Euploid Embryos Research Articles

Embryonic mosaicism: What does it mean clinically?

Embryonic mosaicism continues to represent a complex phenomenon that is considered by many authors as a limiting factor in the interpretation of PGT-A cycles results.
 A mosaic embryo is characterized by the presence of cell populations with at least two different karyotypes. There are controversial aspects related to embryonic mosaicism, such as the great variability in the incidence reported between laboratories and clinics, which in turn is related to the existence of false positives during the diagnosis. These false positives can also affect the results after the transfer of these embryos. Numerous publications have analyzed the results of transferring this type of embryo. The majority agrees that although worse clinical results are achieved that, when transferring a euploid embryo, it is possible to achieve an evolutionary pregnancy with a healthy birth. The objective of this article is to provide tools that allow the professional to advise those patients who perform a PGT-A cycle about this conflictive aspect of it.

Bioaccumulation of Non-Essential Trace Elements Detected in Women's Follicular Fluid, Urine, and Plasma Is Associated with Poor Reproductive Outcomes following Single Euploid Embryo Transfer: A Pilot Study.

This study aims to determine the association of non-essential trace elements present in follicular fluid, plasma, and urine with reproductive outcomes of women undergoing intracytoplasmic sperm injection (ICSI), preimplantation genetic testing for aneuploidies (PGT-A) and single frozen euploid embryo transfer (SET/FET). This single-center, prospective cohort study included sixty women undergoing ICSI with PGT-A and SET/FET between 2018 and 2019. Urine, plasma and follicular fluid samples were collected on the vaginal oocyte retrieval day to simultaneously quantify ten non-essential trace elements (i.e., Ba, Sr, Rb, Sn, Ti, Pb, Cd, Hg, Sb, and As). We found several associations between the levels of these non-essential trace elements and clinical IVF parameters. Specifically, the increased levels of barium in follicular fluid were negatively associated with ovarian function, pre-implantation development and embryo euploidy, while elevated strontium concentrations in this biofluid were negatively associated with impaired blastulation and embryo euploidy. Elevated plasma strontium levels were negatively associated with ovarian function, fertilization and blastulation. Enhanced presence of other trace elements in plasma (i.e., rubidium and arsenic) were associated with a diminished ovarian function and limited the number of recovered oocytes, mature oocytes and zygotes, respectively. Fully adjusted models suggested significantly lower odds of achieving a live birth when increased concentrations of barium and tin were found in urine.

High sperm DNA fragmentation increased embryo aneuploidy rate in patients undergoing preimplantation genetic testing

Is high sperm DNA fragmentation (SDF) associated with a high embryonic aneuploidy rate in patients undergoing intracytoplasmic sperm injection (ICSI)-preimplantation genetic testing (PGT)? This was a retrospective study of 426 couples with normal karyotypes undergoing ICSI-PGT at the authors' centre from March 2017 to March 2021. SDF was assessed using the sperm chromatin structure assay. The population was divided into low and high SDF groups according to cut-off values found by the receiver operating characteristic (ROC) curve. A 1:1 ratio propensity score matching (PSM) method was used to control for potential confounding factors, and a generalized linear mixed model was established to evaluate the relationship between SDF and the embryonic aneuploidy rate. The ROC curve indicated a threshold of 30%. In total, 132 couples were included after PSM, and the high SDF group (>30%) had significantly higher SDF (40.74% ± 9.78% versus 15.54% ± 7.86%, P < 0.001) and a higher embryo aneuploidy rate (69.36% versus 53.96%, P < 0.001) compared with the low SDF group (≤30%). The two pronuclear fertilization rate, cleavage rate, rate of high-quality embryos at day 3 rate, blastocyst rate, biochemical pregnancy rate, clinical pregnancy rate, miscarriage rate, live birth rate, caesarean section rate, preterm birth rate, singleton rate and low birthweight rate were similar in both groups (P > 0.05). After PSM, SDF > 30% was significantly correlated with an increased embryo aneuploidy rate after adjusting for all confounding variables (adjusted odds ratio 1.70, 95% CI 1.00-2.88, P = 0.049). SDF > 30% was associated with an increased embryo aneuploidy rate in couples with normal karyotypes undergoing PGT, but did not affect embryonic and clinical outcomes after transfer of euploid embryos.

Association between endometrial microbiome and implantation success in women with frozen embryo transfer: results of a prospective cohort study

The aim of this prospective study was to compare the endometrial microbiome between pregnant and non-pregnant women after frozen embryo transfer (FET) with euploid embryos. Endometrial biopsies were collected from 30 women during the mid-luteal phase in a natural cycle. FET was performed with euploid embryos up to 3 months after the biopsy. Endometrial microbiota composition was analysed using 16S rRNA (v4-v5 region) next generation sequencing (NGS). The analysis of different clinical outcomes after the biopsy (no pregnancy (n = 14), and ultrasound confirmed pregnancy (n = 16)) revealed differences in the endometrial microbiome composition. In total, 271 distinct bacterial species and 668 bacterial genera were identified. The number of unique species found in non-pregnant women was 62 (22.88%), while in the patients who became pregnant after FET it was 39 (14.39%). Among them, bacteria with high frequency of occurrence such as Bacteroides spp., Cutibacterium granulosum, Isoptericola spp., Acetomicrobium spp., Marivivens spp. and Syntrophomonas spp. were found only in non-pregnant patients, while Bosea spp. was present only in pregnant women. The analysis of bacteria relative abundance revealed that Lactobacillus genus was not significantly different between the studied groups. In contrast, Serratia marcescens, Staphylococcus spp., Glutamicibacter spp. and Delftia spp. were significantly enriched in the non-pregnant group. In conclusion, specific bacteria taxa had higher relative abundance in the endometrium of patients with implantation failure after FET with euploid embryos. We hypothesize that an appropriate treatment for optimization of endometrial microbiome content in women with diagnosed microbiome dysbiosis could be beneficial for improvement of pregnancy rates.

Spontaneous collapse as a prognostic marker for human blastocysts: a systematic review and meta-analysis.

Is spontaneous collapse (SC) by human blastocysts a prognostic factor in IVF treatment? SC in human blastocyst is associated with reduced euploid embryo and pregnancy rates. SC of the human blastocyst is a phenomenon that was revealed relatively recently following the clinical application of time-lapse monitoring in IVF laboratories. The ploidy and clinical prognosis of affected blastocysts are still poorly understood, with inconsistent reports. Systematic reviews and meta-analyses on this topic are currently absent in the literature but its potential as a marker of embryo viability holds great clinical value. In this study, we aimed to comprehensively evaluate the potential of SC as a prognostic factor in regard to ploidy status, and pregnancy, live birth and miscarriage rates. A systematic review and meta-analysis were performed according to PRISMA guidelines, with a protocol registered with PROSPERO (CRD42022373749). A search of MEDLINE, EMBASE, and the Cochrane Library for relevant studies was carried out on 10 October 2022, using key words relevant to 'blastocyst collapse' and 'time-lapse imaging'. Two independent reviewers systematically screened and evaluated each study in terms of participants, exposure, comparator, and outcomes (PECO). The Quality In Prognosis Studies tool was used for quality assessment. Data were extracted according to Cochrane methods. Pregnancy, live birth, ploidy, or miscarriage data were summarized by risk ratios (RRs) or odds ratios and their 95% CIs. All meta-analyses were performed with random-effects models. Following removal of duplicates, a total of 196 records were identified by the initial search. After screening according to PECO, 19 articles were included for further eligibility assessment. For meta-analysis, seven retrospective cohort studies were eventually included. After data pooling, the incidence of blastocyst SC was 37.0% (2516/6801) among seven studies (ranging from 17.4% to 56.2%). SC was associated with significantly lower clinical pregnancy rates (two studies, n = 736; RR = 0.77, 95% CI = 0.62-0.95; I2 = 30%), ongoing pregnancy rates (five studies, n = 2503; RR = 0.66, 95% CI = 0.53-0.83; I2 = 60%), and reduced euploidy rates (three studies, n = 3569; RR = 0.70, 95% CI = 0.59-0.83; I2 = 69%). Nevertheless, live birth rates (two studies, n = 816; RR = 0.76, 95% CI = 0.55-1.04; I2 = 56%) and miscarriage rate (four studies, n = 1358; RR = 1.31, 95% CI = 0.95-1.80; I2 = 0%) did not differ between blastocysts with or without SC. There was, however, significant heterogeneity between the studies included for evaluation of ongoing pregnancy rates (I2 = 60%, P = 0.04), live birth rates (I2 = 56%, P = 0.13), and ploidy rates (I2 = 69%, P = 0.04). Subgroup analyses were conducted according to different definitions of SC, number of collapse events, and whether the transferred blastocyst had undergone preimplantation genetic testing for aneuploidy; with inconclusive findings across subgroups. All studies in the meta-analysis were retrospective with varying levels of heterogeneity for different outcomes. Not all studies had accounted for potential confounding factors, therefore only unadjusted data could be used in the main meta-analysis. Studies employed slightly different strategies when defining blastocyst SC. Standardization in the definition for SC is needed to improve comparability between future studies. Our results indicate that blastocyst SC has negative implications for a pregnancy. Such blastocysts should be given a low ranking when selecting from a cohort for intrauterine transfer. Blastocyst SC should be considered as a contributing variable when building blastocyst algorithms to predict pregnancy or live birth. There is no external funding to report. All authors report no conflict of interest. PROSPERO 2022 CRD42022373749.

Mitochondrial DNA mutations can influence the post-implantation development of human mosaic embryos.

Introduction: Several healthy euploid births have been reported following the transfer of mosaic embryos, including both euploid and aneuploid blastomeres. This has been attributed to a reduced number of aneuploid cells, as previously reported in mice, but remains poorly explored in humans. We hypothesized that mitochondrial function, one of the most critical factors for embryonic development, can influence human post-implantation embryonic development, including a decrease of aneuploid cells in mosaic embryos. Methods: To clarify the role of mitochondrial function, we biopsied multiple parts of each human embryo and observed the remaining embryos under in vitro culture as a model of post-implantation development (n = 27 embryos). Karyotyping, whole mitochondrial DNA (mtDNA) sequencing, and mtDNA copy number assays were performed on all pre- and post-culture samples. Results: The ratio of euploid embryos was significantly enhanced during in vitro culture, whereas the ratio of mosaic embryos was significantly reduced. Furthermore, post-culture euploid and culturable embryos had significantly few mtDNA mutations, although mtDNA copy numbers did not differ. Discussion: Our results indicate that aneuploid cells decrease in human embryos post-implantation, and mtDNA mutations might induce low mitochondrial function and influence the development of post-implantation embryos with not only aneuploidy but also euploidy. Analyzing the whole mtDNA mutation number may be a novel method for selecting a better mosaic embryo for transfer.

Recurrent Implantation Failure: Reality or a Statistical Mirage?: Consensus Statement From the July 1, 2022 Lugano Workshop on Recurrent Implantation Failure

ABSTRACT Despite advancements in assisted reproductive technologies over the past few decades, a 35% failure rate is still observed for unexplained reasons when transferring euploid embryos to an anatomically normal uterus. Recurrent implantation failure (RIF) is defined based on cumulative ART cycles failing; however, there is an absence in consensus on the diagnostic criteria, which leads to the risk of overdiagnosing and overtreating the condition. This consensus statement by the Lugano RIF Workshop aimed to merge variation in the clinical concept and management of RIF, focusing primarily on euploid blastocyst transfers in hormone replacement-primed cycles. The workshop comprised a panel of 27 international experts selected on the basis of overall research activities and publications on the subject. A literature search as conducted for all relevant titles and abstracts published between January 2015 and May 2022 for review. Consensus conclusions were based on the literature search and expert opinions. Consideration of an RIF diagnosis was determined to focus on failure to achieve sustained implantation (defined as a gestational sac identified on ultrasound). Historically, RIF was defined as the failed transfer of &gt;10 embryos; however, today, a common number used is 3, which is also similar to the rate of failure based on a 95% confidence interval. Based on large clinical data sets, approximately 2%–5% of patients pursuing ART treatment may have RIF, and the decline in future implantation rate with each cycle fits more to an exponential delay curve than a linear decrease. The diagnosis of RIF should not be considered based on a single cohort of embryos, but rather on the number of euploid blastocyst transferred adjusted to patient’s age. In the absence of abnormal uterine bleeding and normal uterine size, further evaluation of asymptomatic adenomyosis is not warranted. Testing for biomarkers of endometrial receptivity and the endometrial microbiome have not been validated using RCTs and should not be used. The role of chronic inflammation in RIF remains unclear, but evidence suggests chronic endometritis does not directly play a role in ART outcome as assessed by sustained implantation rate after euploid embryo transfer. Substantial evidence exists that is low progesterone on the day of embryo transfer results in worse ART outcomes, and that IM progesterone may be superior to vaginal progesterone. There are no effective treatments for RIF currently, and immunomodulatory treatments that may be offered including glucocorticoids, IVIG, and GCSF have a lack of comprehensive data. This consensus document finds that RIF is likely present in fewer than 5% of patients undergoing ART and has been overdiagnosed and overtreated without sufficient critical evaluation of the presumed condition. It also highlights areas where future research is needed, both in the pathophysiologic cause of RIF and hypothetical treatment modalities.

Chromosomal, gestational, and neonatal outcomes of embryos classified as a mosaic by preimplantation genetic testing for aneuploidy

To understand the clinical risks associated with the transfer of embryos classified as a mosaic using preimplantation genetic testing for aneuploidy. Analysis of data collected between 2017 and2023. Multicenter. Patients of infertility treatment. Comparison of pregnancies resulting from embryos classified as euploid or mosaic using the 20%-80% interval in chromosomal intermediate copy numbers to define a mosaic result. Rates of spontaneous abortion, birth weight, length of gestation, incidence of birth defects, and chromosomal status during gestation. Implanted euploid embryos had a significantly lower risk of spontaneous abortion compared with mosaic embryos (8.9% [n = 8,672; 95% confidence interval {CI95} 8.3, 9.5] vs. 22.2% [n = 914; CI95 19.6, 25.0]). Embryos with mosaicism affecting whole chromosomes (not segmental) had the highest risk of spontaneous abortion (27.6% [n = 395; CI95 23.2, 32.3]). Infants born from euploid, mosaic, and whole chromosome mosaic embryos had average birth weights and lengths of gestation that were not statistically different (3,118 g and 267 days [n = 488; CI95 3,067, 3,169, and 266, 268], 3052 g and 265 days [n = 488; CI95 2,993, 3,112, and 264,267], 3,159 g and 268 days [n = 194; CI95 3,070, 3,249, and 266,270], respectively). Out of 488 infants from mosaic embryo transfers (ETs), one had overt gross abnormalities as defined by the Centers for Disease Control and Prevention. Most prenatal tests performed on pregnancies from mosaic ETs had normal results, and only three pregnancies produced prenatal test results reflecting the mosaicism detected at the embryonic stage (3 out of 250, 1.2%; CI95 0.25, 3.5). Although embryos classified as mosaic experience higher rates of miscarriage than euploid embryos (with a particularly high frequency shortly after implantation), infants born of mosaic ETs are similar to infants of euploid ETs. Prenatal testing indicates that mosaicism resolves during most pregnancies, although this process is not perfectly efficient. In a small percentage of cases, the mosaicism persists through gestation. These findings can serve as risk-benefit considerations for mosaic ETs in the fertility clinic.

Systematic review and meta-analysis: does pre-implantation genetic testing for aneuploidy at the blastocyst stage improve live birth rate?

PurposeTo establish if preimplantation genetic testing for aneuploidy (PGT-A) at the blastocyst stage improves the composite outcome of live birth rate and ongoing pregnancy rate per embryo transfer compared to conventional morphological assessment.MethodsA systematic literature search was conducted using PubMed, EMBASE and Cochrane database from 1st March 2000 until 1st March 2022. Studies comparing reproductive outcomes following in vitro fertilisation using comprehensive chromosome screening (CCS) at the blastocyst stage with traditional morphological methods were evaluated.ResultsOf the 1307 citations identified, six randomised control trials (RCTs) and ten cohort studies fulfilled the inclusion criteria. The pooled data identified a benefit between PGT-A and control groups in the composite outcome of live birth rate and ongoing pregnancy per embryo transfer in both the RCT (RR 1.09, 95% CI 1.02–1.16) and cohort studies (RR 1.50, 95% CI 1.28–1.76). Euploid embryos identified by CCS were more likely to be successfully implanted amongst the RCT (RR 1.20, 95% CI 1.10–1.31) and cohort (RR 1.69, 95% CI 1.29–2.21) studies. The rate of miscarriage per clinical pregnancy is also significantly lower when CCS is implemented (RCT: RR 0.73, 95% CI 0.56–0.96 and cohort: RR 0.48, 95% CI 0.32–0.72).ConclusionsCCS-based PGT-A at the blastocyst biopsy stage increases the composite outcome of live births and ongoing pregnancies per embryo transfer and reduces the rate of miscarriage compared to morphological assessment alone. In view of the limited number of studies included and the variation in methodology between studies, future reviews and analyses are required to confirm these findings.

The number of prior pregnancy losses does not impact euploidy rates in young patients with idiopathic recurrent pregnancy loss.

Our study aimed to determine the possible factors that might impact the probability of obtaining a euploid blastocyst following intracytoplasmic sperm injection (ICSI) and preimplantation genetic testing for aneuploidy (PGT-A) procedures in idiopathic recurrent pregnancy loss (RPL) patients. This single-center retrospective cohort analysis included 180 oocyte retrieval cycles of 166 women under 35years old and those diagnosed with idiopathic RPL according to American Society of Reproductive Medicine (ASRM) guidelines. Trophectoderm biopsy and next-generation sequencing (NGS) were the techniques used. Patients were stratified by the number of previous losses (Group A: 2, Group B: 3, and Group C: > 3). Baseline and embryological characteristics showed no statistically significant differences. The euploidy rate per analyzed blastocyst was comparable within the groups (63.3%, 58.2%, and 58.5%; p = 0.477). Logistic regression analyses confirmed that only the trophectoderm scores of A and B increased the probability of obtaining a euploid embryo [OR: 1.82, 95% CI (1.120-2.956), p: 0.016]. It is concluded that there was no correlation between the number of previous losses and the chance of finding at least one euploid embryo in ICSI cycles of women younger than 35years.

The mosaic embryo: what it means for the doctor and the patient.

Mosaic embryos are embryos that on preimplantation genetic analysis are found to be composed of euploid and aneuploid cells. Although most of these embryos do not implant when transferred into the uterus following IVF treatment, some may implant and are capable of giving rise to babies. There is currently an increasing number of reports of live births following the transfer of mosaic embryos. Compared to euploid, mosaic embryos have lower implantation rates and higher rates of miscarriage, and occasionally aneuploid component persists. However, their outcome is better than that obtained after the transfer of embryos consisting entirely of aneuploid cells. After implantation, the ability to develop into a full-term pregnancy is influenced by the amount and type of chromosomal mosaicism present in a mosaic embryo. Nowadays many experts in the reproductive field consider mosaic transfers as an option when no euploid embryos are available. Genetic counseling is an important part of educating patients about the likelihood of having a pregnancy with healthy baby but also on the risk that mosaicism could persist and result in liveborn with chromosomal abnormality. Each situation needs to be assessed on a case-by-case basis and counseled accordingly. So far, the transfers of 2155 mosaic embryos have been documented and 440 live births resulting in healthy babies have been reported. In addition, in the literature to date, there are 6 cases in which embryonic mosaicism persisted. In conclusion, the available data indicate that mosaic embryos have the potential to implant and develop into healthy babies, albeit with lower success rates than euploids. Further clinical outcomes should be collected to better establish a refined ranking of embryos to transfer.

Randomised double-blind controlled trial of non-invasive preimplantation genetic testing for aneuploidy in in vitro fertilisation: a protocol paper

IntroductionThe success rate of in vitro fertilisation (IVF) treatment for couples with infertility remains low due to lack of a reliable tool in selecting euploid embryos for transfer. This study...

Deep learning system for classification of ploidy status using time-lapse videos

To develop a spatiotemporal model for de prediction of euploid and aneuploid embryos using time-lapse videos from 10-115 hours after insemination (hpi). Retrospective study. The research used an end-to-end approach to develop an automated artificial intelligence system capable of extracting features from images and classifying them, considering spatiotemporal dependencies. A convolutional neural network extracted the most relevant features from each video frame. A bidirectional long short-term memory layer received this information and analyzed the temporal dependencies, obtaining a low-dimensional feature vector that characterized each video. A multilayer perceptron classified them into 2 groups, euploid and noneuploid. The model performance in accuracy fell between 0.6170 and 0.7308. A multi-input model with a gate recurrent unit module performed better than others; the precision (or positive predictive value) is 0.8205 for predicting euploidy. Sensitivity, specificity, F1-Score and accuracy are 0.6957, 0.7813, 0.7042, and 0.7308, respectively. This article proposes an artificial intelligence solution for prioritizing euploid embryo transfer. We can highlight the identification of a noninvasive method for chromosomal status diagnosis using a deep learning approach that analyzes raw data provided by time-lapse incubators. This method demonstrated potential automation of the evaluation process, allowing spatial and temporal information to encode.

The Impact of Essential Trace Elements on Ovarian Response and Reproductive Outcomes following Single Euploid Embryo Transfer.

Essential trace elements are required in extremely small amounts and obtained through diet. This research focuses on detecting major trace elements in different biofluids of sixty women undergoing ICSI with PGT-A and SET/FET at IVI-RMA, New Jersey, and assessing their impact on their IVF outcomes. Urine, plasma, and follicular fluid samples were collected on the vaginal oocyte retrieval day to measure the concentrations of eight essential trace elements (copper, zinc, molybdenum, lithium, selenium, manganese, chromium, and iron) using ICP-MS. After analysis, ovarian response and preimplantation outcomes had significant positive associations with both copper alone and the copper/zinc ratio in the follicular fluid and plasma, in addition to plasma manganese. Alternatively, elevated follicular fluid lithium concentrations were significantly associated with poor preimplantation outcomes while the urinary molybdenum concentration was significantly associated with a lower probability of implantation, clinical pregnancy, and live birth. Urinary lithium and chromium concentrations were significantly associated with a lower probability of achieving a live birth. Our results suggest that the essential trace elements present in follicular fluid, plasma, and urine of women are directly associated with their reproductive outcomes, with copper and manganese exerting positive effects and lithium and molybdenum exerting negative effects.

Phytoestrogens Present in Follicular Fluid and Urine Are Positively Associated with IVF Outcomes following Single Euploid Embryo Transfer

The impact and safety of phytoestrogens, plant-derived isoflavones with estrogenic activity predominantly present in soy, on female reproductive health and IVF outcomes continues to be hotly debated. In this prospective cohort study, 60 women attending IVI-RMA New Jersey undergoing IVF with single frozen embryo transfer (SET/FET) of good-quality euploid blastocyst after PGT-A analysis were recruited. Concentrations of two phytoestrogens (daidzein and genistein) in follicular fluid (FF) and urine (U) were measured by UPLC–MSMS, both collected on vaginal oocyte retrieval day. These measurements correlated with IVF clinical outcomes. In models adjusted for age, BMI, race/ethnicity, and smoking status, higher FF phytoestrogen concentrations were significantly associated with higher serum estradiol, enhanced probability of implantation, clinical pregnancy, and live birth. Moreover, higher urine phytoestrogen concentrations were significantly associated with improved oocyte maturation and fertilization potential and increased probability of clinical pregnancy and live birth. Finally, higher FF and urine phytoestrogen concentrations were associated with a higher probability of live birth from a given IVF cycle. Our results suggest that dietary phytoestrogens improved reproductive outcomes of women undergoing IVF treatment. However, additional prospective studies are needed to optimize the use of phytoestrogens to further enhance reproductive outcomes and/or protect against reproductive insults.

P-728 Impact of calling mosaicism for a less stringent threshold on clinical pregnancies in more than 6000 PGT-A cycles

Abstract Study question How does calling mosaicism for a higher threshold affect euploidy, the chance of finding at least one euploid embryo and clinical pregnancy? Summary answer Increasing the threshold significantly increased euploidy percentage and the probability of finding at least one euploid embryo, as expected, but also the pregnancy outcomes. What is known already NGS technology has shed light on the occurrence of chromosomal mosaicism at the preimplantation stage of development. Evidence regarding clinical outcomes after transfer of embryos with putative mosaic results are accumulating (ESHRE Working Group on Chromosomal Mosaicism, 2022). A series of studies reporting the birth of healthy babies after the transfer of embryos with a chromosomal mosaic result on PGT-A (Greco et al., 2015; Kahraman et al., 2020; Viotti et al., 2019 and 2021), have been published. Data suggested lower implantation rates and higher miscarriage rates when mosaics were compared with euploid embryo transfers. Study design, size, duration This retrospective study covers a period from January 2017 to October 2022 and includes 6063 PGT-A cycles. A total of 18425 blastocysts were biopsied and analyzed with NGS. From January 2017 to December 2020, the cut-off value for reporting mosaicism and euploidy was %20; the rate of which was increased to 30% after January 2021. Chromosomes with PGT-A profiles that deviate from 2 copies by less than 30% were reported as normal. Participants/materials, setting, methods A total number of 12751 and 5674 blastocysts were biopsied between January 2017-December 2020 and January 2021-October 2022, respectively. PGT-A was performed on Ion Torrent S5 (Thermo Fisher Scientific). The mean female age for 20% and 30% cut-off groups were 36.7+-5.1, 35.9+-5.2, respectively. As this age difference was statistically significant, further analysis was performed in age subgroups &amp;lt; =38 and &amp;gt;38. Main results and the role of chance For PGT-A cycles with female age &amp;lt; =38, cycles with at least one euploid embryo in the 30% cut-off group increased from 74.0% to 77.5% whereas cycles with at least one mosaic embryo decreased from 40.8% to 37.0% (p = 0.0212; p = 0.0274). Cycles with at least one mosaic embryo without any euploid embryos decreased by 2% (9.2% to 7.2%) (p = 0.0522). The euploidy percentage out of all tested biopsies was significantly increased from 40.4% to 45.8% (p = 0.0001). For cycles with female age &amp;gt;38, ratio of PGT-A cycles with at least one euploid embryo increased from 28.1% to 30.3% in the 30% cut-off group but this was not statistically significant (p = 0.28). Cycles with at least one mosaic embryo were decreased from 15.1% to 10.8% and cycles with at least one mosaic embryo without any euploid embryo decreased from 9.5% to 5.6% in the 30% cut-off group (p = 0.006; p = 0.0018). The euploidy percentage was increased by 2.8% from 15.1% to 18.1% (p = 0.0115). The pregnancy outcomes were significantly higher for euploid embryos with the 30% cut-off compared to 20%, as clinical pregnancy was 64.5% vs. 59.1% and clinical pregnancy loss was 7.9% vs. 12.4% (p = 0.0022; p = 0.0011). Similarly, ongoing pregnancy was 5.2% higher, but non-significant. Limitations, reasons for caution These are the results of a single ART center which is using the same protocols for ovarian stimulation and endometrial preparation in the two periods above mentioned. Although not expected, minor changes may have occurred; similar practices have to be reported. Wider implications of the findings Mosaicism is undeniably a true biological phenomenon. However, trophectoderm biopsy and NGS are prone to create an artefactual false mosaicism. Relaxing the mosaicism calling rate by 10% may have added some better-quality blastocysts to the euploid pool giving the opportunity to embryologists of transferring blastocysts with higher implantation potential. Trial registration number Not applicable

P-283 Is it time to abandon the practice of double embryo transfer?

Abstract Study question Can single embryo transfer (SET) with euploid embryos be an effective strategy for improving livebirth rate (LBR) and reducing multiple pregnancy rate (MPR)? Summary answer SET of euploid embryos leads to higher LBR and lower MPR per embryo transfer cycle compared to DET of untested embryos in all age groups. What is known already Multiple pregnancy, IVF, and advanced maternal age are independently associated with adverse obstetric outcomes and their coexistence is likely to lead to aggravation of obstetric risks. DET is often recommended in women aged over 37 with the aim of improving LBR but at the cost of significantly higher MPR. Preimplantation genetic testing for aneuploidy (PGT-A) was pioneered to select euploid embryos for transfer. We evaluate whether utilization of PGTa with a subsequent transfer of a single euploid embryo can be an effective strategy for reducing MPR and achieving higher LBR compared to DET of untested embryos. Study design, size, duration This is retrospective national cohort study using data from the UK Human Fertilisation and Embryology Authority (HFEA). We analysed 137, 186 IVF treatment cycles performed between 2017-2018 in the UK. Other than grouping by maternal age, no further confounders were controlled for. Both fresh and frozen transfers were included. Participants/materials, setting, methods We included all IVF treatment cycles. Cycles undertaken for donation or embryo storage were excluded. Cycle parameters including age, ethnicity, the number of previous cycles, previous infertility history, previous pregnancy outcomes, gestational age at delivery, birthweight and early pregnancy outcomes were evaluated. We compared the LBR and MPR per embryo transfer cycle following a SET of a euploid embryo (PGTa SET) vs. a DET of untested embryos. Main results and the role of chance PGTa was utilised in 1990 IVF treatment cycles, of which 1521 had a SET of a euploid embryo (76%, 95% CI 75-78%). Of 135, 195 IVF cycles in which PGTa was not utilised, there 37, 281 had DET of untested embryos (28%, 95% CI 27-28%). There were 319 IVF treatment cycles in which there were no euploid embryos to transfer following PGTa (16%, 95% CI 14-18%). In the untested group, there were 16, 680 IVF treatment cycles in which there were no embryos to transfer (12%, 95% CI 12-13%). Per embryo transfer cycle, the LBR was significantly higher and the MPR was significantly lower in all age groups in PGTa SET group vs. DET of untested embryos group (including those aged under 35) (43.7% vs. 31.4%, p &amp;lt; 0.001 and 0.3% vs. 8.4%, p &amp;lt; 0.0001, respectively). Limitations, reasons for caution It is unclear whether the HFEA dataset includes all intended collections when assessing outcomes, thus we were unable to report on outcomes per cycle started. Wider implications of the findings Advanced maternal age is associated with increased perinatal risk and maternal morbidity. This population are particularly vulnerable to multiple pregnancy. PGTa followed by SET of a euploid embryo is an effective strategy for reducing MPR and improving LBR in all age groups. Trial registration number not applicable

P-634 Is duo stim a better approach than non-consecutive cycles in patients undergoing PGTA?

Abstract Study question Are there differences in the results of cycles and in the time to achieve an euploid embryo in cycles with DuoStim compared to non-consecutive cycles? Summary answer A DuoStim strategy does not compromise ovarian response, oocyte competence or euploidy rate, and reduces time to pregnancy. What is known already In patients with advanced maternal age undergoing PGT-A, the risk of not reaching embryo transfer is relatively high. Increasing the number of oocytes available should increase the chances of finding a healthy euploid embryo and reach embryo transfer. Recently, DuoStim has been proposed as an alternative to accumulate oocytes prior to the final cycle in which oocytes will be fertilized, cultured, and biopsied Duostim strategy allows to reduce the time between ovarian stimulations, which may reduce the stress that waiting for assisted reproduction treatments entails for patients Study design, size, duration Retrospective study carried out at our institution during 2015-2020 with 451 patients &amp;gt; 38 years undergoing PGT-A and whose previous cycles were distributed as follows: n = 101 performed DuoStim (first OS with oocyte vitrification, second was reinitiating OS five days after OPU) and a control group of 350 patients who underwent two standard non-consecutive cycles, the first to accumulate oocytes and from the second cycle, and then performed PGT-A with all oocytes, fresh and vitrified. Participants/materials, setting, methods All patients undergoing PGT-A performed 1 or 2 ovarian stimulations (OS) cycles to accumulate oocytes. OS cycles were re-initiated after a 5-day interval. Trophectoderm biopsies were studied by NGS. The variables were expressed as mean ± SD and comparison among groups by using ANOVA. Main results and the role of chance Mean age was comparable in both groups, DuoStim vs control 39.3±3.1 vs 39.2±3.0; p:0.76 AMH was lower in DuoStim group compared with control group (1.07±0.56 vs 2.64±4.1 ng/mL; p:0.006, and AFC were 4.3±2 vs 7.1±3; p:0.013 respectively. Gonadotropin doses were higher in DuoStim group compared control group 4576.8±1847 vs 4045±1857 IU; p:0.01, and total stimulation days were significantly higher in DuoStim compared with control group (22.7±4.85 vs 18.5±10,74; p &amp;lt; 0.001. The mean of total eggs retrieved in both OS were similar between DuoStim and control group (12.9 vs 13.1; p = 0.78) The ratio of euploid embryos found was similar 35.99 % vs 38.6% When we compared pregnancy rates per embryo transfer, were similar between both groups 66.6% (36/54) vs 65.3% (132/211); p value=0.98. We confirmed differences in the time to achieve the first euploid embryo in favour of DuoStim group 30.2±6 vs 36±9 with a p value: 0.001. Limitations, reasons for caution The retrospective nature is the major limitation of this study. The study population reflects the indication of PGT-A in a particular setting, mainly advanced maternal age and in DuoStim group a poor prognosis profile Wider implications of the findings Accumulating oocytes through vitrification prior to PGT-A with DuoStim offers similar clinical outcomes in both groups, it may reduce time to reach an euploid embryo, which is relevant for advanced maternal age group of patients. Trial registration number Not applicable

P-188 Cleavage stage morphokinetic timings can predict the likelihood of aneuploidy in blastocysts

Abstract Study question Can morphokinetic analysis be used as a tool to predict the ploidy status of blastocysts? Summary answer A blastocyst is more likely to be aneuploid when it divides slowly to two cells and eight cells but quickly to three cells. What is known already Existing data suggests morphokinetic analysis and PGT-A both have advantages and limitations when used independently. However, the hope is that if strong associations can be found linking morphokinetic qualities of embryos and their ploidy status, then this can be used as a predictive tool. Currently, morphokinetic analysis cannot be used as a predictive tool for euploidy. However, there is some existing data that suggests that clinics can correlate morphokinetics and ploidy status using their in-house data. Study design, size, duration The study was retrospective and involved the assessment of blastocysts that had undergone PGT-A with next generation sequencing (NGS) and had a confirmed outcome of aneuploid or euploid between January 2021 and June 2022. Only patients who obtained genetic results showing they had at least one aneuploid embryo and at least one euploid embryo were included to allow for a matched case-control study – this resulted in 89 aneuploid and 89 euploid embryos being analysed. Participants/materials, setting, methods All embryos were cultured in the same conditions, including the same incubators, media (both from Vitrolife, SWE), and standard operating procedures. Morphokinetic data was exported and analysed from annotations made on the EmbryoScope annotation software. Embryos that could not be fully annotated were excluded from analysis. A conditional logistic regression was performed in order to ascertain the effects of each morphokinetic parameter on the likelihood that the embryo will be aneuploid or euploid. Main results and the role of chance From 89 patients, 178 embryos were analysed, of which half were euploid and half were aneuploid. The morphokinetic time points assessed were time to pronuclei fading (tPNF), time to 2-cell (t2), time to 3-cell (t3), time to 4-cell (t4), time to 5-cell (t5), time to 8-cell (t8), time to start of blastulation (tSB) and time to blastulation (tB). The duration of the second cell cycle (cc2) and its synchrony (s2) were also analysed. When euploid and aneuploid case-control matched embryos were compared, t2, t3 and t8 were the best predictors of aneuploidy when analysed together (P = 0.06, P = 0.074, P = 0.071 respectively). There are higher odds of a blastocyst being aneuploid if a later t2 (OR = 1.361), an earlier t3 (OR = 0.784) and a later t8 (OR = 1.047) have occurred. A later t2 (P = 0.044, OR = 1.361) is statistically significant when alongside an earlier t3 (P = 0.122, OR = 0.828). A later t8 is statistically significant, irrespective of t2 and t3 (P = 0.05, OR = 1.043). In addition to these results, there is a general trend for aneuploid embryos to develop to each time point slower than euploid embryos. Furthermore, there is more variation in the range of values for each time point in aneuploid embryos compared to less variation in euploid embryos. Limitations, reasons for caution There was a small number of embryos included in the study, so a larger cohort would increase the statistical power. A multi-centre study would also be beneficial to incorporate factors such as larger patient demographics, multiple embryo culture environments and variable clinical protocols. Wider implications of the findings Utilising morphokinetic data to predict aneuploidy can help us to counsel the patient on selecting embryos for biopsy, and to make a predictive tool that would be applicable within our laboratory and patient settings. A secondary impact could be supporting the use of non-invasive methods to predict embryo ploidy. Trial registration number Not applicable

P-701 Human embryos diagnosed as mosaic and aneuploid after preimplantation genetic testing for aneuploidy are associated with increased levels of apoptosis in trophectoderm cells

Abstract Study question Are there cell lineage-related differences in the apoptotic rates of human blastocysts classified as euploid, mosaic and aneuploid after preimplantation genetic testing for aneuploidy (PGT-A)? Summary answer In human blastocysts, apoptosis is more frequent in trophectoderm (TE) cells and is associated with the presence of mosaic and uniform chromosomal alterations. What is known already Embryos diagnosed as mosaic after PGT-A can develop into healthy infants. However, the reason why these embryos achieve reproductive competence needs further research. One hypothesis is that aneuploid cells are negatively selected during embryo development through cell competition mechanisms, such as apoptosis or differential proliferation. While these mechanisms have been demonstrated in mouse embryos, in which apoptosis occurs more frequently in the inner cell mass (ICM), evidence in human embryos is scarce. In fact, only one previous study has shown an association between mosaicism and aneuploidy after PGT-A and increased levels of apoptosis, which was more frequent in TE cells. Study design, size, duration Prospective cohort study performing colocalization of cell-lineage and apoptotic expression markers by immunofluorescence (IF). A total of 53 vitrified human blastocysts with a previous PGT-A diagnosis on day 5 (D5) or day 6 (D6) of development were analysed: n = 13 euploid embryos (n = 11 on D5; n = 2 on D6), n = 22 mosaic embryos (n = 16 on D5; n = 6 on D6), and n = 18 aneuploid embryos (n = 11 on D5; n = 7 on D6). All embryos were donated for research purposes. Participants/materials, setting, methods Following warming and re-expansion, blastocysts were fixed in 4% paraformaldehyde, processed for IF and imaged by confocal microscopy. Primary antibodies: 1:80 goat anti-human Nanog (AF1997, R&amp;D-Systems), 1:100 mouse anti-human Gata3 (MAB6330, R&amp;D-Systems) and 1:1000 rabbit anti-human Caspase-3 (NB10056113, Novus Biologicals). 165 μM DAPI was used for nuclei staining. Total cells: DAPI+ cells. TE: Gata3+ cells. ICM: Gata3-/Nanog+ cells. Incidence of apoptosis: % Caspase-3+ cells (per embryo). Data were analysed using Chi-square or ANOVA (p &amp;lt; 0.05 significant). Main results and the role of chance Total cell number was similar among euploid embryos (59.2±20.6 on D5; 78.5±0.7 on D6), mosaic embryos (49.6±15 on D5; 58.8±16.9 on D6) and aneuploid embryos (48.6±14.2 on D5; 59.6±23.5 on D6) (P &amp;gt; 0.05). Nanog and Gata3 expression evidenced the establishment of ICM and TE cell lineages during the blastocyst stage: while the proportion of Nanog+ cells decreased from 38.2% (755/1979) on D5 to 20.3% (188/927) on D6 (P &amp;lt; 0.0001), the proportion of Gata3+ cells increased from 77.8% (1540/1979) on D5 to 83.1% (770/927) on D6 (P &amp;lt; 0.01). The persistence of Nanog expression in TE cells was associated with the chromosomal status of the embryo, as evidenced by the significantly higher proportion of Gata3+/Nanog+ cells found in aneuploid embryos (27.9%=149/534 on D5; 4.6%=19/417 on D6) compared to mosaic embryos (13.1%=104/794 on D5; 3.4%=12/353 on D6) and euploid embryos (9.7%=63/651 on D5; 0%=0/157 on D6) (P &amp;lt; 0.05). The incidence of apoptosis in the TE was significantly higher in aneuploid embryos (45.9%±16.1 on D5; 49%±15.1 on D6) and mosaic embryos (44.1%±19.6 on D5; 43%±16.8 on D6), compared to euploid embryos (26.6%±16.6 on D5; 17.5%±14.8 on D6) (P &amp;lt; 0.05). Conversely, apoptosis was much less frequent in the ICM (2.2%±7.7), and no differences were found among the study groups (P &amp;gt; 0.05). Limitations, reasons for caution This is a descriptive, single-centre study with a limited sample size. The karyotype concordance between ICM and TE cells was not tested. Wider implications of the findings Our findings demonstrate that apoptosis is common in human blastocysts, particularly in TE cells. The increased apoptotic rates found in the TE of embryos with chromosomal abnormalities support its role as a mechanism to eliminate aneuploid cells. This mechanism may indeed be related to the reproductive potential of mosaic embryos. Trial registration number Not applicable