution The main limitations of the study are: its retrospective design, the possibility that the trigger method was chosen based on the ovarian response and the limited number of patients in both the hCG trigger and women ≥ 41 years groups. Wider implications of the findings Our results suggest that triggering with GnRHa, hCG or dual trigger leads to comparable euploidy rates. Triggering with GnRHa should be recommended in older patients since it has been associated with a higher rate of embryonic euploidy. Trial registration number Not Applicable

P-145 Impacts of double biopsy and double vitrification on the clinical outcomes following euploid blastocyst transfer: a systematic review and meta-analysis

Abstract Study question Do “double biopsy + double vitrification” and “single biopsy + double vitrification” compromise subsequent clinical outcomes following euploidy blastocyst transfer? Summary answer Both “double biopsy + double vitrification” and “single biopsy + double vitrification” led to reduced ongoing and clinical pregnancy rates and elevated miscarriage rates What is known already It is not uncommon to have inconclusive results following blastocyst biopsy and preimplantation genetic testing for aneuploidy (PGT-A). One option for these blastocysts is a second biopsy for re-test after warm, followed by a second vitrification. Furthermore, for better workflow, laboratories may choose to routinely vitrify all blastocysts formed followed by a subsequent biopsy post warm at a preferred timing, after which a second vitrification would be required. However, there is currently a lack of systematic evaluation in the literature regarding potential clinical risks associated with the above-mentioned applications in reference to the conventional “single biopsy + single vitrification” approach. Study design, size, duration A systematic review and meta-analysis were performed, with a protocol registered with PROSPERO (CRD42023469143). A search of PUBMED, EMBASE, and the Cochrane Library for relevant studies was carried out on 30 August 2023, using the keywords ‘biopsy’ and ‘vitrification’ and associated variations respectively. Only studies involving frozen transfers of euploid blastocysts tested via PGT-A were included, with those tested via PGT-M or PGT-SR excluded. Participants/materials, setting, methods Study groups included blastocysts having undergone “double biopsy + double vitrification” or “single biopsy + double vitrification”, with “single biopsy + single vitrification” used as control. The primary outcome was live birth rate while secondary outcomes included ongoing pregnancy, clinical pregnancy, miscarriage, and post warm survival rates. Random effects meta-analysis was performed with risk ratios (RR) and 95% confidence interval (CI) used to present outcome comparisons. Main results and the role of chance Following duplicate removal, a total of 607 records were identified after the initial search. After screening of titles and abstracts, 37 records were included for further eligibility assessment. For meta-analysis, 9 studies (5 full articles and 4 abstracts) were eventually included. Compared to “single biopsy + single vitrification”, “double biopsy + double vitrification” was associated with significantly reduced live birth rates (5 studies, n = 10,068; RR = 0.57, 95% CI = 0.35–0.94; I2=82%), ongoing pregnancy rates (2 studies, n = 4,659; RR = 0.69, 95% CI = 0.51–0.94; I2=2%), clinical pregnancy rates (5 studies, n = 17,673; RR = 0.79, 95% CI = 0.71–0.89; I2=0%), and elevated miscarriage rates (7 studies, n = 22,332; RR = 1.64, 95% CI = 1.02–2.64; I2=53%). However, no significant changes were found in post warm survival rates (p > 0.05, respectively). Furthermore, “single biopsy + double vitrification” was also linked with significantly decreased ongoing pregnancy (3 studies, n = 5,980; RR = 0.84, 95% CI = 0.70–1.00; I2=61%), clinical pregnancy rates (5 studies, n = 12,136; RR = 0.84, 95% CI = 0.72–0.99; I2=71%), and increased miscarriage (5 studies, n = 17,781; RR = 1.25, 95% CI = 1.02–1.53; I2=3%) rates. But live birth rate and post warm survival rate were not affected (p > 0.05, respectively). Limitations, reasons for caution All studies included in this meta-analysis were retrospective with varying levels of heterogeneity for different outcomes. Not all studies have accounted for potential confounding factors, therefore only unadjusted data could be used in the main meta-analysis. Only one study reported neonatal outcomes, making it impossible to perform meta-analysis. Wider implications of the findings Our data clearly indicated adverse impacts by “double biopsy + double vitrification” and “single biopsy + double vitrification” on clinical outcomes following euploid blastocyst transfers. If possible, such approaches should be avoided in routine practice, and patients should be carefully consulted about the risks when offered with such approaches. Trial registration number CRD42023469143

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

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

O-073 CD163 in follicular fluids can be the novel marker to predict clinical outcome of oocyte

Abstract Study question Can we find novel marker in follicular fluid at the oocyte retrieval to evaluate oocyte condition besides karyotype by proteomics? Summary answer We found significant higher concentration of CD163 in the follicle fluids, from those oocytes resulted in pregnancy or live birth. What is known already Although preimplantation genetic testing for aneuploidy (PGT-A) is effective for embryo selection, only karyotype is considered in PGT-A. If we can evaluate other conditions of oocytes besides karyotype, the pregnancy rate can be improved. In addition to changes in the oocyte per se, factors affecting the quality of oocytes, such as ageing, change the microenvironment surrounding the oocyte. For example, it has been reported that the follicle itself becomes more fibrotic with age, resulting in increased oxidative stress, and that the concentration of cytokines, amino acids, fatty acids, and other substances in the follicle also changes. Study design, size, duration Proteomic analyses were performed on follicular fluid collected at the oocyte retrieval, and the proteins showed differences in relation to clinical outcome. We focused on one of those candidates, CD163, and evaluated the concentration by ELISA and checked for possible relevance to clinical outcome of the oocytes derived from each follicles. We also investigated the effect of this protein on oocyte maturation. Participants/materials, setting, methods Under the ethical review of Yokohama City University and informed consent with patients in Reproduction center of Yokohama City University Medical Center, we aspirated human follicular fluids individually at the oocyte retrieval. The oocytes were retrieved from the follicular fluid and remained fluid was cryopreserved for further analysis. We followed clinical outcome of oocyte from each follicle, compared the concentration of the protein in each follicle. Main results and the role of chance Proteomic analysis revealed CD163 in follicular fluids seemed to have relation with the clinical outcome. We collected 120 follicular fluid samples from 93 patients from 2017 to 2021, and evaluated the concentration of CD163 of them. Follicles which had contained oocytes derived are cryopreserved or fresh embryo transferred had higher than other follicles; 140.6 ± 73.1 vs 95.2 ± 30.7 ng/ml (mean ± SD, p < 0.01). Similarly, follicles, which had contained oocytes led to clinical pregnant and live birth after single embryo transfer, had higher concentrations of CD163; 140.5 ± 71.8 vs 101.6 ± 38.4 ng/ml (pregnant vs not pregnant, mean ± SD, p < 0.01), 138.7 ± 74.1 vs 109.2 ± 45.3 ng/ml (live birth vs not live birth, mean ± SD, p < 0.01). These results suggest that CD163 concentration in follicular fluid might be a novel marker of the clinical outcome. Limitations, reasons for caution Although we tried to investigate the relationship of CD163 and oocyte maturation, unfortunately we have not yet been able to clarify them. However, we believe that CD163 may act as an indirect marker representing other factors directly involved in oocyte maturation and development. We would like to investigate this further. Wider implications of the findings When CD163 concentration in follicular fluids is evaluated, we might be able to predict the clinical outcome of each oocyte at the day of oocyte retrieval. This may contribute to further improvement of pregnancy rate without invasion to embryo. Trial registration number the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant-in-Aid for Scientific Research C (Grant Number 22K07920)

P-228 Competence of injected MI versus MII sibling oocytes in terms of normal fertilization, blastocyst development and euploidy

Abstract Study question Is the injection of immature MI oocytes as effective as injection of their siblings mature MII oocytes regarding normal fertilization, blastocyst development and euploidy? Summary answer MI oocytes show a lower rate of fertilization, a similar blastulation rate, and a slightly lower rate of euploidy compared to their MII sibling oocytes. What is known already Although several studies have been performed on immature MI oocytes evaluating their developmental competence after intracytoplasmic sperm injection (ICSI), all have been performed on oocytes that extruded the first polar body by the time of injection. ICSI is typically performed on MII oocytes which have completed the process of maturation. Some laboratories also inject MI oocytes, nevertheless, they are generally not used due to the lack of required molecular and cellular changes to support normal development. However, there is limited research on the reproductive potential of oocytes in MI. Consequently, our current practices are founded on assumptions. Study design, size, duration This is a retrospective, observational study of 168 oocytes injected in MI and 839 sibling oocytes injected in MII from 82 ICSI cycles with Preimplantation Genetic Testing for Aneuploidies (PGT-A) performed in a single IVF clinic between April 2018 and December 2023. All PGT-A cycles included in this study contained at least one MI oocyte injected. Participants/materials, setting, methods After oocyte retrieval and denudation, the oocytes were classified as MII, MI or GV. MI oocytes with extruded polar body by the moment of ICSI were reclassified as MII. All MII and MI oocytes were inseminated and the embryos that reached blastocyst stage were biopsied on day 5, 6 or 7. Normal fertilization rate, blastocyst formation rate and euploidy rate were compared between MI-derived embryos and their MII-derived siblings. Main results and the role of chance A total of 1,094 oocytes were retrieved from 83 ICSI cycles with PGT-A, comprising 839 (76.7%) MII, 168 (15.4%) MI y 87 (7.9%) VG. The normal fertilization rate was significantly higher in oocytes injected at MII compared to MI (76.9% vs 53.0%, P < 0.001). The rates of abnormal fertilization in MII and MI oocytes were as follows: 1 PN 0.8% vs 0.6%, P = 0.342; 3PN 2.6% vs 1.2%, P = 0.075; 0 PN 19.7% vs 45.2%, P < 0.001. Interestingly, the blastulation rate of zygotes with normal fertilization was very similar between both groups (46.4% vs 43.8%, P = 0.708), as well as the proportion of biopsied blastocysts on day 5 (74.2% vs 71.8%, P = 0.858), on day 6 (23.1% vs 25.6%, P = 0.946), and on day 7 (2.7% vs 2.6%, P = 0.594). The euploidy rate of the 298 biopsied MII-derived blastocyst was 36.2%, compared with 23.1% of the 39 MI-derived blastocysts biopsied (P = 0.077). Despite the limited sample size, it is noteworthy that three out of the four euploid blastocysts derived from MI oocytes, which were transferred in single embryo transfers, successfully led to pregnancy resulting in one healthy live birth, one ongoing pregnancy and one miscarriage at 9 weeks of pregnancy. Limitations, reasons for caution While this study provides valuable insights into the developmental competence of MI oocytes, it is not exempt from certain limitations, such as its retrospective nature, limited sample size and ethical considerations regarding the injection of putative immature oocytes. Wider implications of the findings MI oocytes showed lower fertilization rates compared to their MII siblings. However, once fertilized, MI-derived embryos had similar blastulation rates and a trend towards lower euploidy rates. Our study suggests that MI oocytes may result in successful pregnancies and live birth, but further research is needed to validate these findings. Trial registration number Not applicable

P-107 Sperm DNA fragmentation impacts embryonic developmental potential but is not associated with PGT-A outcomes

Abstract Study question Does sperm DNA Fragmentation Index (DFI) affect embryonic development and influence Preimplantation Genetic Testing for Aneuploidy (PGT-A) outcomes in IVF? Summary answer DFI adversely affects early embryonic development, diminishing blastocyst potential. Nonetheless, PGT-A outcomes are unaffected, underscoring the importance of selecting optimal sperm for enhanced IVF results. What is known already Several major factors are thought to cause DFI. The relationship between sperm DFI and IVF results remains debated. Study design, size, duration This study was conducted from January 2018 to March 2023, included 56 married couples undergoing IVF after performing a sperm DFI test. The DFI was assessed at the first semen examination and categorized into three groups: low (≤15%), moderate (15-30%), and high (≥30%). Participants/materials, setting, methods Intracytoplasmic sperm injection (ICSI) was performed on 1006 mature oocytes, followed by blastocyst culture. A total of 278 blastocysts meeting freezing criteria were vitrified. Trophectoderm biopsies from 216 blastocysts underwent Next-Generation Sequencing for aneuploidy analysis. Statistical analysis utilized the Cochran-Armitage test to compare outcomes among the DFI groups. Main results and the role of chance The average age of the husbands and wives who underwent IVF after the DFI examination was 42.3±4.2 and 41.4±2.4 years old. Normal fertility rates after ICSI in low, moderate, and high DFI were 85.5%, 84.5%, and 83.1%, with no significant difference. Similarly, the blastocyst freezing rates were 35.4%, 26.3%, and 29.6%, and tended to decrease as the DFI value increased (p < 0.05). However, the euploid rates for PGT-A were 22.4%, 16.3%, and 18.2%. There were no statistically significant differences between the DFI groups in terms of euploidy. Limitations, reasons for caution Cases declining ICSI due to patient preference or historical medical reasons were excluded from this study. Wider implications of the findings This suggests that sperm DNA imperfections have a negative impact on the developmental process during the early stages of division, but during late stages of division, the ability to repair DNA works to differentiate into blastocysts with normal developmental potential. Trial registration number not applicable

P-510 The impact of the severe male factor in the egg donation cycles using preimplantation genetic test for aneuploidy

Abstract Study question Does severe male factor affect the euploidy rate and IVF outcomes in donor egg ICSI cycles using PGT-A? Summary answer Severe male factor affects the euploidy rate in egg donation cycles, however, no statistical differences were reported in live birth and cumulative live birth rate. What is known already Around 50% of couples seeking fertility treatment have male-factor infertility. Intracytoplasmic sperm injection (ICSI) relies on evaluating sperm morphology, but the direct relationship between semen quality and embryonic potential remains unclear. Evidence indicates that poor semen characteristics correlate with an increase in aneuploidy rate of spermatozoa. Therefore, pregenetic testing for aneuploidies (PGT-A) is recommended in cases of male factor infertility. The relationship between aneuploidy rates and abnormal sperm remains a debated topic. The use of the egg donation model offers a valuable chance to isolate the male-related factor and evaluate its influence on the occurrence of embryonic aneuploidies. Study design, size, duration This is a retrospective, observational, cohort study including all European IVI clinics. The study included egg donation cycles using pre-implantation genetic testing for aneuploidies (PGT-A) generated in IVI clinics from January 2017 until December 2023. 690 egg donation cycles were included in the study, N = 202 in the group with sperm < 5mil/ml, N = 102 in the sperm group with 5-15 mil/ml and N = 386 in the group with sperm >15 mil/ml. Participants/materials, setting, methods The study included women between 30-49 years undergoing egg-donor cycles, using fresh/frozen sperm Sperm samples were derived after 2-5 days of abstinence. The cycles of egg donation were divided into three groups according to sperm concentration, severe oligozoospermia (SO, < 5mil/ml), moderate oligozoospermia (MO, between 5-15 mil/ml), and control group(>15 mil/ml). Trophectoderm biopsy was performed on day 5 or 6 and subsequent analysis used the next sequencing generation (NGS). Single euploid blastocyst transfers were included. Main results and the role of chance The fertilization rate was decreased in SO (73.33 ± 16.19) than in the MO group (79.07 ±12.45) and control group (77.80 ±14.72) p = 0.0047. The number of blastocysts was higher in the control (5.80 ± 2.72) and MO (5.97± 2.78) than in the SO group (4.99 ± 2.40), p = <0.001. The number of euploid blastocysts was decreased in SO (3.21 ± 2.07) than in the control (4.09 ± 2.41) and MO group (4.05 ± 2.15), p = <0.001. The euploidy rate of SO decreased (63.32 ±26.39) compared to the control (69.67 ± 25.08) and MO group (68.55 ± 20.37), p = 0.012. The logistic regression model included possible confounders (male age, age donor, male BMI, sperm motility, sperm morphology and sperm concentration) that showed no effect on the euploidy rate. The pregnancy rate was similar across the groups 69.77% vs 66.05% vs 66.23%, p = 0.54. No statistical differences were reported for the clinical pregnancy rate 62.79% vs 61.73% vs 58.40%, p = 0.44, and the live birth rate 62.79% vs 61.73% vs 58.40%, p = 0.44. 48.05 vs 46.38% vs 42.64%, p = 0.37. No differences were reported in the miscarriage rate (p = 0.37) and CLBR (p = 0.26). Limitations, reasons for caution The retrospective nature of the study and the sample size represent the principal limitations of the study. The clinical reason for severe male factors was not considered in the study. Wider implications of the findings SO affects the quality affect the early embryonic potential through an increase in aneuploidy, despite no clinical effects.The use of PGT-A in case of severe male factor remains not justified in the egg donation.Future prospective multi-center studies are needed to highlight the effect of sperm quality on early embryonic potential. Trial registration number not applicable