Embryo Aneuploidy Research Articles

Aligning genotyping and copy number data in single trophectoderm biopsies for aneuploidy prediction: uncovering incomplete concordance.

To what extent can genotype analysis aid in the classification of (mosaic) aneuploid embryos diagnosed through copy number analysis of a trophectoderm (TE) biopsy? In a small portion of embryos, genotype analysis revealed signatures of meiotic or uniform aneuploidy in those diagnosed with intermediate copy number changes, and signatures of presumed mitotic or putative mosaic aneuploidy in those diagnosed with full copy number changes. Comprehensive chromosome screening (CCS) for preimplantation genetic testing has provided valuable insights into the prevalence of (mosaic) chromosomal aneuploidy at the blastocyst stage. However, diagnosis of (mosaic) aneuploidy often relies solely on (intermediate) copy number analysis of a single TE biopsy. Integrating genotype information allows for independent assessment of the origin and degree of aneuploidy. Yet, studies aligning both datasets to predict (putative mosaic) aneuploidy in embryos remain scarce. A single TE biopsy was collected from 1560 embryos derived from 221 couples tested for a monogenic disorder (n = 218) or microdeletion-/microduplication syndrome (n = 3). TE samples were subjected to both copy number and genotyping analysis. Copy number and SNP genotyping analysis were conducted using GENType. Unbalanced chromosomal anomalies ≥10 Mb (or ≥20 Mb for copy number calls <50%) were classified by degree, based on low-range intermediate (LR, 30-50%), high-range intermediate (HR, 50-70%) or full (>70%) copy number changes. These categories were further subjected to genotyping analysis to ascertain the origin (and/or degree) of aneuploidy. For chromosomal gains, the meiotic division of origin (meiotic I/II versus non-meiotic or presumed mitotic) was established by studying the haplotypes. The level of monosomy (uniform versus putative mosaic) in the biopsy could be ascertained from the B-allele frequencies. For segmental aneuploidies, genotyping was restricted to deletions. Of 1479 analysed embryos, 24% (n = 356) exhibited a whole-chromosome aneuploidy, with 19% (n = 280) showing full copy number changes suggestive of uniform aneuploidy. Among 258 embryos further investigated by genotyping, 95% of trisomies with full copy number changes were identified to be of meiotic origin. For monosomies, a complete loss of heterozygosity (LOH) in the biopsy was observed in 97% of cases, yielding a 96% concordance rate at the embryo level (n = 248/258). Interestingly, 4% of embryos (n = 10/258) showed SNP signatures of non-meiotic gain or putative mosaic loss instead. Meanwhile, 5% of embryos (n = 76/1479) solely displayed HR (2.5%; n = 37) or LR (2.6%; n = 39) intermediate copy number changes, with an additional 2% showing both intermediate and full copy number changes. Among embryos with HR intermediate copy number changes where genotyping was feasible (n = 25/37), 92% (n = 23/25) showed SNP signatures consistent with putative mosaic aneuploidy. However, 8% (n = 2/25) exhibited evidence of meiotic trisomy (9%) or complete LOH in the biopsy (7%). In the LR intermediate group, 1 of 33 (3%) genotyped embryos displayed complete LOH. Furthermore, segmental aneuploidy was detected in 7% of embryos (n = 108/1479) (or 9% (n = 139) with added whole-chromosome aneuploidy). These errors were often (52%) characterized by intermediate copy number values, which closely aligned with genotyping data when examined (94-100%). N/A. The findings were based on single TE biopsies and the true extent of mosaicism was not validated through embryo dissection. Moreover, evidence of absence of a meiotic origin for a trisomy should not be construed as definitive proof of a mitotic error. Additionally, a genotyping diagnosis was not always attainable due to the absence of a recombination event necessary to discern between meiotic II and non-meiotic trisomy, or the unavailability of DNA from both parents. Interpreting (intermediate) copy number changes of a single TE biopsy alone as evidence for (mosaic) aneuploidy in the embryo remains suboptimal. Integrating genotype information alongside the copy number status could provide a more comprehensive assessment of the embryo's genetic makeup, within and beyond the single TE biopsy. By identifying meiotic aberrations, especially in presumed mosaic embryos, we underscore the potential value of genotyping analysis as a deselection tool, ultimately striving to reduce adverse clinical outcomes. L.D.W. was supported by the Research Foundation Flanders (FWO; 1S74621N). M.B., K.T., F.V.M., S.J., A.V.T., V.S., D.S., A.D., and S.S. are supported by Ghent University Hospital. B.M. was funded by Ghent University. The authors have no conflicts of interest.

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.

Basal FSH values are positively associated with aneuploidy incidence in pre-advanced maternal age (AMA) but not in AMA patients.

To assess the impact of maternal age on the association between maternal basal FSH and aneuploidy. A retrospective study including data from 1749 blastocysts diagnosed as euploid or aneuploid by PGT-A (preimplantation genetic testing for aneuploidy). Aneuploidy incidence was compared between embryos from mothers with high vs. low basal FSH levels (above and below the group median, respectively) in total, pre-AMA (advanced maternal age; < 35years, 198 embryos) and AMA (≥ 35years, 1551 embryos) patient groups, separately. To control for the interference of potentially confounding variables, the association between aneuploidy and high basal FSH levels was assessed by multivariate logistic analysis in overall, pre-AMA and AMA patient groups. Overall, aneuploidy rate was 9% higher (p = 0.02) in embryos from patients with high basal FSH (63.7%) compared to those with low basal FSH (58.4%). In the pre-AMA subgroup, aneuploidy incidence was 35% higher (p = 0.04) in embryos from patients with high basal FSH (53.5%) compared to those with low basal FSH (39.4%). Differently, aneuploidy occurrence did not vary between embryos from AMA patients with low (61.0%) and high (64.8%) basal FSH (p = 0.12). The multivariate analysis revealed that, in pre-AMA embryos, the association between aneuploidy occurrence and high basal FSH is independent of potential confounding variables (p = 0.04). Maternal basal FSH values are associated with embryo aneuploidy in pre-AMA but not in AMA patients. The present findings suggest that basal FSH is a useful parameter to assess aneuploidy risk in pre-AMA patients and reinforce the hypothesis that excessive FSH signalling can predispose to oocyte meiotic errors.

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.

HIF1A contributes to the survival of aneuploid and mosaic pre-implantation embryos.

Human fertility is suboptimal, partly due to error-prone divisions in early cleavage-stages that result in aneuploidy. Most human pre-implantation are mosaics of euploid and aneuploid cells, however, mosaic embryos with a low proportion of aneuploid cells have a similar likelihood of developing to term as fully euploid embryos. How embryos manage aneuploidy during development is poorly understood. This knowledge is crucial for improving fertility treatments and reducing developmental defects. To explore these mechanisms, we established a new mouse model of chromosome mosaicism to study the fate of aneuploid cells during pre-implantation development. We previously used the Mps1 inhibitor reversine to generate aneuploidy in embryos. Here, we found that treatment with the more specific Mps1 inhibitor AZ3146 induced chromosome segregation defects in pre-implantation embryos, similar to reversine. However, AZ3146-treated embryos showed a higher developmental potential than reversine-treated embryos. Unlike reversine-treated embryos, AZ3146-treated embryos exhibited transient upregulation of Hypoxia Inducible-Factor-1A (HIF1A) and lacked p53 upregulation. Pre-implantation embryos develop in a hypoxic environment in vivo, and hypoxia exposure in vitro reduced DNA damage in response to Mps1 inhibition and increased the proportion of euploid cells in the mosaic epiblast. Inhibiting HIF1A in mosaic embryos also decreased the proportion of aneuploid cells in mosaic embryos. Our work illuminates potential strategies to improve the developmental potential of mosaic embryos.

P-090 Is preimplantation genetic testing for aneuploidy (PGT-A) necessary in the treatment strategy for ICSI with testicular sperm?

Abstract Study question Does the combination of testicular sperm extraction (TESE) and ICSI increase the risk of embryo aneuploidy? Summary answer Embryo aneuploidy occurrence is not increased when using testicular sperm and is not affected by the etiology of azoospermia. What is known already TESE-ICSI, especially in patients with non-obstructive azoospermia (NOA), has been reported to result in poor outcomes and difficulty in obtaining good blastocysts, which may be due to immature sperm retrieved by TESE. In addition, patients who require TESE have been shown to be at particularly high risk of chromosomally abnormal sperm. These factors may increase the risk of embryo aneuploidy in TESE-ICSI and contribute to poor outcomes. However, few reports have investigated the results of PGT-A following TESE-ICSI. This study was therefore designed to investigate whether using testicular sperm is more likely to result in embryo aneuploidy. Study design, size, duration This retrospective study included 81 couples and 197 TESE-ICSI cycles undergoing PGT-A (average maternal age: 38.1±5.4 years) between September 2016 and November 2023. Of the total cycles, 94 cycles were case of obstructive azoospermia (OA) and 53 cycles were cases of NOA. The etiologies of NOA included were unexplained (22 cycles), Klinefelter’s syndrome (KS) (10 cycles), post-chemotherapy (3 cycles), post-orchiopexy (11 cycles), and microdeletion of azoospermia factor (AZF) c on the Y chromosome (7 cycles). Participants/materials, setting, methods ICSI outcomes were compared between different etiologies (OA vs. NOA). A total of 356 blastocysts acquired were analyzed using PGT-A with next-generation sequencing (NGS). The PGT-A results were classified according to maternal age (&amp;lt;38, 38-41, &amp;gt;41 years) and paternal age (&amp;lt;40, ≥40 years). In addition, TESE-ICSI cycles were limited to the maternal age of &amp;lt; 38 years to remove the effect of maternal factors, the euploidy rates were compared between different etiologies or morphological grade. Main results and the role of chance The rates of fertilization and blastocyst formation for TESE-ICSI were significantly lower in NOA than OA (47.4% vs. 61.6%, P &amp;lt; 0.001 and 37.3% vs. 50.7%, P &amp;lt; 0.01, respectively). The rate of good-quality blastocyst (16.6% vs. 21.2%) was also lower, but not statistically significant. The euploidy rate of all evaluated TESE-ICSI embryos decreased with increasing maternal age (52.9% in &amp;lt; 38, 29.3% in 38-41, 10.0% in &amp;gt; 41 years), but was not affected by the paternal age (40.6% in &amp;lt; 40, 42.5% in ≥ 40 years). For cycles with the maternal age of &amp;lt; 38 years, the euploidy rate was not significantly different between OA and NOA patients (55.3% vs. 49.3%). In addition, the association between embryo euploidy and morphological grade (using Gardner’s classification, ≥BB for good-quality vs. &amp;lt;BB for poor-quality embryos) was evaluated, and the euploidy rate was significantly higher in good-quality embryos compared to poor-quality embryos (60.3% vs. 33.3%, P &amp;lt; 0.01). These results indicate the frequency of embryo aneuploidy in TESE-ICSI is comparable to commonly used ejaculated sperm. Furthermore, it suggests that embryo aneuploidy is dependent on maternal age or morphological grade of embryo, but not on paternal age or the etiology of azoospermia. Limitations, reasons for caution Patients with chromosomal abnormalities, i.e. KS, could not be evaluated due to the small number of cases and patients with chromosomal translocation were not included in this study. Therefore, the effects of male factors in NOA on embryo aneuploidy require extended investigation. Wider implications of the findings In TESE-ICSI, especially in the case of NOA, it is difficult to obtain good quality embryos due to poor ICSI outcomes. However, if good quality embryos are obtained, the euploidy rate is high and there is no need to perform PGT-A on the sole basis of using testicular sperm. Trial registration number not applicable

P-199 The development time of good quality blastocysts is dependent on their genetic status in patients over 36 years old

Abstract Study question Is there any difference in PGT-A results between good quality blastocysts depending on whether they reach this stage on the fifth or sixth day? Summary answer In patients over 36 years old, there is a significantly higher rate of aneuploidy in embryos that reach the blastocyst stage on Day 6. What is known already The level of chromosomal abnormalities in embryos increases with maternal age, as indicated by PGT-A data. Embryo quality also correlates with aneuploidy level. The normal timeframe for blastocyst formation is considered to be between the fifth and sixth day of development. In this study, the genetic status of good quality blastocysts was examined to determine a difference in the aneuploidy rate between blastocysts formed on the fifth day compared to those formed on the sixth day.This information could provide insights into the optimal timing for blastocyst formation and its relationship to genetic abnormalities. Embryo quality also correlates with aneuploidy level Study design, size, duration The retrospective study included 513 good quality blastocysts (3-4, A-B according to the Gardner classification) after trophectoderm biopsy and obtained from fresh oocytes of 141 patients aged 23-45 years. The PGT-A data were analyzed. The analysis considered the age of the patients, with one group of patients younger than 36 years old (35 patients, 174 blastocysts), and another group aged 36 years and older (106 patients, 339 blastocysts). Participants/materials, setting, methods Trophectoderm biopsy was performed on good quality embryos as they progressed in development, either on the fifth or sixth day. PGT-A was conducted using the NGS method, and the data was processed using the chi-square test. Main results and the role of chance Among patients younger than 36 years old, 58.5% of good quality blastocysts formed by the fifth day of development. In comparison, among patients 36 years old and older, 43.0% of good quality blastocysts formed by the fifth day. The difference in the growth rate of blastocysts of the same quality between the age groups was statistically significant (p &amp;gt; 0.01). In patients younger than 36 years old, 75.0% of blastocysts formed by the fifth day and 66.7% of blastocysts formed by the sixth day were euploid (with no significant difference). In patients 36 years old and older, 50.5% of blastocysts formed by the fifth day were euploid, while only 19% of blastocysts formed by the sixth day were euploid. This difference in euploidy rates between the fifth- and sixth-day blastocysts was statistically significant (p &amp;gt; 0.01). Limitations, reasons for caution The study was conducted with a relatively small sample size, which may limit the generalizability of the results. A larger and more diverse sample would be beneficial to confirm the findings. Wider implications of the findings In patients over 36 years of age, the majority of blastocysts develop by Day 6, and there is a significant increase in aneuploidy level. This suggests that there may be a higher risk of chromosomal abnormalities in embryos that take longer to develop into blastocysts in this group. Trial registration number Not applicable

P-120 Morphokinetic behavior in embryos leading to biochemical pregnancy

Abstract Study question Does embryos leading to biochemical pregnancy behave morphokinetically different than those leading to positive or negative pregnancy result? Summary answer Embryos that resulted in a biochemical pregnancy did not display evidence of abnormal morphokinetics on time-lapse imaging. What is known already One of the possible outcomes of the pregnancy test is a biochemical pregnancy; where the pregnancy test is positive but does not progress into a clinical pregnancy. Despite its unknown etiology, several associated factors have been suggested such as embryo aneuploidy, abnormal uterine lining, sperm DNA damage and so on. Embryo monitoring with timelapse imaging (TLI) may be a valuable approach to evaluate whether embryonic morphokinetics can predict the occurrence of a biochemical pregnancy. The aim of this study was to investigate whether embryos leading to biochemical pregnancy behave morphokinetically different than those leading to positive or negative pregnancy result. Study design, size, duration This cohort study was performed in a private university–affiliated IVF center between March/2019 and April/22. 753 women undergoing ICSI were split into three groups according to the pregnancy outcome: Biochemical Group (n = 30 cycles and 54 transferred embryos); Positive Group (n = 255 cycles and 444 embryos); and Negative Group (n = 468 cycles and 750 embryos). Generalized mixed models followed by Bonferroni post-hoc test were used to compare morphokinetics among the groups (post hoc achieved power &amp;gt; 90%). Participants/materials, setting, methods Injected oocytes were cultured in the EmbryoScope+ incubator, which recorded the following kinetic markers: timing to pronuclei appearance and fading (tPNa and tPNf), two (t2), three (t3), four (t4), five (t5), six (t6), seven (t7), and eight cells (t8), morulae (tM), start of blastulation (tSB) and blastulation (tB). Durations of second and third cell cycles (cc2 and cc3) and timing to complete synchronous divisions s1, s2, and s3 were calculated. The KIDScore ranking was recorded. Main results and the role of chance Embryos resulting in biochemical pregnancy behaved similarly to those embryos resulting in a clinical pregnancy in terms of tPNa, tPNf, t2, t3, t4, t5, t6, t7, t8, tM, tsB, cc2, cc3, s1, s3 and KIDScore ranking, but tended to present slower tB (p = 0.089) and s2 (p = 0.084). Embryos resulting in a negative pregnancy showed significantly slower embryo development in terms of tPNa, tPNf, t2, t3, t4, cc2, and KIDScore ranking compared to both Biochemical and Positive groups and differed from the Positive group only in terms of t6, t7, t8, s1, s2, and s3. Limitations, reasons for caution The use of historical cohort groups is a drawback. Despite the eligibility criteria for inclusion in the analysis, potential differences in the baseline characteristics cannot be ruled out. Wider implications of the findings Biochemical pregnancy is likely multifactorial, including both embryo and endometrial factors. Further research is needed to identify factors that can predict and prevent biochemical pregnancy. Trial registration number N/A

P-009 Impact of sperm preparation methods on embryo development rate and embryo ploidy status: ICSI egg donation as a model

Abstract Study question Do different sperm separation techniques result in distinguishable contributions to embryo development rates and ploidy status? Summary answer CA0 sperm separation device, devoid of centrifugation, yielded a higher euploidy rate than conventional swim-up (50% vs. 74%, p = 0.02), notably observed in abnormozoospermic cases. What is known already Sperm DNA fragmentation (SDF) emerges as a key paternal risk factor associated with embryo aneuploidy, increasing the risk of ART failures. Factors like advanced male age, prolonged ejaculatory abstinence, and the detrimental effects of centrifugation during sperm separation jeopardize sperm DNA integrity. A recent centrifugation-free sperm separation device (CA0), employing live sperm sorting technology, exhibits efficiency in producing highly motile sperm with minimized SDF. This retrospective cohort study solely focusing on ICSI egg donation cycles to assess the influence of sperm separation methods (conventional swim-up vs. CA0) on early embryo development, and embryo ploidy. Study design, size, duration This retrospective study included 83 couples who underwent ICSI egg donation cycles at Chachava Clinic, Tbilisi, Georgia. Thirty-three semen specimens were processed using conventional swim-up (SU) and 427 Mii were inseminated. Forty-four semen samples were separated by CA0 and 519 Mii were inseminated. Clinical outcome measures included fertilization rate (number of 2PN over number of inseminated oocyte), good embryo rate (embryo number with more than 8 cells over inseminated oocyte), and euploidy rate using PGT-A. Participants/materials, setting, methods Male age and basic semen parameters via CASA (CEROS II, Hamilton-Thorne) were recorded. SU involved layering 1 mL neat semen to 1.2 mL HTF medium, inclining the tube at 45° for 60 min at 37 °C, collecting 1 mL uppermost, adding 2 mL HTF for centrifugation at 500×g for 5 min, and resuspending sperm pellet with 0.5 mL HTF. Semen samples were sorted using LensHooke® CA0 device following the manufacturer’s instructions. Main results and the role of chance In comparing demographic characteristics between SU and CA0 arms, CA0 exhibited higher median total motility (SU: 40% vs. CA0: 54%, p = 0.02), with no significant differences in female age and Mii oocyte count. This study assessed the impact of sperm separation methods on early embryo development, finding no significant differences in fertilization rate (SU: 79% vs. CA0: 77%) and good embryo rate (SU: 64% vs. CA0: 62%). However, CA0 demonstrated a significantly higher euploidy rate than SU (SU: 59% vs. CA0: 69%, p = 0.03). Further analysis within subpopulations, including normozoospermic and asthenozoospermic (total motility &amp;lt;40%), revealed equivalent euploidy rates between SU and CA0 for normospermic specimens (SU: 66% vs. CA0: 68%). However, a significant differentiation in euploidy rate emerged for asthenozoospermic cases (SU: 59% vs. CA0: 74%, p = 0.02). Specifically, comparing outcomes in normozoospermic, a significant reduction was observed in asthenozoospermic cases using SU (66% vs. 50%, p = 0.02), whereas CA0 provided improved euploidy rates regardless of semen quality (68% for normozoospermic and 74% for asthenozoospermic). In conclusion, this study suggests that the non-invasive live sperm sorting device enhances embryo euploidy rates, serving as a reliable sperm preparation method, ensuring selection consistency despite sample variations. Limitations, reasons for caution This study’s limitations include its retrospective nature and single-center trial design. To more effectively assess CA0 efficiency, a randomized controlled trial targeting diverse patient eligibility criteria is warranted. Wider implications of the findings Attributed to the improved embryo euploidy with CA0, we anticipate enhanced ART outcomes, such as increased embryo transfer rate and prevention of ART failures related to embryo aneuploidy. Additionally, CA0 offers practical benefits by streamlining and standardizing the sperm separation process with only three steps involved. Trial registration number not applicable

P-257 Building on a commercialized time-lapse algorithm to improve the predictive power for embryo ploidy status

Abstract Study question Would incorporating oocyte morphometric parameters into a predefined algorithmic model (KIDscore) enhance its ability to predict embryo ploidy status? Summary answer While larger oocyte area is significantly associated with embryo euploidy, its integration into a commercial algorithm did not add a clinical value to its predictivity. What is known already Currently, the genetic status of embryos is primarily assessed using preimplantation genetic testing for aneuploidy (PGT-A) following embryo biopsy. Concerns about potential harm to the embryo have spurred research into less invasive predictive tools, including time-lapse imaging (TLI). While numerous time-lapse data points have been explored, the literature is scarce when assessing the value of oocyte morphometric characteristic in predicting embryo euploidy. Study design, size, duration The study design was observational and retrospective. A total of 414 embryos from 98 cycles of preimplantation genetic testing for aneuploidies were included (January 2019 to December 2023). Participants/materials, setting, methods Trophectoderm samples were analyzed by preimplantation genetic testing for aneuploidies (PGT-A). Manually annotated time-lapse data points were recorded from which predictive models of embryo aneuploidy were developed. Measurements of the oocyte area were recorded and the automatic embryo KID scores calculated using an in-built commercial software. Multiple regression analyses were performed and a Receiver Operating Characteristic (ROC) analysis conducted. Main results and the role of chance The mean age of female participants included in the study was 34.5 ± 4.6 years. The rate of embryo aneuploidy detected was 41.8%. When a univariate analysis of different time-lapse data was performed, a large number of variables showed statistically significant differences between the euploid and aneuploid embryo groups. The mean oocyte area (µm2) in the euploid group was found to be significantly larger than in the aneuploid group (10151.4 ±621.6 vs 9915.3 ± 950.4; P &amp;lt; 0.01). There was also a significant positive association between oocyte area and embryo euploid status after adjusting for confounders (P &amp;lt;0.01). The Area Under the Curve (AUC) value was 0.62 for the aneuploidy explanatory model when oocyte area was combined with the KID score, compared to an AUC of 0.60 for the KID score alone. Although significant associations were detected, the predictive power of oocyte area measurement did not translate into a clinically useful value. Limitations, reasons for caution The limitations of this study stem from its retrospective nature. The low number of observations also limits the strength of the findings. Wider implications of the findings Although it is possible to predict embryo aneuploidy from some oocyte characteristics, the discriminatory power of designed time-lapse predictive models remained suboptimal for use in current clinical practice. Trial registration number Not applicable

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

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

P-527 Low sperm motility and elevated sperm DNA fragmentation index (DFI) are associated with increased rates of embryo aneuploidy in patients with advanced reproductive age

Abstract Study question Is there a correlation between the paternal age, sperm characteristics, and the prevalence of chromosomal abnormalities in embryos generated through in vitro fertilization (IVF)? Summary answer A correlation exists between advanced paternal age, suboptimal sperm parameters, and elevated rates of embryo aneuploidy in individuals of advanced reproductive age. What is known already The exact mechanisms underlying the relationship between paternal age, sperm quality and embryo ploidy, although a subject of considerable research in assisted reproduction, are not fully elucidated. Some reports, including our group’s, suggest that paternal age may influence sperm quality and segmental embryo aneuploidy, while others suggest a potential association between high DFI(DNA Fragmentation Index) and an increased likelihood of embryo aneuploidy. Studies also show that sperm with optimal motility and fertilization competence are more likely to contribute to the development of euploid embryos, while data on the effect of abnormal sperm parameters on embryo ploidy is scarce and conflicting. Study design, size, duration This was a retrospective cohort study performed in the Reproductive Genetics Department, CReATe Fertility Centre, Toronto, Canada, between January 2019-December 2023. A total of 7628 blastocysts from 1512 cycles (1392 patients) were analyzed using high resolution NGS PGT-A. Sperm motility, concentration and DFI data, as well as, IVF cycle parameter data were collected. In addition, age of gamete providers and any relevant clinical, laboratory and demographic data were obtained for statistical analysis. Participants/materials, setting, methods Embryos were allocated into two groups by oocyte-provider age: OP-≤33 years and OP-≥34 years. Each group was divided into two sub-groups by paternal age: SP-≤39 years (n = 4570); SP-≥40 years (n = 3058). Multiple logistic/ordinal regressions were conducted to evaluate Segmental abnormalities and Segmental mosaicism between these age groups in relation to sperm DFI (GOOD ≤15%; FAIR 15-30%; POOR ≥30%), Motility (HIGH &amp;gt;40%; LOW &amp;lt;40%) and Count (LOW &amp;lt;5 mil/ml; MEDIUM 5-15 mil/ml; HIGH &amp;gt;15 mil/ml). Main results and the role of chance PGT-A analysis of 7628 embryos showed 51.5% euploidy, 36.7% aneuploidy and 11.8% mosaicism. In the OP-≤33 group, there were no significant differences between the two paternal age groups for euploidy, aneuploidy or mosaicism rates. In the OP-≥34 group, decreasing euploidy rates (p &amp;lt; 0.0001 and increasing aneuploidy rates (p &amp;lt; 0.0001) were observed between the two paternal age groups, while mosaicism rates were not different, an expected oocyte age-effect result. No significant differences in aneuploidy rates were observed for each of the sperm parameters (DFI, motility, count) in the groups of the OP-≤33 with SP-≤39, OP-≤33 with SP-≥40 and OP-≥34 with SP-≤39. In the group of OP-≥34 with SP-≥40, in patients with LOW motility vs. HIGH motility the aneuploidy rates were higher (56.8% vs.50.6%, p = 0.005) and euploidy rates were lower (33.8% vs.38.4%, p = 0.03). Each sperm parameter in each age group was further analyzed for chromosomal segmental abnormalities and segmental mosaicism. Higher rates of segmental abnormalities were seen in LOW motility groups with POOR-DFI compared to GOOD DFI in OP-≤33 with SP-≤39 (3.2% vs. 7.4%, p = 0.03). Limitations, reasons for caution The main limitation was the retrospective nature of this study; however, multiple regression analyses were conducted to limit potential bias by adjusting for sperm and oocyte characteristics. In addition, PGT-A analysis cannot provide information on origin of the aneuploidy and all conclusions are deduced from indirect associations to paternal age. Wider implications of the findings The identified associations could stimulate further research into exploring the molecular/cellular processes involved in fertilization, early embryonic development, and the potential impact of sperm-related factors on the chromosomal stability of developing embryos. The broader implications of these findings extend to clinical practice, patient counseling, technological advancements, and ethical considerations. Trial registration number not applicable

P-137 A deep learning model using time-lapse imaging data to predict successful clinical pregnancy and embryo euploidy

Abstract Study question Are the deep learning models helpful in selecting embryos with a high probability of clinical pregnancy and euploidy from time-lapse imaging during embryo growth? Summary answer Our deep learning models using time-lapse imaging data predicted the probabilities of successful clinical pregnancy and euploidy of embryos with high accuracy. What is known already Recent studies have attempted to select the most viable embryo using deep learning of time-lapse imaging during incubation. However, most studies included embryos that were not transferred due to low evaluation by the embryologist as embryos with unsuccessful pregnancies, or they mixed single and multiple embryo transfer cases in the analyses. Some studies’ algorithms rely on humans’ subjective interpretation of morphological and morphokinetic features, leading to embryologist-to-embryologist and within-embryologist variabilities. Some reports, including the study predicting embryo aneuploidy, used a snapshot image for prediction. These approaches have restricted the potential of deep learning in embryo selections. Study design, size, duration Retrospective analyses of time-lapse videos of 2,192 embryos with clinical pregnancy outcomes, defined as confirmed gestational sacs in utero, and 1,467 embryos with karyotype outcomes examined by preimplantation genetic testing for aneuploidy (PGT-A). The data were obtained from two different in-vitro fertilization clinics between November 2017 and December 2021. All transfers were performed as a single embryo transfer. Participants/materials, setting, methods The deep learning model was trained using time-lapse videos with known clinical pregnancy outcomes for predicting the probability of clinical pregnancy with given time-lapse video sequences and patient ages (CP model). Another deep learning model was trained using time-lapse videos with known karyotype outcomes by PGT-A for predicting the probability of euploidy (AP model). The models’ predictive powers were measured using the average area under the curve (AUC) of the receiver operating characteristic curve. Main results and the role of chance The CP model predicted clinical pregnancy with an AUC of 0.693. The AP model predicted embryo aneuploidy with an AUC of 0.702. Limitations, reasons for caution The CP model is based on the embryos that were transferred because the embryologist judged them as having a high probability of pregnancy. Clinical pregnancy may be associated with maternal factors besides embryo factors. Wider implications of the findings Our two deep learning models, the CP and AP models, will be helpful for selecting the embryos to be transferred. The CP model may increase pregnancy rates in patients undergoing assisted reproductive technology. The AP model may reduce the miscarriage caused by embryo aneuploidy. Trial registration number not applicable

P-552 Unveiling associated factors to in vitro embryonic ploidy: Insights from 8194 blastocysts by Next Generation Sequencing

Abstract Study question What factors are associated with an increased probability of having aneuploid or mosaic embryos in vitro? Summary answer Maternal age and biopsy day significantly influence aneuploidy, while trophectoderm quality impacts both aneuploidy and embryo mosaicism. What is known already Embryonic aneuploidy and mosaicism are critical factors affecting the success of in vitro fertilization. Several studies have demonstrated that advanced maternal age is the most important variable associated with chromosomal abnormalities. However, the influence of other factors, including the day of the blastocyst biopsy and the quality of specific embryonic components, such as the trophectoderm, on the likelihood of aneuploidy and mosaicism is less understood. Study design, size, duration This retrospective study involved the analysis of 8,194 blastocyst embryos derived from 2,180 IVF/ICSI cycles at a specialized assisted reproduction clinic. The study spanned 7 years, from 2017 to 2023, focusing on identifying factors influencing embryonic ploidy Participants/materials, setting, methods Retrospective analysis of 8194 blastocysts from 2180 IVF/ICSI cycles at a single fertility center. Evaluated maternal age, paternal age, day of embryo biopsy, inner cell mass and trophectoderm quality, embryo biopsy operator, fertilization technique, using logistic regression to assess impact on ploidy (euploid, aneuploid, mosaic). Analysis identified significant factors influencing ploidy, adjusting for confounders. Statistical significance at p &amp;lt; 0.05. Main results and the role of chance The analysis of 8,194 blastocyst embryos highlighted significant findings. Maternal age showed a considerable impact on aneuploidy with a p-value &amp;lt;0.001 and an odds ratio (OR) of 1.74, indicating a strong association. Similarly, the day of embryo biopsy was a significant factor (p &amp;lt; 0.001, OR = 1.51) emphasizing its influence on embryo aneuploidy. The quality of the trophectoderm was a crucial factor, with a notable correlation with both aneuploidy (p &amp;lt; 0.001, OR = 1.78) and mosaicism (p &amp;lt; 0.001, OR = 1.28). Further analysis was also performed to find the variables associated with mosaicism, and the only significant variable was quality of trophectoderm increasing the probability of mosaicism by 28%. These statistical values underscore the robustness of the study’s conclusions. The large sample size and the application of logistic regression models further minimized the role of chance, enhancing the reliability of these findings. The research provides significant insights into the factors influencing embryonic ploidy, extending beyond the well-recognized impact of maternal age. It underscores the importance of considering these variables in clinical assessments during fertility treatments, particularly in evaluating the risk of chromosomal abnormalities. Limitations, reasons for caution The study’s retrospective nature limits causal inferences. The findings need validation in prospective, multicenter studies to establish broader applicability and account for potential biases. Wider implications of the findings These results contribute to the understanding of the factors associated with aneuploidy and embryonic mosaicism, beyond maternal age. Integrating the day of embryo biopsy and trophectoderm quality as key factors into clinical evaluation that could improve clinical decision-making in fertility treatments, especially in assessing the risk of chromosomal abnormalities. Trial registration number Non-Clinical trials

O-099 Implantation 2024 - what do we know?

Abstract Three major factors are involved in implantation, the embryo, the endometrium and the microbiome. The interactions between these major participants need to, support normal pregnancies and reject abnormal ones and have an appropriate pro/anti-inflammatory response to the genital tract microbiome. Implantation is controlled by a series biological checks and balances in the context of an endometrium that regenerates monthly. Our understanding of these checks and balances has been advanced with the use of sequencing technologies. • The high incidence of aneuploidy in early human embryos has been described in detail with low-coverage sequencing- based PGT-A. • The contribution of karyotype abnormality to miscarriages has been clarified with the use of a microarray-based comparative genomic hybridisation and the cell free DNA. • New insights into endometrial composition have been made through single cell sequencing that has revealed the existence of new subsets of cells at the maternal fetal interface. • The genital tract microbiome has been investigated using high-throughput next-generation sequencing of bacterial 16S rRNA genes. This rich tapestry of information has revealed the complexity of cellular and molecular process controlling implantation. The challenge is to identify specific mechanisms and therapeutic targets for couples suffering reproductive failure.

O-270 Clinical-grade whole genome sequencing-based haplarithmisis enables all forms of preimplantation genetic testing

Abstract Study question Can a simple, and scalable assay be developed to concurrently perform all forms of preimplantation genetic testing (PGT), effectively addressing the increasing demand? Summary answer Whole genome sequencing-based haplarithmisis offers a single-assay solution for all forms of PGT, enhancing resolution and diagnosis in complex cases. What is known already State-of-the-art preimplantation genetic testing (PGT) methods, including genotyping-by-sequencing (GBS)-based haplotyping, enable PGT for monogenic disorders (PGT-M), structural rearrangements (PGT-SR), and aneuploidies (PGT-A) in a single assay. However, they are labour intensive, only partially cover the genome, require a close relative to determine meiotic and mitotic origin of aneuploidies, and are troublesome for consanguineous families and in complex genomic contexts. Furthermore, the prevailing PGT approach for mitochondrial DNA disorders (PGT-MT) involves a PCR-based method. This underscores the need to develop a universal PGT method that streamlines laboratory protocols and provides genome-wide coverage to address genetic disorders, even in complex genomic loci. Study design, size, duration In a retrospective pilot study, we compared whole genome sequencing (WGS) at 30-40X with GBS (6 embryos, 2 families, 3 indications). We carried out subsampling of WGS data and determined 10X as the optimal diagnostic depth-of-coverage. We clinically validated our approach by comparing WGS to GBS for PGT-M and PGT-SR (31 embryos, 16 families, 22 indications), to shallow sequencing for PGT-SR (10 embryos, 3 families) and to PCR-based PGT for PGT-MT (10 embryos, 2 families). Participants/materials, setting, methods DNA from parents and close relative(s) and whole genome amplified (WGAed) DNA from embryos, underwent WGS with subsequent haplarithmisis. Quality metrics, such as Mendelian inconsistency rates, number of informative SNPs, and haplotype concordance were assessed. Translocation breakpoints were identified utilizing aberrantly aligned paired sequencing reads. Additionally, single-nucleotide variants (SNVs), including mitochondrial DNA SNVs, were detected by allele counting. Finally, segregational origin was determined via a parents-only haplarithmisis approach that uses the embryo as phasing reference. Main results and the role of chance WGS-PGT alleviates technical limitations by decreasing WGA artifacts by 68.4%, increasing breadth of coverage by 4-fold, and decreasing wet-lab turnaround time by 2.5-fold. Owing to its inherent higher resolution and improved genomic coverage, 5 out of 13 – previously inconclusive – embryos with genetic disorders in complex genomic regions could be assigned a diagnosis. Furthermore, we show the ability to directly detect single- and few-base pair genetic variants in any region of interest. Importantly, we show trio-based PGT-A for aneuploidy origin, an approach we term PGT-AO that – using the parents-only genotype calls – can detect the segregational origin of aneuploidies without the need of a close relative as reference for phasing. The segregational origin detected by parents-only haplarithmisis was concordant with the segregational origin determined from haplarithmisis when using a close relative. Unbalanced translocations could be detected indirectly via haplotyping or directly at single base resolution using a structural variant caller. Mitochondrial heteroplasmy level calculation from WGS yielded comparable results to PCR-based methods, both in blastomere and trophectoderm biopsy samples. Limitations, reasons for caution Direct variant detection with an average coverage of 10X may occasionally be inadequate, as certain locations may lack sufficient coverage (&amp;lt;5 reads). Inherently to the limited sample size of PGT-MT families, WGS-PGT needs further validation. The need for invasive trophectoderm biopsies of preimplantation embryos remains the key limitation of WGS-PGT. Wider implications of the findings Our approach enables all forms of PGT in a single assay. Direct variant detection complements haplarithmisis, addressing inconclusive findings and aiding families with a de novo variant or lacking a close relative for phasing. Finally, PGT-AO facilitates research on the origin of aneuploidies in preimplantation embryos and their clinical outcome. Trial registration number not applicable

O-008 Sperm-derived centrioles play a key role in ensuring correct chromosome segregation in the first mitotic division of mammalian zygotes

Abstract Study question What is the role of the centrosomes during mitotic division in the mammalian zygote? Does the atypical structure of sperm-derived centrioles affect their function? Summary answer Correct centrosome positioning guarantees spindle stability and correct chromosome segregation. Centrosome malposition or inactivity predisposes to chromosome segregation errors and failure of cytokinesis. What is known already The centrosomes of somatic cells comprise two centrioles surrounded by pericentriolar material, and are major microtubule-organizing centres with an essential role in spindle assembly and chromosome segregation. Mammalian oocytes lack centrosomes and, although two centrioles are introduced by the spermatozoon at fertilization, they are structurally different to the canonical centrioles found in somatic cells. We recently demonstrated that centrosomes play a minor role during zygotic spindle assembly, when they are often mal-positioned (not at the spindle poles). However, the causes and consequences of abnormal centrosome positioning in zygotes are not known. Study design, size, duration We combined systematic immunofluorescence of bovine zygotes, fixed at different stages, with real-time imaging of live zygotes to investigate the implications of centrosome mal-positioning during zygotic mitosis on the fidelity of chromosome segregation and cytokinesis. To study the functionality of the sperm-derived centrioles alone, we inhibited the formation of daughter centrioles using the PLK4 inhibitor (PLK4i), Centrinone. Bovine zygotes were used because the structure of bovine and human sperm-derived centrioles are very similar. Participants/materials, setting, methods Bovine oocytes were retrieved from slaughterhouse ovaries and matured in vitro. Matured oocytes were fertilized in vitro with semen from a bull of proven fertility and, either fixed at 27-29h post-fertilization, or used for time-lapse light-sheet imaging after injection of mRNA for microtubule and chromatin reporters. Fixed bovine zygotes were immunostained for chromatin, alpha-tubulin, centriolar and pericentriolar components, and imaged with a confocal microscope. Microtubule nucleation ability was evaluated using a microtubule regrowth assay. Main results and the role of chance Live imaging showed that in 52% of bovine zygotes (24/46) the centrosomes engaged with the spindle but were malposition, while in 26% (12/46) the centrosomes were not visible (inactive). All zygotes with normally positioned centrosomes showed correct chromosome segregation. Centrosome malposition and inactivity induced chromosome segregation errors in 44% and 82% of the zygotes respectively. PLK4i treatment successfully inhibited centriole duplication, since the volume of core-centriole protein (POC5) in treated zygotes was half that in non-treated zygotes. When centriole duplication was prevented, the sperm-derived centrioles showed a similar ability to recruit pericentrosomal material (NEDD1) and nucleate microtubules. However, live imaging showed that 82% (14/17) of PLK4i treated zygotes the spindle, although bipolar, was unusually elongated and chromosomes either failed to align correctly on the metaphase plate or showed anaphase lag. Moreover, in treated zygotes where both centrioles were positioned at the same spindle pole, the spindle failed to anchor correctly to the cell membrane and resulted in rotational behavior of the zygote and an increase of cytokinesis failure from up to 47% (8/17). Therefore we conclude that centrosomes are likely to play a key role in correct chromosome segregation in mammalian zygotes. Limitations, reasons for caution For ethical reasons, we could not manipulate and study human zygotes. Instead, we chose an animal model with a rate of embryonic aneuploidy similar to man. It is, however, possible that zygotic centrosome function differs between these two species. Wider implications of the findings A better understanding of the key regulators of centrosome function might lead to the development of interventions to reduce the risk of malfunction, thereby reducing the incidence of embryonic aneuploidy. Moreover, our study unveiled an mechanism by which impaired PLK4 function induces aneuploidy in mammalian embryos. Trial registration number not applicable

O-153 Clinical utility of preimplantation genetic testing for aneuploidy origin (PGT-AO) to enhance embryo selection for monogenic disorders: a retrospective pan-country integrative genomic and clinical study

Abstract Study question What is PGT-AO’s impact on implantation and pregnancy rate per embryo transfer, by determining the prevalence and effect of (mosaic) aneuploidies and their segregational origin? Summary answer PGT-AO potentially enhances the efficiency of PGT and increases the pregnancy rate per embryo transfer by minimizing transfers with poor outcomes and improving implantation rates. What is known already Demographic factors such as advanced parental age increases risk of aneuploid offspring, driving demand for advanced PGT. Randomized controlled trials revealed that PGT-A does not increase live birth rates after IVF. PGT-A, the most frequent form of PGT, overlooks the mosaic nature and segregational origin of aneuploidies in IVF embryos. This possibly leads to avoiding the transfer of mosaic embryos and reduces the pregnancy rate per cycle. Prompting this study, five embryos transferred based on PGT-M, led to trisomic pregnancies terminated following invasive prenatal diagnosis. Subsequent genome haplarithmisis revealed that these embryos carried (meiotic) trisomies not previously detected by PGT-M. Study design, size, duration In this retrospective study, we included 1944 embryos (816 PGT-M embryos, 323 couples, 224 indications). Currently, 284 have been processed by genome haplarithmisis-based PGT-AO and analyzed to assess if (mosaic) aneuploidies and segregational origin would have altered transfer decisions and subsequent clinical outcomes. We evaluated our novel PGT- A approach, termed PGT-AO, providing a nuanced understanding of early embryonic development in clinical practice and more responsible PGT-A, potentially leading to viable mosaic embryo transfer. Participants/materials, setting, methods The couples were counseled by clinical geneticists at Dutch University Medical Centers (UMCs), in Maastricht, Amsterdam, Utrecht, and Groningen, and enrolled in the diagnostic PGT-M procedure after signing an informed consent form. Embryos (n = 816) eligible for transfer based on absence of the monogenic aberration of interest by PGT-M were analyzed by haplarithmisis-based PGT-AO including parental information. This information allows for the detection of (mosaic) chromosomal abnormalities and their parental and segregational origin. Main results and the role of chance Of 816 included embryos (323 couples, 224 indications), 284 were found to be suitable for transfer by PGT-M and were analyzed by PGT-AO. Of these, 183 were transferred. Of the transferred embryos, 65 (35.5%) had a positive HCG and 48 (26.3%) resulted in a liveborn. 14.6% (7/48) carried abnormalities following our PGT-AO approach. These abnormalities consisted of mitotic mosaic trisomies, mosaic monosomies, and mosaic duplications and a deletion. Of the transferred embryos, 132 (72.1%) did not result in a liveborn and 115 (62.8%) had negative HCG tests. Of the transferred embryos with negative HCG, 38.3% (44/115) contained a total of 90 abnormalities, consisting of 37 monosomies, 35 trisomies, 12 deletions, 2 duplications, 2 UPD and 1 ring chromosome. The 35 trisomies occurred in 20 embryos, of which 9 were of meiotic origin and 5 of mitotic origin, 1 of both meiotic and mitotic, and 5 could not be determined. Based on these preliminary results, PGT-AO can avoid the transfer of embryos with aneuploidy of meiotic origin and non-mosaic status. In contrast, embryos with aneuploidy of mitotic origin are often mosaic and the abnormality may not be uniformly distributed throughout the blastocyst and could potentially be considered for transfer. Limitations, reasons for caution Haplarithmisis relies on the inclusion of parental DNA to phase the embryonic genome and thus determine the segregational origin of aberrations. Additionally, mitosis and meiosis II trisomies cannot be discerned where a crossover event is absent for a chromosome with trisomy. Wider implications of the findings Implementation of PGT-AO will re-define the current embryo selection system. This enhances PGT by increasing pregnancy rate per embryo transfer and minimizing transfers with poor outcomes, disposal of potentially viable embryos, spontaneous pregnancy losses, and termination of aneuploid pregnancies. This reduces the emotional and financial burdens on families and healthcare. Trial registration number Not applicable

O-156 Trophectoderm biopsy for pre-implantation genetic testing (PGT) does not increase rates of placental abnormalities among singleton pregnancies conceived with in-vitro fertilization (IVF)

Abstract Study question Does trophectoderm biopsy for PGT affect placental pathology? Summary answer Trophectoderm biopsy does not increase rates of placental abnormalities among singleton pregnancies conceived with in-vitro fertilization (IVF). What is known already PGT, routinely performed at the blastocyst stage, is used to screen embryos for aneuploidy, structural rearrangements, and single-gene disorders. The association of ART with abnormal placentation and adverse pregnancy outcomes is known. Whether, in a similar fashion, the disruption of the trophectoderm contributes to increased rates of abnormal placentation, remains unknown. Study design, size, duration Data from 987 singleton livebirths with available placental pathology, who conceived following IVF/±PGT at a single academic fertility center between 01/2004 and 12/2019, were retrospectively reviewed. Placental abnormalities were classified as anatomic, inflammatory, infectious, and vascular (any features of fetal or maternal vascular malperfusion), by one expert perinatal pathologist using the Amsterdam Workshop Consensus definitions. Placental pathology was compared between PGT (n = 76) and non-PGT (fresh and frozen, n = 911) cycles. Participants/materials, setting, methods Primary Outcome Measures: incidence of anatomic, vascular (maternal and fetal side), infectious, and inflammatory placental abnormalities. Odds ratios (OR) and 95% confidence intervals (95%CI) were calculated using generalized estimating equations regression to adjust for multiple deliveries per patient and for potential confounders [maternal age, body mass index (BMI:kg/m2), race, parity, infertility diagnosis, gestational age, infant gender, hypertensive disorders of pregnancy, IVF and FET protocol]. Main results and the role of chance Patients undergoing PGT were older than the non-PGT group [36.2(3.5) vs. 34.9(3.8) years, p = 0.006] and did not otherwise differ in AMH, BMI, and race. Overall, adjusted (Adj) OR (95%CI) did not reveal any differences between groups in anatomic [0.92(0.82-1.03)], inflammatory [1.03 (0.94-1.14)]), infectious [1.02 (0.9-1.16)], or vascular abnormalities [1.02(0.88-1.18)]. When limiting analysis to blastocyst biopsies/FET cycles only, adjOR did not show any differences in inflammatory [1.01 (0.89-1.13)]), infectious [1.02 (0.88-1.17)], or vascular abnormalities [1.01 (0.84-1.2)] between the PGT and non-PGT group. Interestingly, adjOR (95%CI) revealed lower odds for anatomic abnormalities in the PGT group (0.86 (0.76-0.98), p = 0.021, non-PGT: ref). A sub-analysis of programmed FET cycles revealed persistently lower odds of anatomic abnormalities among the PGT group [adjOR (95%CI): 0.77 (0.67-0.89), p &amp;lt; 0.001, non-PGT: ref]. Among anatomic abnormalities studied in the programmed FET cycles, marginal cord insertion showed a difference between the groups [adjOR(95%CI): 0.83 (0.73-0.95), p:0.007, non-PGT: ref ]. AdjOR(95%CI) showed no differences in inflammatory [ 1.01 (0.87-1.17)], infectious [1.01 (0.86-1.2)], and vascular [0.9 (0.73-1.1)] abnormalities between groups. Limitations, reasons for caution This study is limited by its retrospective nature. Although a large number of IVF placentas was included, the number of patients in the PGT compared to the non-PGT group was smaller potentially limiting our ability to detect subtle differences between groups. Wider implications of the findings Trophectoderm biopsy for PGT does not appear to increase risk for placental abnormalities, a reassuring finding given the increased incidence of abnormal placentation and adverse perinatal outcomes among IVF pregnancies. Trial registration number not applicable

P-630 Assessing Bologna and POSEIDON criteria as predictors of PGT-A in Poor Ovarian Response: A comparative analysis

Abstract Study question Do Bologna or POSEIDON criteria more accurately predict PGT-A outcomes in poor ovarian response, considering aneuploidy rates and clinical outcomes? Summary answer Comparing Bologna and POSEIDON criteria highlights higher aneuploidy rates in Bologna patients, with no significant differences in mosaicism rate or clinical outcomes in either group. What is known already Ovarian reserve stands out as the primary factor influencing reproductive senescence. Approximately 9-24% of individuals undergoing ovarian stimulation are categorized as ‘low-responders’. Key indicators of Poor Ovarian Response (POR) include age, antral follicle count (AFC), Anti-Müllerian Hormone (AMH), and response to controlled ovarian stimulation. While some studies link ovarian reserve to embryo quality, others dispute any correlation with embryo abnormalities. The Bologna criteria classify patients meeting at least two of the following: advanced maternal age, low AFC, and AMH. The more precise POSEIDON criteria combine these factors, forming four distinct groups for classification. Study design, size, duration We conducted a retrospective case-control study, examining PGT-A results of 2740 embryos derived from 797 IVF cycles conducted in our clinic between 2021 and 2022. Trophoectoderm biopsies, taken at D + 5/6, underwent Next-Generation Sequencing. Embryos were vitrified and transferred after analysis. Regarding the interpretation of the results, embryos with ≤25% cells aneuploids were considered euploid, between 25% and 50% were classified as mosaic and those with &amp;gt;50% cells were considered aneuploid. Participants/materials, setting, methods All patients used their own oocytes. Bologna and POSEIDON criteria were used for the classification of POR and non-POR patients. Univariate analysis was performed using Pearson’s chi-square statistical test for categorical variables and binary logistic regression to compare the rates of aneuploidy and embryonic mosaicism, pregnancy rate miscarriage, ongoing pregnancy rate and implantation rate with the above variables taking into account possible confounding factors. Main results and the role of chance Patient ages ranged from 22 to 47 years, with a mean of 38.9 years. In Bologna groups, mean ages were 41.15 years for Bologna and 38.43 years for the control. For POSEIDON: 32.47 group I, 39.85y group II, 32.11y group III, 40.41y group IV and 38.24y control group of the classified by POSEIDON criteria. The mean AFC was 10.95 and the AMH value was 13.15pmol/L. Among 797 cycles, 59.16% (449/797) met POR criteria by POSEIDON, distributed across groups: 1.19% (9/797) in group I, 1.32% (10/797) in group II, 22.66% (172/797) in group III and 33.99% (258/797) in group IV. Using Bologna criteria, 17.74% (486/797) were classified as POR. Our findings reveal that individuals identified with POR by Bologna criteria exhibit a significantly elevated incidence of embryo aneuploidy (78.8% POR patients vs. 59.9% control patients; p = 0.004). However, no such distinction was observed when employing the POSEIDON criteria for classification. In terms of embryo mosaicism, no notable differences were detected between POR and non-POR patients under both Bologna and POSEIDON criteria. Furthermore, no significant variations in clinical data were observed between the two groups, even after correcting for maternal age. Limitations, reasons for caution The study is constrained by the inherent limitations of a retrospective design and a restricted sample size. Moreover, the complexity and heterogeneity involved in classifying patients with POR contribute to additional challenges in the interpretation of results. Wider implications of the findings The study indicates that diminished ovarian reserve does not significantly augment the probability of embryo aneuploidies. Particularly noteworthy is the observation that young women with low ovarian reserve do not exhibit an increased susceptibility to aneuploidies. This finding holds implications for understanding reproductive outcomes among ovarian reserve variations. Trial registration number No aplicable