Embryo Culture Research Articles

The synergy of morphokinetic parameters and sHLA-G in cleavage embryo enhancing implantation rates.

Objective: This study aimed to assess the relationship between implantation and soluble HLA-G (sHLA-G) expression in cleavage embryo culture medium (ECM) in conjunction with early developmental kinetics determined by time-lapse imaging (TLI). Methods: A retrospective, single-center study was conducted involving 238 embryos from 165 patients who underwent Frozen-thawed embryo transfer (FET) using autologous oocytes, with either single or double embryo transfer. TLI morphokinetic parameters (t2, t3, t4, t5, t6, t7, t8, cc2, s2, cc3, s3) of embryos were analyzed, and sHLA-G levels in D3 ECM were measured using an enzyme-linked immunosorbent assay (ELISA). A hierarchical classification model was developed to categorize embryos into five groups (A, B, C, D, E). The correlation between sHLA-G levels, TLI classification of embryos, and embryo implantation was investigated to establish a non-invasive method for evaluating implantation potential. Multivariate logistic regression analysis was performed to identify potential influencing factors, and receiver operating characteristic (ROC) curves were used to evaluate the predictive value for implantation. Results: Multivariate unconditional logistic regression analysis indicated that TLI parameters t5 and s3 and sHLA-G level in ECM were independent risk factors affecting embryo implantation. The implantation rate decreased from TLI classification A to E. The proposed classification model effectively assessed the implantation potential of embryos. The implantation rate was higher in the sHLA-G positive group compared to the sHLA-G negative group (p < 0.001). The expression of sHLA-G in D3 ECM, combined with the TLI classification model, accurately evaluated the implantation potential of embryos with an AUC of 0.876. Conclusion: The integration of cleavage kinetics and embryonic sHLA-G expression could reliably identify embryos with a high likelihood of successful implantation.

Systemic interindividual DNA methylation variants in cattle share major hallmarks with those in humans

BackgroundWe recently identified ~ 10,000 correlated regions of systemic interindividual epigenetic variation (CoRSIVs) in the human genome. These methylation variants are amenable to population studies, as DNA methylation measurements in blood provide information on epigenetic regulation throughout the body. Moreover, establishment of DNA methylation at human CoRSIVs is labile to periconceptional influences such as nutrition. Here, we analyze publicly available whole-genome bisulfite sequencing data on multiple tissues of each of two Holstein cows to determine whether CoRSIVs exist in cattle.ResultsFocusing on genomic blocks with ≥ 5 CpGs and a systemic interindividual variation index of at least 20, our approach identifies 217 cattle CoRSIVs, a subset of which we independently validate by bisulfite pyrosequencing. Similar to human CoRSIVs, those in cattle are strongly associated with genetic variation. Also as in humans, we show that establishment of DNA methylation at cattle CoRSIVs is particularly sensitive to early embryonic environment, in the context of embryo culture during assisted reproduction.ConclusionsOur data indicate that CoRSIVs exist in cattle, as in humans, suggesting these systemic epigenetic variants may be common to mammals in general. To the extent that individual epigenetic variation at cattle CoRSIVs affects phenotypic outcomes, assessment of CoRSIV methylation at birth may become an important tool for optimizing agriculturally important traits. Moreover, adjusting embryo culture conditions during assisted reproduction may provide opportunities to tailor agricultural outcomes by engineering CoRSIV methylation profiles.

Temporal Evaluation of a Minimally Invasive Method of Preimplantation Genetic Testing for Aneuploidy (mi-PGT-A) in Human Embryos

Preimplantation genetic testing for aneuploidy (PGT-A) has become a useful approach for embryo selection following IVF and ICSI. However, the biopsy process associated with PGT-A is expensive, prone to errors in embryo ploidy determination, and potentially damaging, impacting competence and implantation potential. Therefore, a less invasive method of PGT-A would be desirable and more cost-effective. Noninvasive methods for PGT-A (ni-PGT-A) have been well-studied but present limitations in terms of cf-DNA origin and diagnostic accuracy. Minimally invasive pre-implantation genetic testing (mi-PGT-A) for frozen-thawed embryo transfer is a promising, less studied approach that utilizes a combination of spent culture media (SCM) and blastocoelic fluid (BF)-derived cell-free (CF)-DNA for genetic testing. This study aimed to optimize the effectiveness of mi-PGT-A for aneuploidy diagnosis by investigating the optimal temporal sequence for this protocol. SCM+BF was collected at either 48 or 72 h of culture after thawing day 3 preimplantation embryos. cf-DNA in the SCM+BF was amplified, analyzed by next-generation sequencing (NGS) and compared with results from the corresponding whole embryos (WEs) obtained from human embryos donated for research. Fifty-three (42 expanded blastocysts, 9 early blastocysts, and 2 morula) WE and SCM+BF samples were analyzed and compared. The overall concordance rate between SCM+BF and WE was 60%. Gender and ploidy concordance improved with extended culture time from 48 h (73% and 45%) to 72 h (100% and 64%), respectively. These results demonstrate that SCM+BF-derived cf-DNA can be successfully used for mi-PGT-A. Our findings indicate that longer embryo culture time prior to SCM+BF-derived cf-DNA analysis improves DNA detection rate and concordance with WEs and decreases the proportion of false positive results.

Non-invasive preimplantation genetic testing for aneuploidy: is the promise real?

Recent advances in preimplantation genetic testing for aneuploidy (PGT-A) have significantly enhanced its application in ART, providing critical insights into embryo viability, and potentially reducing both the time spent in fertility treatments and the risk of pregnancy loss. With the integration of next-generation sequencing, PGT-A now offers greater diagnostic precision, although challenges related to segmental aneuploidies and mosaicism remain. The emergence of non-invasive PGT-A (niPGT-A), which analyzes DNA in spent embryo culture media, promises a simpler aneuploidy screening method. This mini review assesses the methodological criteria for test validation, the current landscape of PGT-A, and the potential of niPGT-A, while evaluating its advantages and potential pitfalls. It underscores the importance of a robust three-phase validation process to ensure the clinical reliability of PGT-A. Despite initial encouraging data, niPGT-A not only confronts issues of DNA amplification failure and diagnostic inaccuracies but also has yet to meet the three-prong criteria required for appropriate test validation, necessitating further research for its clinical adoption. The review underscores that niPGT-A, like traditional PGT-A, must attain the high standards of precision and reliability expected of any genetic testing platform used in clinical settings before it can be adopted into routine ART protocols.

P-219 Does a dry or humid incubation environment affect continuous and undisturbed culture media protocols?

Abstract Study question Can continuous embryo culture medium maintain a safe and effective osmolality in dry and humidified incubators under both light viscosity and heavy viscosity oil overlay? Summary answer Undisturbed embryo culture is possible in 25uL and 100uL media volumes with both light and heavy oil and in both dry and humidified environments. What is known already Ideal osmolality for pre-implantation embryos is 255-295 mOsm/kg. Much debate surrounds continuous embryo culture, without refreshing of the medium, providing a suitable concentration of osmoles during embryo culture in both humidified and dry incubators. Meanwhile, ideal pH or measure of free hydrogen ions available in the embryo culture medium is 7.20-7.50. Contemporary knowledge that undisturbed embryos are able to regulate their own needs during culture has replaced belief that embryos require specific and varying environmental pH induced at different developmental stages. Thus, environmental pH needs to be maintained and stable in order to optimize health and outcomes of resulting blastocysts. Study design, size, duration Osmolality, pH, and mass were measured in humidified and dry incubators over 8 days of continuous embryo culture conditions. Four dishes each of 25uL and 100uL drops of embryo culture medium (CSCM-NXC) were analyzed per incubator type (Non-humidified – Miri, Humidified – Astec). Additionally, each micro-volume was analyzed under both Light Oil for Embryo Culture and Heavy Oil for Embryo Culture (Irvine Scientific). In total, 2352 data points for osmolality, pH, and mass were examined. Participants/materials, setting, methods A private IVF laboratory conducted the testing with no patient samples introduced to the embryo culture media. To mitigate early evaporative loss, dish preparation was strictly time and temperature controlled. Osmolality was measured in triplicate on days 0-7 using Advanced Micro-Osmometer Model 330 (Advanced Instruments). Mass measures were taken in triplicate on days 0-7 using the Torbal AGZN120 Analytical Balance. pH was measured in triplicate using the i-STAT 300 (Abbott) on days 0,3,5,7. Main results and the role of chance The pH in 25uL and 100uL drops under light oil and heavy oil remained well within suitable ranges and stable when compared day to day, from day 0 (M = 7.276, σ = 0.011) through day 7 (M = 7.303, σ = 0.011). The average aggregate pH overall was 7.290 (σ = 0.012). Aggregate osmolality, measured as milliosmoles per kilogram (mOSM/kg), over the course of incubation in all conditions also remained well within acceptable safety range (M = 277mOsm/kg, σ = 5). Dry incubator incubation exhibits a 7% increase from day 0 to day 6 (M = 270mOsm/kg, σ = 4 and M = 289mOsm/kg, σ = 5) compared to a 2% increase in humidified incubation (M = 269mOsm/kg, σ = 5 and M = 274mOsm/kg, σ = 5) regardless of oil type or microdrop size, both remaining within accepted limits. Additionally, when monitoring mass in grams (g) in 25uL as a function of evaporative loss, humidified incubation reduces from 12.6346g to 12.5566g (day 0 to day 7). In dry incubation, there is negligible change day 0 to day 7 (12.6543g and 12.6538g). Similarly, in 100uL, the change in humidified incubators is 13.37222g to 13.25025g (day 0 to day 7) and dry incubation reduces from 13.3236g to 13.2963g. The changes in mass of prepared culture dishes maintains consistency with the study findings for osmolality. Limitations, reasons for caution As CSCM-NXC is for use through day 6 of embryo culture, the extended simulated embryo culture length is for data collection purposes only. There is no developmental data to include in this study. Additional studies must be conducted in order to explore any differences when embryos are present in culture. Wider implications of the findings The ability to culture embryos continuously in dry incubator environments with micro-volumes of culture medium is widely debated. These many data point study has shown that safe and efficacious embryo culture can be undertaken undisturbed, therefore relaxing some of the burdensome and labor-intensive processes in laboratories employing this equipment. Trial registration number not applicable

O-214 The effect of constant 5% versus gradient 8%, 5%, 2% oxygen concentration on the development of sibling human embryos in vitro to the blastocyst stage

Abstract Study question Does a physiological gradient of oxygen concentration in embryo culture atmosphere yield a higher percentage of fertilized oocytes developing into morphologically optimal blastocysts? Summary answer Study favours sustaining a static 5% oxygen level, instead of a gradient, during extended embryo culture, resulting in more viable embryos for cryopreservation and transfer. What is known already To date, there’s ongoing research about the optimal oxygen concentration for embryo culture. The available data from studies based on animal models show that the O2 level in the fallopian tube is around 8%, and the in-utero O2 level is at around 2% (Wale &amp; Gardner, 2010). Data on oxygen measurement in the human uterus is scarce, yet aligns with similar findings in other mammalian species (Ng et al., 2018). These findings support the use of sequential O2 levels for embryo culture. Our study stands as the first to implement such dynamic oxygen culture conditions within an in vitro setting. Study design, size, duration A prospective sibling-split oocyte study, analyzing 44 ICSI cycles from January 2022 to January 2023, with alternate allocation into a static 5% (Control group) during entire culture period or under an oxygen gradient (intervention group), starting with 8% from day-0 to day-3, continuing with 5% on day-3 and following with 2% of oxygen from the end of day-3 to day-5/6. Participants/materials, setting, methods 44 ICSI cycles, which produced a total of 521 metaphase II oocytes. A total of 353 2PN embryos were cultured and annotated in time-lapse for morphokinetic and morphometric analysis. Main results and the role of chance While morphological outcomes slightly favored the intervention group for day 2 and day 3 embryos, the trend reversed by day 5. The control group (5% O2) showed a notably higher proportion of zygotes developed to clinically utilized blastocysts (47% vs. 37%, p = 0.016) and consequently more of them were used for transfer on day 5 (10% vs. 4%, P = 0.008) compared to the intervention group. Additionally, though not statistically significant, the control group showed a higher percentage of morphologically optimal blastocysts on day 5 (10.3% vs. 14.7%, p = 0.1233). Morphokinetic timings, as revealed by time-lapse analysis, indicated a delayed development in the intervention group, notably seen in the initiation of blastulation (p = 0.0024), initiation of expansion (p &amp;lt; 0.0001), and full blastocyst formation (p &amp;lt; 0.0001). The speed of blastulation, however, does not necessarily reflect differences in implantation potential, as implantation (58.6% vs 57.1% p &amp;gt; 0.92) and clinical pregnancy rates (55.2% vs 57.1%, p &amp;gt; 0.92) after transferring blastocysts from either group were comparable. Limitations, reasons for caution Measuring the in vivo O2 levels in female reproductive tract should take into account possible oscillations and dynamic nature of gas and nutrient exchange. It’s also important to acknowledge the complexity of embryo’s adaptive response, making it challenging to attribute observed outcomes solely to the impact of targeted oxygen concentration. Wider implications of the findings Based on these clinical results, we will analyze the metabolomic footprint in spent embryo culture under static 5% or gradient 8-5-2% oxygen concentration, employing non-invasive NMR and chromatography methods. A randomized control trial should be conducted to explore the effect of different oxygen regimes on clinical outcomes. Trial registration number NCT05898178

O-019 Outcomes and correlations between traditional Intracytoplasmic Sperm Injection (ICSI) method and a new AI-enhanced system

Abstract Study question In the context of ICSI, how does the Sperm Tracer Deep Neural Network analysis compare to the embryologist’s sperm selection technique? Summary answer Sperm Tracer AI system significantly improves fertilization rates, offering a standardized, objective approach to sperm selection, but nuanced correlations highlight complexities in sperm parameters. What is known already Traditional sperm selection during ICSI relies on subjective, manual methods, introducing variability and affecting pregnancy success. In response, AI applications, aim to enhance objectivity, potentially improving pregnancy rates. Study design, size, duration Between September and December 2023, the study examined 26 fresh ICSI cycles in a total of 268 sibling oocytes. Men aged 18 years and above with 34.4 mean women’s age. The Sperm Tracer group underwent single embryo culture, while the Embryologists' group underwent group embryo culture. The study analyzes statistical differences in fertilization rates, day 3 and day 5 embryo development. The study duration aligns with the analyzed cycles. Participants/materials, setting, methods The study compared real-time AI-assisted sperm evaluation with the Sperm Tracer system to the traditional ICSI method by embryologists. Correlation analysis was performed for the following kinematic parameters: Amplitude of Lateral Head displacement (ALH), Dance (DNC), Linearity (LIN), Straightness (STR), Wobble (WOB), Curvilinear Velocity (VCL), Linear Velocity (VSL) and Average Path Velocity (VAP). Statistical analysis involved Odds Ratios (OR) with 95% confidence intervals, Mann-Whitney U tests and correlation analyses by Pearson’s coefficient. Main results and the role of chance The Sperm Tracer system exhibited a noteworthy enhancement in fertilization rates (OR: 0.46, 95% Confidence Intervals (CI) [0.23, 0.89], p = 0.0224) but no significant differences in cleavage stages, blastocyst formation, or the production of good-quality embryos compared to conventional methods. For correlation analysis used two groups of sperm population. Group A comprises spermatozoa subjected to ICSI with the Sperm Tracer system, resulting in unsuccessful oocyte fertilization and Group B consists of spermatozoa that achieved successful fertilization. In Group A, correlation analysis identified significant negative associations between DNC and linear parameters (LIN, STR, WOB) (p &amp;lt; 0.001), highlighting the clinical relevance in sperm motility. Group B displayed a distinct negative correlation between DNC and LIN (p = 0.03941), indicating potential effects on sperm linear velocity. LIN in Group A exhibited strong negative correlations with WOB (p = 0.0001), moderate negative correlations with STR (p = 0.0028), and VCL (p = 0.0773). Moreover, ALH analysis in Group A unveiled robust negative correlations with LIN, STR, and WOB, suggesting clinical significance in sperm motility. These intricate findings underscore the Sperm Tracer system's primary advantage in optimizing fertilization rates, offering valuable insights for refining sperm selection strategies in assisted reproduction. Limitations, reasons for caution The study faces limitations due to a small sample, impacting generalizability. Divergence in culture embryo strategies introduces bias, necessitating validation in larger studies. The system includes morphology analysis, but no data analysis has been conducted. Exploring the correlation between morphology, considering embryo culture variations, and motility could provide valuable insights. Wider implications of the findings This study highlights AI applications like Sperm Tracer, showcasing their potential to improve assisted reproduction outcomes. Recognizing AI's impact on IVF success is vital for adoption. Integrating AI into clinic protocols may boost IVF efficiency, requiring training for embryologists to ensure effective utilization. Trial registration number Not applicable

P-241 The in vivo human uterine pH and uterine fluid composition differs from pH and composition of clinically used in vitro preimplantation embryo culture media

Abstract Study question To study the pH and uterine fluid composition in the human uterus three days after a positive LH test or ovum pick-up. Summary answer The uterine pH three days after a positive LH test or ovum pick-up was 6.8. Concentrations of 37 components were determined in uterine fluid. What is known already In assisted reproductive technologies (ART), the in vitro culture of human preimplantation embryos is known to play a crucial role in the success of In Vitro Fertilization (IVF) treatments. It affects live birth rates and child outcomes such as birth weight. Numerous commercial embryo culture media are routinely used in clinical practice worldwide, but their composition is primarily based on laboratory-based clinical insights and experimental studies with animal models. Studies on the natural human preimplantation embryo environment during the physiologically relevant time of the menstrual cycle are limited. Study design, size, duration A cross sectional clinical study was conducted to study the uterine pH and the composition of human uterine fluid. The uterine pH was measured in 53 women on the third day after LH surge (N = 46) or ovum pickup (N = 7), as that time in the menstrual cycle is associated with endometrial receptivity for embryo implantation. Twenty-two samples of aspirated uterine fluid, that were collected at the same time, were analyzed for the concentrations of thirty-seven components. Participants/materials, setting, methods Healthy women of reproductive age with normal functional uterine anatomy were included. Uterine pH was determined using pH probe inserted through outer sheath of an Embryo Transfer (ET) catheter at the lithotomy position. Uterine fluid was then aspirated using a syringe connected to outer sheath of this ET catheter. Collected samples were analyzed using a Cobas 8000 chemistry analyser and ultra-performance liquid chromatography-tandem mass spectrometry. Main results and the role of chance The mean pH in the uterine cavity on the third day after a positive LH test (6.75 ± 0.22) or ovum pick-up (6.82 ± 0.18) was clearly lower than the pH used in routine clinical practice for preimplantation embryo culture (pH 7.3 ± 0.1). Calcium, phosphate, lactate(L), pyruvate, albumin and 21 amino acids were present at different concentrations in human uterine fluid compared to most commonly used preimplantation embryo culture media. Immunoglobulins, citrulline, ornithine and uric acid were present in uterine fluid, but not in clinically used culture media. Limitations, reasons for caution As the uterine cavity is only one part of the reproductive tract, our measurements do not fully represent the complete in vivo environment of preimplantation embryo development. We only studied 37 components, recognizing that uterine fluid likely contains additional components. Wider implications of the findings The observed differences between the uterine pH and the composition of uterine fluid and the pH and composition of preimplantation embryo culture media used in routine clinical practice could be a starting point to further optimize in vitro embryo culture in ART treatments. Trial registration number Not applicable

O-040 Pro -better late than never

Abstract In IVF, blastocysts developed on Day5-6 are typically selected for transfer, cryopreservation, or biopsy, while embryos that do not reach this developmental stage before 144 hours post-insemination (hpi) are often discarded. Recently, there has been an increased adoption of blastocyst culture aimed at enhancing embryo selection potential and supporting a single embryo transfer approach. Consequently, the clinical significance of Day7 blastocysts (embryos reaching this stage beyond 144 hpi) has become a prominent topic of discussion, particularly in the context of treating patients with poor prognosis. Numerous studies worldwide have reported lower competence for these embryos, as evidenced by poorer morphological quality, higher aneuploidy rates, and lower implantation rates following both untested and euploid transfer. Nonetheless, a notable proportion of these blastocysts are euploid and result in healthy live births. While their clinical utilization may entail lower efficiency per transfer, it ultimately increases the cumulative live birth rate per cycle. However, most studies have encountered challenges due to diverse definitions of Day7, varying clinical settings, culture strategies, incubators, and local regulations. Presently in IVF, leveraging time-lapse technology (TLT), artificial intelligence (AI), and preimplantation genetic testing for aneuploidies (PGT-A) could standardize and enhance Day7 blastocyst characterization and clinical assessment. Consequently, we designed a study exclusively in freeze-all PGT-A cycles with vitrified-warmed euploid single embryo transfer after continuous culture in time-lapse incubators, trophectoderm biopsy without zona pellucida drilling on day3, comprehensive chromosome testing, confirming embryo morphological assessments through an AI-powered software, and utilizing this tool also to automate developmental timing annotations. Our analysis confirmed that all blastocysts were biopsied only after reaching an expansion level suitable for the procedure (approximately 24,000 µm²) and consistent across all embryos clustered every 12 hours in 6 groups (&amp;lt;120, 120-132, 132-144, 144-156, 156-168, and &amp;gt;168 hpi). Day7 blastocysts accounted for approximately 15% of all blastocysts obtained over 8 years, derived from older oocytes, exhibiting poorer quality according to both Gardner’s score and AI score, slower development from early stages, and accumulating delays across various intermediate processes like blastocyst expansion from tEB to the time of biopsy. They were less euploid and resulted in a lower live birth rate per euploid transfer. However, only the latter association was significant when adjusted for confounders (e.g., blastocyst quality). Comparing only embryos biopsied before 144 hpi versus embryos biopsied after this threshold, neither the euploidy nor the live birth rate were significantly different, supporting the extension of embryo culture beyond Day6. Overall, we estimated that Day7 embryos contribute to a relative increase of 4.4%, 13.7%, and 5.2% in the number of patients who achieved a live birth, who were not pregnant after the transfer of faster embryos, and who have supernumerary transferable embryos after a live birth, respectively. The main limitations of our investigation were its retrospective nature and the fact that retrospective associations do not provide evidence of causation. There might be a bias towards poor prognosis patients being subject to the transfer of Day 7 embryos, for instance. Lastly, more data are required regarding the putative impact of extended embryo culture including post-natal outcomes (reassuring to date), as well as the cost-effectiveness of this clinical choice. Considering the increasing personalization and patient-centeredness of IVF, where permitted by local regulations, the decision to culture embryos to Day7 should be based on a careful evaluation of couples’ reproductive history and future prognosis, and should be subject to a patient-centered decision-making process. In the future, AI can be harnessed to support IVF practitioners by objectifying the assessment of poor-quality and slow-growing embryos with measurable information and reliable predictions, thereby mitigating the inherent subjectivity of embryologists’ decisions.

O-096 Human fallopian tube organoids provide a favorable environment for sperm motility

Abstract Study question Does a human fallopian tube (HFT) organoid model offer a favorable apical environment for human sperm survival and motility? Summary answer The apical compartment of a new model of human fallopian tube organoids provides after differentiation a favorable environment for sperm motility. What is known already Human fallopian tubes are the site of major events that are crucial for achieving an ongoing pregnancy, such as gamete survival and competence, fertilization steps, and preimplantation embryo development. In order to better understand the tubal physiology and tubal factors involved in these reproductive functions, and to improve still suboptimal in vitro conditions for gametes preparation and embryo culture during In Vitro Fertilization IVF, we sought to develop a human fallopian tube organoid model from isolated adult stem cells allowing spermatozoa coculture in the apical compartment. Study design, size, duration Over a two-year period, fallopian tube tissues were collected for organoid culture purposes from 10 “donor” patients undergoing bilateral salpingectomy by laparoscopy for definitive sterilization. After tissue digestion, adult stem cells from the isthmus and ampulla regions were separately seeded in 3D Matrigel and cultured with conventional growth factors for organoid culture and specific factors for differentiation of the female genital tract. Participants/materials, setting, methods HFT organoids were characterized by light microscopy, scanning and transmission electron microscopy, immunofluorescence and transcriptomic analysis. Following simultaneous organoid culture on specific inserts, spermatozoa from five donors were placed either in control media or in the apical compartment of colon or HFT organoids (isthmus and ampulla separately) for 96 hours. Vitality and motility were assessed at 0, 48, and 96 hours on 200 spermatozoa in each condition and in duplicate and compared using Wilcoxon test. Main results and the role of chance Specific fallopian tube differentiation of our model was confirmed by immunofluorescence, transcriptome analysis and electron microscopy observations that exhibited ciliary and secretory cells. We succeeded in releasing spermatozoa in the apical compartment of HFT organoids and in recovering them for sperm analysis. Sperm vitality values are similar in HFT organoids and in commercial sperm media. We demonstrated a superiority of HFT organoid apical compartment for sperm motility compared with other controls (colon organoids, organoid culture media, and conventional commercial sperm washing and fertilization media). At 48 hours of incubation, progressive sperm motility was higher in the apical compartment of HFT organoids (ampulla 30.6% ±17.3, isthmus 29.3% ±14.8) than in commercial fertilization media (15.3% ±14.6) (p &amp;lt; 0.05) and compared with all other conditions. At 96 hours progressive sperm motility was almost nil (&amp;lt;1%) in all conditions except for spermatozoa within HFT organoids (p &amp;lt; 0.05): 11.5% ±14.7 and 12.9% ±16.9 in ampulla and isthmus organoids, respectively. Limitations, reasons for caution This was an in vitro study in which conditions of organoid culture could not exactly mimic the in vivo environment of extracellular matrix and the vascularization of fallopian tubes. Wider implications of the findings This work opens up perspectives for better understanding of HFT physiology. For the first time, it highlights the possibility of developing HFT organoids for reproductive purposes. In the future, it could help us to improve gametes fertilizing abilities and culture conditions of embryos during human assisted reproductive technologies. Trial registration number not applicable

O-211 Exploring risk factors of mismatch in the ART laboratory: An analysis based on 73719 electronic witnessing points

Abstract Study question What are the risk factors associated with mismatch (MM) and genuine mismatch (GMM) in the ART laboratory? Summary answer MM is associated with individual operators, oocyte denudation (OD) and embryo transfer (ET) processes, while GMM related to OD and extended work hours (&amp;gt;4pm). What is known already Mix-up of gametes or embryos in the ART laboratory causes dramatic consequences to both the patients and clinics. Recent studies using electronic witnessing system (EWS) equipped with radio frequency ID (RFID) tags, have revealed that most MM events only involved empty cell culture vessels, while the true mix-up risk arose from a subgroup of GMM involving culture vessels carrying gametes or embryos belonging to different patients. Exploration of risk factors associated with MM and GMM occurrence, separately, is still currently lacking in the literature. Study design, size, duration A total of 73719 witnessing points were retrospectively extracted from an EWS database, representing 11210 consecutive treatment cycles performed at Fertility North between January 2016 and July 2023. The 11210 treatment cycles comprised 5472 fresh IVF, 3732 frozen embryo transfer, 93 oocyte freeze, and 1913 intrauterine insemination cycles. Participants/materials, setting, methods All laboratory work areas were equipped with RFID readers. A MM was triggered by detection of RFID tagged cell culture vessels (either carrying biological material or not) and/or ID cards that belong to different patients in the same work area. A GMM was defined when a MM had involved biological materials and/or patients during procedure, with a potential mix-up. Risk factors evaluated included time of the day, weekdays, weekend, public holiday, operators, and laboratory processes. Main results and the role of chance A total of 138 (0.19%) MMs and 16 (0.02%) GMMs were identified during the study period, without actual mix-up. MM rate was the highest during 12-2pm (0.28%, p &amp;lt; 0.001) compared to other periods of the day, whilst GMM was the highest after 4pm (0.13%, p = 0.004). Neither MM nor GMM was associated with any specific weekday, weekend or public holiday (p &amp;gt; 0.05, respectively). Amongst 8 laboratory processes investigated, ET (0.36%, p &amp;lt; 0.001) led to the highest MM rate, followed by OD (0.35%, p = 0.021), but no increase was observed in oocyte collection, sperm preparation, insemination, embryo culture, cryopreservation and Assign ID. OD was the only process associated with higher GMM (0.13%, p &amp;lt; 0.001). Furthermore, Two of the 20 assessed operators were identified to produce more MMs (0.59%, p &amp;lt; 0.001, and 0.40%, p = 0.004; respectively) while GMM rates were similar among all operators (p &amp;gt; 0.05). Multivariate logistic regression indicated that the OD (adjusted odds ratio or aOR=2.147, p = 0.011) and ET (aOR=2.118, p &amp;lt; 0.001) processes as well as 2 individual operators (aOR=3.511, p &amp;lt; 0.001; aOR=2.538, p = 0.002; respectively) were independent risk factors to MM, but not the 12-2pm period of the day (aOR=1.341, p &amp;gt; 0.05). Finally, both extended work hours beyond 4pm (aOR=8.651, p = 0.005) and OD (aOR=9.538, p &amp;lt; 0.001) were independent risk factors to GMM. Limitations, reasons for caution Each individual ART laboratory has a unique team of scientists and workflow arrangement so findings in our study may not be universally applicable. GMM occurs at an extremely low frequency and is often calculated based on large numbers, therefore caution ought to be taken at statistical interpretation of data. Wider implications of the findings GMMs should be separated from MMs for analysis considering the very different clinical implications. Sufficient staffing with regular break and streamlined workflow are highlighted in preventing mix-up in the ART laboratory. Using electronically logged data, EWS enables identification of site-specific risk factors, which facilitates the development of corresponding preventative protocols. Trial registration number not applicable

P-513 Non-invasive PGT (niPGT) of vitrified blastocysts cultured for 24 hours after thawing

Abstract Study question Is niPGT on culture medium exposed for 24 hours to vitrified-thawed blastocysts useful? Summary answer niPGT using a culture medium from vitrified blastocysts cultured for 24hours after thawing showed a high concordance rate with the PGTa results. What is known already Conventional PGTa, which is widely practiced around the world, requires specialized techniques and involves embryonic trophectoderm (TE) biopsy. Recently, niPGT using cell free DNA released from embryos into culture medium has been reported as an alternative to conventional PGT, and has been found to correlate well with TE-biopsy analysis results (Carmen. Rubio, 2020). Although there have been numerous reports of niPGT accuracy based on the timing of culture medium collection, there have been few comparisons of its accuracy based on different time points of medium collection from the same embryo. Study design, size, duration From March to April 2023, 128 vitrified pronuclear stage embryos from 33 patients with consent for research use, were thawed and cultured, and from Day 4, they were individually cultured for over 40 hours. 60 embryos that reached blastocyst by Day 6 were vitrified, and the spent culture medium (SCM1) was collected. The vitrified blastocysts were later thawed and cultured individually for 24 hours, and then the embryos and spent culture medium (SCM2) were collected. Participants/materials, setting, methods Embryos were cultured in a 30 μl droplet until Day 4 and a 10 μl droplet for individual culture. Culture medium was collected and embryos were subjected to chromosomal analysis by NGS. We compared the results of niPGT taken from the same embryos on days 4- 6 (SCM1) and after thawing of vitrified blastocysts (SCM2), with PGTa analysis of the embryos. Main results and the role of chance Among all media samples analyzed, results were obtained for 80.0% (48/60) SCM1 and 96.7% (58/60) SCM2 samples. The ploidy concordance rate was 81.3% (39/48) for SCM1 and 98.3% (57/58) for SCM2. The overall concordance rate, defined as the combined rate of the total concordance for all chromosomes and the partial concordance for some chromosomes was 72.9% (35/48) for SCM1 and 94.8% (55/58) for SCM2 respectively. Significant differences were identified in the informative sample rate, the ploidy concordance rate, and the overall concordance rate(p &amp;lt; 0.05) between SCM1 and SCM2. Fisher’s exact test was used for statistical analysis. Limitations, reasons for caution In this study, embryo culture was continued to day 6, regardless of morphology of the embryo at day 5, which differs from the usual clinical culture process. Additionally, the timing of sample collection differed between the day 4-6 culture medium sample and that for a blastocyst. Wider implications of the findings The results demonstrated that accuracy of ploidy status was higher in the SCM2 compared to the SCM1 sample, suggesting that analysis of thawed vitrified blastocyst medium maybe a more precise predictor and therefore a non-invasive alternative to conventional PGTa. Trial registration number not applicable

P-611 Quantity or quality? Comparative study on the effectiveness of recombinant, purified and mixed gonadotropins on the outcome of ICSI treatments from an embryologist perspective

Abstract Study question Are oocyte retrieval and embryo development affected by the origin of the gonadotropins used for controlled ovarian stimulation (COS), when controlling fertilization and culture conditions? Summary answer Patients stimulated with recombinant gonadotropins obtained similar number of oocytes than those using purified hormones, but more blastocysts and better embryo morphology, needing lower doses. What is known already Many authors have questioned the safety of purified urinary gonadotropins used for COS prior to an IVF treatment, and have demonstrated the presence of impurities and a high heterogeneity between batches. Another strategy involves using recombinant follicle-stimulating hormone with/without luteinizing hormone (rFSH+/-rLH) synthetized in vitro. Many studies have compared the cost-effectiveness of these drugs; however, a conclusion has not been reached. These studies have varying inclusion criteria and focus on final endpoints such as pregnancy and live birth, sometimes lacking control over the many variables introduced during the fertilization and culture procedures, which affect embryo development and potential. Study design, size, duration Preliminary retrospective observational study performed over 453 patients subjected to COS prior to autologous ICSI treatments in a private clinic over 3 years, using Time-lapse incubator. The total dose employed, the number and maturity rate of the oocytes retrieved of patients subjected to stimulation with recombinant (RH; n = 46) or mixed hormones (MH; n = 332) were compared with those using purified hormones (PH; n = 75). After ICSI, fertilization and embryo development were also compared between the three groups. Participants/materials, setting, methods The stimulation protocol was adjusted for each patient following the standard protocols of the clinic. Fertilization was performed by ICSI and embryo culture was carried out in Geri time-lapse incubators using Geri Medium (Genea Biomedx). When available (1,097 embryos), Eeva-Xtend Test was applied. Statistical analysis was performed by generalized estimating equations (significance: P≤0.05). Considered confounders include age, body mass index, days of stimulation, down-regulation protocol, and other variables relevant to embryo development. Main results and the role of chance Proper follicle stimulation was achieved with lower doses of RH (1,606.26±1,110.52 IU) than PH (2,941.73±1,011.14 IU) and MH (3,629.68±1,080.79 IU). No association was found between the use of different gonadotropins and the number of oocytes collected (Odds ratio (OR, RH vs PH)=2.51, P=0.411; OR (MH vs PH)=1.26, P=0.754) or their maturity rate (OR (RH vs PH)=2.38, P=0.328; OR (MH vs PH)=1.01, P=0.988). No significant association was found between the odds of a correct fertilization and the type of hormone used. However, the odds of reaching blastocyst stage were statistically higher for embryos from patients treated with RH: OR (RH vs PH)=1.72, P=0.006; OR (MH vs PH)=1.45, P=0.005; blastocyst rate=68.7% RH, 61.9% MH and 54.6% PH. The mean morphological classification by ASEBIR criteria of the embryos was significantly better in the RH group than in the PH group (OR = 1.55, P=0.032), but no association was found in the MH-PH comparison (OR = 1.26, P=0.092). No association was found between the type of hormone and the distribution of the Eeva-Xtend scores. Significantly lower values of the morphokinetic parameter s2 (synchrony between the second and third cell divisions) were also associated with the use of RH (OR (RH vs PH)=0.311, P=0.004). Limitations, reasons for caution The reduced sample size and the heterogeneous number of patients in the three groups limit the statistical power of the comparisons. Different hormones and brands were included in each group. Wider implications of the findings Although not altering the number of oocytes retrieved, the use of RH may improve the chances of success of ICSI cycles by increasing the number of available blastocysts. The positive outcomes paired with the lower dose needed, could make the use of recombinant hormones a cost-effective strategy. Trial registration number Not applicable

P-058 The role of sperm selection in improving embryo euploidy rate: a comparison between Swim-up and Microfluidic Sperm Selection

Abstract Study question Does Microfluidic Sperm Selection (MSS) improve the blastocysts’ development rate and the euploidy rate compared to Swim-up sperm selection? Summary answer MSS doesn’t improve the rate of blastocyst development and euploidy compared to Swim-up. Furthermore, in women age ≥ 37 years D6 aneuploid blastocyst rate increases What is known already Sperm defects have been shown to have a negative impact on embryo quality and development. MSS was introduced as an alternative to traditional centrifugation-based sperm preparation techniques to efficiently isolate highly motile and healthy spermatozoa. Centrifugation has been suggested to compromise membrane integrity, motility, mitochondrial energy production, and DNA structure. Less DNA fragmentation and ROS formation was observed in spermatozoa isolated with MSS compared to standard techniques. Our study aims to analyze whether MSS also improves biological outcomes. Study design, size, duration In a prospective study of 51 ICSI/PGT-A cycles performed between September 2022 and November 2023, freshly ejaculated semen sample was divided into two aliquots and processed by Swim-up and MSS. Split sperm sample was used to inject siblings’ mature oocytes (249 vs 274 oocytes in Swim-up vs MSS groups). Inclusion criteria: retrieved MII oocytes ≥8, oligoteratozoospermic and normozoospermic semen. Results were analyzed according to women’s age &amp;lt;37 years (21 cycles) and ≥37 years (30 cycles). Participants/materials, setting, methods Spermatozoa were selected using either conventional Swim-up or a microfluidics device (ZyMōtTM). Post treatment sperm parameters (sperm count; total sperm motility; progressive motility and hyperactivated motility) were examined for both. Embryo culture was performed in Irvine Continuous Culture-NX media until day5 or day6 and biopsy was performed at the blastocyst stage by next generation sequencing. Primary endpoint was fertilization rate, blastocyst development and usable blastocysts rate. Chi-square analysis was performed and p &amp;lt; 0.05 was considered significant Main results and the role of chance There is no significant differences overall between Swim-up and MSS in fertilization rate (73% vs 75%), blastocyst development rate (47% vs 54%) and ploidy rate: aneuploid (47% vs 50%), usable blastocyst (i.e. euploid+mosaics) (53% vs 50%). When considering female age, no significant difference is observed in the primary endpoints in the &amp;lt;37 years group. However, in women aged ≥37 years, the rate of blastocyst development is similar in the two sperm preparation groups, but a significant difference is observed in the timing of blastocyst development: The percentage of blastocysts formed on day 6 in the MSS group is significantly higher than in the Swim-up group (43% vs. 19% p &amp;lt; 0.05). In addition, the aneuploidy rate of blastocysts on day 6 is also significantly higher than in the Swim-up group (79% vs. 50% respectively, p &amp;lt; 0.05). The concentration of sperm recovered post-treatment was similar in Swim-up vs MSS (23 x106 vs 19 x106); total motility and progressive motility were similar in Swim-up compared to MSS (92.7% vs. 95.3% and 85% vs. 87% respectively). Only hyperactivated motility assessed with the SCASCOPE CASA System (Microptic) was significantly higher in MSS vs Swim-up 41% vs 4% respectively (p &amp;lt; 0.0005). Limitations, reasons for caution Our study included couples with different infertility diagnosis and semen parameters. Further studies with an increased number of patients may provide more comprehensive data to better evaluate biological and clinical outcomes to eventually suggest the use of MSS in laboratory routines Wider implications of the findings Recently, some studies reported that microfluidic-sorted sperm showed significantly less ROS formation and DNA fragmentation compared to those treated with conventional Swim-up method. Nevertheless, our data does not currently show the expected increase in embryo euploidy rate to justify the routine use of these expensive devices in male infertility treatment Trial registration number non applicabile

O-296 The benefits of higher concentrations of synthetic serum substitutes in post-warming embryo culture and transfer media: a retrospective study of 864 frozen blastocyst transfer cycles

Abstract Study question Does a higher concentration of synthetic serum substitutes (SSS) in post-warming embryo culture and transfer media improve clinical outcomes in frozen blastocyst transfer (FBT) cycles? Summary answer Increased SSS concentration in post-warming embryo culture and transfer media reduces miscarriage rate in FBT cycles and improves ongoing pregnancy rate in euploid blastocyst transfer. What is known already Several studies showed increased clinical pregnancy rates with the use of complex protein supplementation such as SSS in embryo culture media. Nevertheless, there is a lack of standardization on their concentrations. The range of concentrations employed is strikingly wide, spanning from 5% to 50%. Moreover, no studies investigating different SSS supplement concentrations in post-warming embryo culture and transfer media in FBT cycles are available. These uncertainties warrant further investigation in this field. Study design, size, duration This retrospective study included 864 patients undergoing vitrified-warmed euploid (n = 102) and untested (n = 762) blastocyst transfers between September 2022 and August 2023. The cohort was divided based on the concentration of SSS used in post-warming embryo culture and transfer media: 10% (n = 415) and 20% (n = 449). The exclusion criteria were ICSI-cycles performed with surgically retrieved or frozen sperm, gamete donation and vitrified-warmed oocyte cycles. Prior to embryo transfer, warmed blastocysts were cultured for 2-3 hours. Participants/materials, setting, methods Treatment allocation to 10% or 20% SSS groups followed an alternating regimen. To reduce any pre-existing differences between the two groups, a propensity-score (PS) analysis was performed, targeting unbiased treatment effect estimates. Variables included in the PS were: maternal age, maternal BMI, infertility type, transfer day, endometrial preparation, blastocyst quality, and the operators for biopsy, warming, and transfer. Main results and the role of chance Patient baseline characteristics were evenly distributed in the two study groups. After the PS-based analysis, no statistically significant differences in ongoing pregnancy rates for both overall and untested blastocyst transfers were observed between 10% and 20% SSS groups. However, the 20% SSS group exhibited a significantly lower miscarriage rate in overall FBTs [OR = 0.61, 95% CI (0.37-1.00), p-value=0.050] and a higher ongoing pregnancy rate in euploid blastocyst transfers [OR = 3.48, 95% CI (1.30-9.35), p-value=0.013]. A mild trend of an increased miscarriage rate was also observed in both euploid and untested blastocyst transfers in the 10% SSS group compared to the 20% SSS group [OR = 2.834, 95% CI (0.651-12.337), p-value=0.165; OR = 1.558, 95% CI (0.897-2.705), p-value=0.115 respectively]. Limitations, reasons for caution The study is limited by its retrospective design and a lack of randomization, although a PS-based analysis may mitigate such a weakness. Not considering live birth rate as outcome and protein concentrations higher than 20% and lower than 10% might represent other study limitations. Wider implications of the findings To our knowledge, this is the first study investigating the impact of varying protein supplementation concentrations in post-warming embryo culture and transfer media on clinical outcomes in FBTs. Our findings suggest that higher serum concentrations improve the likelihood of ongoing pregnancy, especially in blastocysts undergoing stressful manipulations such as biopsy. Trial registration number not applicable

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

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

P-214 Direct unequal cleavage-1 (DUC1) in IVF/ICSI cycles: relationship with patient characteristics and insemination method

Abstract Study question Does the incidence of DUC1 in IVF/ICSI cycles vary with the maternal profile and insemination method? Summary answer The incidence of DUC1 cycles is higher in patients with a more favourable prognosis. DUC1 embryos are more frequent in ICSI than in IVF cycles. What is known already The emergence of Time-Lapse Microscopy (TLM) has enabled a dynamic and thorough evaluation of in vitro embryo development, unveiling the occurrence of morphological phenomena unobserved with static observation, such as direct unequal cleavage (DUC), which is associated with a drastically reduced competence to implant. The incidence of DUC-1 (DUC immediately after syngamy) has been reported to vary from 8.3% to 26%, but the reasons underlying its occurrence and variability are unknown. There is evidence that the maternal profile and insemination method can impact embryo developmental competence, but whether they impact the incidence of DUC is also unknown. Study design, size, duration The study includes 2920 first IVF/ICSI cycles (2018 to 2022) providing 12492 embryos. Cycles presenting male infertility were excluded from the analysis. The percentages of DUC1 embryos in relation to total embryos and of cycles providing at least one DUC1 embryo in relation to total cycles were compared between AMA (advanced maternal age; ≥35) and pre-AMA (&amp;lt;35) cycles. Patient/cycle characteristics were compared between cycles with and without DUC1 occurrence in AMA and pre-AMA cycles, separately. Participants/materials, setting, methods Ovarian stimulation was induced with rFSH or with a combination of FSH and LH using classic antagonist or short agonist protocols. Oocytes were inseminated with either IVF or ICSI standard methodology. Embryo culture was performed in an integrated embryo culture Time-lapse System (EmbryoScope; Vitrolife). Differences between percentages were assessed by the Fisher’s exact test, while differences between continuous variables were assessed with the Wilcoxon sum rank test. Main results and the role of chance The percentage of DUC1 embryos in relation to total embryos did not vary between pre-AMA (5.4%) and AMA (5.2%; p &amp;gt; 0.05) cycles. Alternatively, the incidence of cycles providing at least one DUC1 embryo was higher in pre-AMA (21.7%) as compared with AMA patients (16.0%; p &amp;lt; 0.0001). In both pre-AMA and AMA cycles, DUC1 occurrence was associated with higher oocyte yield (13.5±7.7 vs. 10.6±6.7 and 11.0±6.3 vs. 7.5±5.5, respectively; p &amp;lt; 0.0001), higher AMH serum levels (4.4±4.7 vs. 3.4±3.2 and 3.0±3.6 vs. 2.1±2.1ng/mL, respectively; p &amp;lt; 0.004) and lower basal FSH serum levels (7.0±2.1 vs. 7.6±2.9 and 7.8±3.3 vs. 8.5±3.4mU/mL, respectively; p &amp;lt; 0.0001). Overall, the percentage of DUC1 embryos was higher in ICSI (5.6%) as compared to IVF (4.4%, p &amp;lt; 0.01) cycles. However, when the data was stratified by maternal age, DUC1 incidence was significantly higher in ICSI vs. IVF in AMA (5.6 vs 4.0%; p = 0.002) but not in pre-AMA (5.7 vs 5.0%; p = 0.19). All 21 DUC1 embryos transferred during the study failed to implant. Limitations, reasons for caution Our study is limited by its retrospective nature. The present conclusions must be confirmed in other patient populations with different race/genetics and habits. Wider implications of the findings The occurrence of cycles providing DUC1 embryos is paradoxically associated with a more favourable patient prognosis, and the percentage of DUC1 embryos is higher in ICSI as compared to IVF cycles in AMA patients. Our findings represent new valuable clues for the clarification of DUC1 underlying causes. Trial registration number NA

P-533 Impact on clinical outcomes of day-6 extended culture and reduced culture media volume applied to non-invasive PGT-A

Abstract Study question Impact on clinical outcomes of day-6 extended culture and reduced culture media volume applied to non-invasive PGT-A Summary answer The implementation of changes in culture conditions to accommodate niPGT-A has no impact on blastocysts viability or pregnancy outcomes. What is known already The recent identification of embryo cell-free DNA (cfDNA) in the culture media has created the possibility of a non-invasive approach for aneuploidy testing in blastocysts. Concerns have however arisen at two levels. Firstly, the concordance rates that cfDNA results have with the full blastocyst or a trophectoderm (TE) biopsy and secondly, the impact on clinical outcomes of the changes in the embryo culture protocol that are required to implement niPGT-A in the IVF laboratory. Concordance rates of niPGT-A to invasive PGT-A have gradually improved, however the impact of culture protocol changes is not as well understood. Study design, size, duration Six IVF clinics participating in a trial examining concordance rates for invasive and non-invasive PGT-A on day-6/7 blastocysts were involved (Rubio et al., 2020). Patients were recruited from April 2018 to December 2020. In the study group, 428 PGT-A cases following the niPGT-A protocol for embryo culture were included. The control group consisted of 1,392 regular PGT-A cases that did not participate in the study and underwent the standard PGT-A culture protocol. Participants/materials, setting, methods In the control group, embryos were biopsied and vitrified on day 5, 6 or 7. In the study group, niPGT-A culture conditions started on day 4, when embryos were washed and cultured individually in 10μl of fresh media. On day 6/7 blastocysts were biopsied, vitrified and media collected for niPGT-A analysis. Clinic A cultured all embryos up to day 6/7, whereas in clinics B-F a minority of day-5 blastocysts were biopsied and transferred. Main results and the role of chance Statistical comparisons were performed between the study and the control groups. Mean female age, number of oocytes, fertilization rates and number of blastocysts biopsied were not significantly different for the study and the control group. Overall, the niPGT-A protocol did not have any detrimental effect on blastocyst quality, euploidy rates or informativity rates. Deferred single embryo transfers (SET) were performed in both groups. Regarding the overall pregnancy outcomes, no significant effect was observed on clinical pregnancy rate, miscarriage rate or ongoing pregnancy rate (OPR, ≥12 weeks) in the study group compared to the control group when stratified by day of biopsy. Clinic A showed no difference in OPR per transfer regardless of culture protocol (62.3% in control group day 5/6/7; 60.8% in control group day 6/7; and 65.6% in study group day 6/7). For the remaining clinics (B-F), a comparison was made separately between day-5 and day-6 blastocysts that were transferred and cultured according to the standard and to the niPGT-A protocol. No significant differences in OPR were observed, neither between control group day 5 (60.6%) and study group day 5 (58.7%), nor between control group day 6 (44.7%) and study group day 6 (41.9%). Limitations, reasons for caution The limitations are intrinsic to the retrospective nature of the study, and also to the fact that the study was conducted in invasive PGT-A patients and not in niPGT-A cases alone. Wider implications of the findings This study shows that changing current IVF laboratory practice to adopt niPGT-A protocols has no impact on the number of blastocysts available for transfer and overall clinical outcomes. Whether removal of the actual invasive biopsy step leads to further improvements in pregnancy rates awaits further studies. Trial registration number ClinicalTrials.gov (NCT03520933)

O-182 To err is human, also for those working in ART

Abstract Medically assisted reproduction (MAR) centers combine clinical and technical expertise with a very high complexity. Over the years, MAR centers have undergone increased intricacy due to incorporating laboratory techniques, such as cryopreservation of oocytes and embryos and micromanipulation, embryo culture, and pre-implantation genetic testing. Not only the technicality of the MAR laboratory profession is challenging, but the complexity of each phase has increased over the years; the patient population undergoing treatment has expanded, where not only the intended parents’ gametes are involved but also the utilization of gametes from egg and sperm donors, as well as including a gestational carrier receiving the embryo. These complexities have led to the implementation of rigorous control measures to mitigate the risk of errors. Nevertheless, a zero-risk fertility clinic is an illusion, given the many necessary interventions and manipulations in each treatment cycle. Errors in fertility practice involving gametes or embryos can be devastating to patients and clinical personnel, often raising psychological, legal, ethical, economic, and practical concerns. The most critical cases of risk of harm in MAR procedures involve misidentifying a patient, gamete, or embryo, called ‘mix-up,’ which could lead to dramatic consequences. Although the frequency is low, mix-up cases have been described worldwide. Other adverse events derive from preventable human actions, e.g., overlooking an embryo when carrying out assessments, dropping a dish, incorrect labeling, improper pipetting technique, unusually poor thawing, and inadvertent discard of the sample. Human error cannot be eradicated. In the old view, human error was perceived as the root cause of problems, with individuals being viewed as variables requiring control. Conversely, contemporary perspectives consider human error a symptom of deeper systemic issues. As suggested by the Institute of Medicine in 2000, which published the report “To Err Is Human,” the core of the problem lies not with the fallibility of healthcare personnel but the flawed systems within which they operate. Preventing errors is possible by incorporating safety into system processes and hospital structures. Latent errors are errors that arise due to system inefficiencies. In the context of MAR, these latent conditions can, amongst others, be work-related pressure, high workload, lack of communication between team members, distraction, a suboptimal setup of the lab, a lack of clarity in the procedures, and insufficient time for thorough communication. In the famous Reason’s Swiss cheese theory of error, these latent conditions are weaknesses (or gaps/ holes) in the system’s defense layers. When all the holes from each layer (i.e., policy, planning, design, communication) align, they will permit an error to cause harm. Understanding the theory of human errors and building safety into various layers of care processes in MAR are effective ways of reducing errors rather than blaming sentinel individuals. Errors within MAR centers can be prevented by designing the healthcare system at all levels to make it safer, making it hard for people to do the wrong thing and easy for people to do the right thing.

O-196 Men who fathered embryos with poor pre-implantation development rates exhibit an increased frequency of sperm with high DNA accessibility

Abstract Study question Do differing levels of DNA accessibility in sperm affect the ability of embryos generated by ART to undergo pre-implantation development? Summary answer Individuals whose sperm generated embryos that failed to efficiently form blastocysts tend to have more sperm with high levels of DNA accessibility. What is known already Chromatin is a complex composed of nucleic acids and proteins that serves to condense and regulate the genome in all eukaryotic cells. Sperm chromatin is packaged into a structure with higher density than that found in precursor germ cells or somatic cells. Samples from individuals with decreased normal semen parameters have previously been shown to possess an increased frequency of sperm with aberrant chromatin packaging. However, existing methodologies for measuring sperm chromatin packaging are indirect and non-quantitative. Study design, size, duration Prospective cohort study, registered January 2020. We adapted an enzymatic methodology for measuring DNA accessibility known as NicE-view (Nicking Endonuclease-mediated visualization), for use in human sperm. DNA accessibility was quantified in three millions of individual sperm from semen obtained from 60 individuals. Both total sperm and sperm selected for motility via swim-up were analyzed for each participant. Participants/materials, setting, methods Participants sought treatment for infertility at Universitätsspital Basel and possessed normospermic levels in conventional semen analysis. Participant partners had normal endocrine parameters and ovulation response to hormonal stimulation. 20 couples obtained pregnancies without the need for ART, while 40 individuals who were treated using ART and divided into two groups differing in pre-implantation development rates: (1) 20 with blastocyst development of &amp;gt; 50% of all zygotes and (2) 20 produced 0-1 blastocyst following embryo culture. Main results and the role of chance We quantified DNA accessibility and the presence of single stranded DNA nicks in more than 3 million sperm derived from 117 samples. We found that while DNA nicks were only present in a small fraction of the sperm analyzed, DNA accessibility levels varied dramatically with the data indicating a bimodal distribution. At a single sperm level, DNA accessibility and the presence of single stranded DNA damage both correlated with nuclear size. We found that DNA nicking was more abundant in total sperm samples compared to sperm selected for motility via swim-up preparation. In contrast, sperm with high levels of DNA accessibility were more abundant following swim-up, perhaps resulting from capacitation. We observed that individuals with a history of low blastocyst development were more likely to have high frequencies of sperm harboring DNA nicks and high DNA accessibility. We also measured increased frequencies of sperm with high DNA accessibility to negatively correlate with conventional semen parameter values. Limitations, reasons for caution Our study cohort consists of only 60 individuals. Variation is also observed between individuals within each participant category, so further analysis with increased participant numbers is required to strengthen these conclusions. Wider implications of the findings Our results suggest measuring DNA accessibility in sperm from normospermic individuals provide additional information about the efficiency of pre-implantation development following ART. Future work examining the sources of increased DNA accessibility in sperm will also be interesting to identify possible treatments for such individuals. Trial registration number SNCTP000003654 | NCT04256668