Single Euploid Blastocyst Transfer Research Articles

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

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

Does Recurrent Implantation Failure Exist? Prevalence and Outcomes of Five Consecutive Euploid Blastocyst Transfers in 123 987 Patients

(Abstracted from Hum Reprod 2024;39(5):974–980 Recurrent implant failure (RIF) has been defined as repeated failed implantations following multiple embryo transfers. A landmark paper on RIF reported the cumulative sustained implantation and live birth rates achieved with 3 consecutive single euploid blastocyst transfers and found a similar live birth rate across the first 3 consecutive transfers with a “true” unexplained RIF rate of <5% in the in vitro fertilization population.

O-293 Mastering consistency: a similar training allows to standardize embryo transfer performance, but experience does not involve further improvements

Abstract Study question Is there any variation in the live birth rate (LBR) per vitrified-warmed single euploid blastocyst transfer (SEBT) among clinicians trained at the same IVF center? Summary answer The LBR per SEBT (overall: 53%) remained consistent among equally trained transfer operators (range: 48%-60%) regardless of the number of prior procedures performed. What is known already The number of ART treatments and practitioners has increased over the years. To ensure high standards, an efficient quality control system should monitor and standardize operators’ performance by leveraging statistically-sound key-performance-indicators (KPIs). The embryo transfer procedure is a clinically relevant step, particularly susceptible to variations among clinicians, irrespective of their experience. However, the outcome of “LBR per transfer” is influenced by various confounders, such as embryo stage, morphology, reproductive history, uterine conditions, and others. Our objective was to assess potential differences among transfer practitioners who underwent the same training at a single IVF center in the least biased scenario possible. Study design, size, duration Retrospective single center study. Out of 8663 transfers performed by 4 operators (all trained by operator-1) between 2013 and 2021, we selected 421 first vitrified-warmed transfers with day5 euploid blastocysts whose morphology was >Gardner’s BB-grade in non-obese women, thereby excluding all main confounders on our primary outcome (= LBR per SEBT across different operators). The study was powered to exclude a 18%-decrease (β = 0.8, α = 0.05) in the LBR versus operator-1 (ratio comparator:control=1:2). Participants/materials, setting, methods We conducted ICSI, blastocyst biopsy without day3 zona-drilling, and aneuploidy testing to exclude full-chromosome uniform aneuploidies. All blastocysts were transferred ∼2 hours after warming. Uterine assessment was conducted to exclude putative conditions associated with lower LBR. Endometrial preparation protocol (modified-natural cycle versus hormone-replacement-therapy), maternal age at transfer, experience of prior implantation(s) (yes/no), the embryologists involved in the transfer procedure, were assessed as putative further confounders. All significant differences were confirmed through regression analyses. Main results and the role of chance The overall LBR per vitrified-warmed SEBT was 53.4% (N = 225/421). Operator-1 conducted 3960 transfer procedures during the study period, with 167 eligible for this study, achieving a 55.7% LBR (N = 93/167). Operator-2,-3, and -4 respectively conducted 2533, 1087, and 1083 procedures during the study period, with 134, 62, and 58 eligible for this study. Their LBRs were 50% (N = 67/134, p-value versus operator-1=0.2), 48.4% (N = 30/62, p-value=0.2), and 60.3% (N = 35/58, p-value=0.3). Multivariate logistic regression analysis, adjusted for endometrial preparation protocol and embryologists involved in the transfer procedure, confirmed the absence of an association (Operator-2 vs. 1 multivariate-OR: 0.83, 95%CI 0.5-1.39, adjusted-p=0.49; Operator-3 vs. 1 multivariate-OR: 0.75, 95%CI 0.40-1.40, adjusted-p=0.37; Operator-4 vs. 1 multivariate-OR: 1.40, 95%CI 0.72-2.70, adjusted-p=0.32). Importantly, the number of prior transfer procedures conducted by each clinician during the study period also showed no association with the primary outcome (OR = 1.0, p-value=0.82). From an IVF center perspective, the LBR per SEBT remained stable across the 9-year study period, showing minimal non-significant fluctuations. These outcomes describe an IVF center where equally-trained clinicians adopt the same standard-operating-procedures to manage vitrified-warmed embryo transfers. In this context, the LBR per SEBT represents an ideal KPI to assess the learning curve of operators in training. Limitations, reasons for caution Over 2500 SEBTs are required to rule out a 5%-decrease in the LBR. Yet, such a substantial number is better suited for comparing centers’ rather than practitioners’ performance. It would be intriguing to investigate potential differences among operators who underwent different training regimens but operate within the same clinic. Wider implications of the findings No correlation exists between the number of prior embryo transfer procedures conducted and further improvements, therefore initial operators’ training is crucial for standardizing their performance. LBR per SEBT is a strong KPI, though poorly-actionable in the short-term. It’s better suited for center comparisons, prompting internal procedure reviews when differences arise. Trial registration number not applicable

P-135 Impact of TE-biopsy practitioners on PGT-A results and live-birth outcomes after single euploid blastocyst transfer: comparison of 19 TE-biopsy practitioners in a single center

Abstract Study question Can TE-biopsy practitioners impact pre-implantation genetic test for aneuploidy (PGT-A) results, pregnancy, and live-birth outcomes in a single center that has nineteen biopsy practitioners? Summary answer Yes. Trophectoderm (TE)-biopsy practitioners, especially micromanipulation experience, can impact PGT-A results but do not impact pregnancy or live-birth outcomes after single euploid blastocyst transfer (SeBT). What is known already Recently, PGT-A has become important in in vitro fertilization treatment. However, several factors can impact the accuracy and interpretation of PGT-A results. Several studies have suggested that TE-biopsy practitioners can impact PGT-A results. However, these findings are controversial because these previous studies used data from a low number of TE-biopsy practitioners and from multiple centers. There is, therefore, still limited knowledge of the impact of TE-biopsy practitioners on PGT-A results and their clinical outcomes. Study design, size, duration A total of 3,589 PGT-A cycles (average maternal age, 40.9±3.2; 1,168 patients; February 2020 to November 2022) were retrospectively analyzed. Furthermore, 779 single frozen euploid blastocyst transfer (SeBT) cycles were analyzed. First, we investigated the association between TE-biopsy practitioners and transferable blastocysts that included euploid and low-level mosaic blastocysts (LLM, 20 to 40% abnormal cells). Second, we analyzed the impact of TE-biopsy practitioners on pregnancy and live-birth outcomes after SeBT. Participants/materials, setting, methods The nineteen practitioners (Years of intracytoplasmic sperm injection (ICSI) experience: 2 to 20 years) who received the same training performed a total of 5,613 TE-biopsies for PGT-A. The same practitioner performed both the biopsy and the tubing procedures for each blastocyst they biopsied. The Wald test for multivariable logistic regression analysis (mLR) was used to determine the impact of TE-biopsy practitioners. The confounding factors were selected by univariable logistic regression analysis before mLR. Main results and the role of chance TE-biopsy was carried out by the flicking method with laser assistance and it was taken five to seven TE cells. Overall, 22.9% of blastocysts were transferable blastocysts (euploid: 21.1%, LLM: 1.8%). The results were inconclusive for 0.1% of the blastocysts. Maternal/paternal age, number of previous egg retrieval/embryo transfers, anti-Müllerian hormone, sperm characteristics, indication for PGT-A, number of previous deliveries/chemical abortions, blastocyst quality (the day of vitrification, inner diameters of blastocysts, and Gardner grading) were used as confounding factors on the mLR. TE-biopsy practitioners were significantly associated with both ensuring transferable blastocysts and LLM in transferable blastocysts (p < 0.05). Additionally, the practitioner characteristics that impact ensuring transferable blastocysts or LLM in transferable blastocysts were assessed in a sub-analysis. Years of ICSI experience (p < 0.05), number of ICSI performed during the last years (p < 0.05), and number of TE-biopsies at that point (p < 0.05) were associated with ensuring transferable blastocysts and LLM in transferable blastocysts. On the other hand, TE-biopsy practitioners did not have a significant impact on biological pregnancy, chemical abortion, pregnancy, or live-birth rates after SeBT. Limitations, reasons for caution This study did not determine the influence of TE biopsy itself and the laser assistance on PGT-A and clinical results. And this study was based on a minimal stimulation cycle. Moreover, the patient cohort in this study only included cases involving recurrent implantation failure or repeated pregnancy loss. Wider implications of the findings This study showed that TE-biopsy practitioners could impact decreasing transferable blastocysts, and this depends on their micromanipulation experience. This result suggests TE-biopsy training should be started when embryologists have gained several years of experience with ICSI procedures. This could prevent the decline in the efficacy of PGT-A for clinical results. Trial registration number not applicable

P-318 Reproductive ageing: the seed, the soil, or both?

Abstract Study question Is it all about the embryo? Can a single euploid blastocyst transfer sustain reproductive outcomes in women with advanced reproductive age? Summary answer Despite euploid embryo transfer, endometrial senescence appears to play a crucial role in determining reproductive success, particularly in older patients undergoing in vitro fertilization (IVF). What is known already Advanced reproductive age significantly impairs oocyte and embryo quality. Effective crosstalk and synchronization between embryo and endometrium are pivotal for successful pregnancies, both in vivo and in vitro. Implantation failure remains a persistent challenge in IVF, exacerbated by delayed family planning trends. The transfer of euploid blastocysts through preimplantation genetic testing for aneuploidy (PGT-A) is a potential strategy to address implantation issues, decrease miscarriages, and shorten time-to-pregnancy. However, a significant proportion of IVF treatments remain unsuccessful, suggesting critical factors beyond the embryo. Study design, size, duration This retrospective study analyzed 457 single euploid blastocyst transfers performed by two doctors at a private IVF center from January to November 2023. Inclusion criteria encompassed cases with PGT-A indications (advanced maternal age, repeated implantation failure, recurrent pregnancy loss, etc.) or cases where embryo biopsy was patient-requested. The primary outcome was ongoing pregnancy rate (OPR), with secondary outcomes including pregnancy rate (PR) and clinical pregnancy rate (CPR). Participants/materials, setting, methods Participants were categorized into four groups: Group 1 (< 35 years old; N = 100), Group 2 (35-37 years old; N = 102), Group 3 (38-40 years old; N = 118), and Group 4 (> 41 years old; N = 87). A control group (oocyte donation; N = 50) with single euploid embryo transfers showed a mean recipient age of 43 years. Donors were always less than 32 years old. Endometrial preparation protocols were consistent across all groups. Chi-square test was used to compare groups. Main results and the role of chance Among the 457 transfers analyzed, with a mean female age of 37 years, the overall PR was 71.3%, CPR was 64.8%, and OPR was 60.2%. Groups showed no differences in demographic variables and cycle parameters. Group 1 demonstrated higher PR (83.3%), CPR (78.4%), and OPR (73.5%) compared to Group 2 (PR 70.9%, p = 0.034; CPR 66%, p = 0.047; OPR 62.1%, p = 0.081), Group 3 (PR 68.2%, p = 0.009; CPR 61.2%, p = 0.005; OPR 56.6%, p = 0.008), and Group 4 (PR 65%, p = 0.002; CPR 56.1%, p < 0.001; OPR 51.2%, p = 0.001). Furthermore, the control group (oocyte recipients) showed significantly lower PR (68%, p = 0.031), CPR (54%, p = 0.002), and OPR (46%, p < 0.001) compared to Group 1, but not with the other groups. These findings demonstrate that even with euploid embryos originating from young donors, older women exhibited lower pregnancy rates than young women transferring single euploid blastocysts from their own oocytes and similar rates compared to those with advanced reproductive age transferring their own euploid blastocysts, implying that endometrial ageing may disproportionately influence IVF treatment outcomes. Limitations, reasons for caution The retrospective design introduces inherent bias, notably selection bias and unmeasured confounders. This preliminary data did not account for male infertility factors and their impact on embryo quality. Wider implications of the findings Reproductive aging appears to be influenced by factors beyond the embryo itself, especially in women above 35 years. Further research is warranted to elucidate the causes and impact of reproductive aging on implantation potential, aiming to refine clinical practices and enhance the success of IVF treatments. Trial registration number not applicable

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

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

O-316 Clinical application of PGT-A for recurrent pregnancy loss, what effects live birth after single euploid blastocyst transfer?

Abstract Study question Which factors effect the likelihood of live birth after euploid frozen embryo transfer (FET) in recurrent pregnancy loss (RPL)? Summary answer Female age and number of previous pregnancy loss do not effect the live birth rates after euploid blastocyst FET. What is known already PGT-A is helpful to discard the aneuploid embryos and has been proven to improve pregnancy outcomes and reduce miscarriage rate per embryo transfer. As approximately 50% of clinical miscarriages have chromosomal abnormalities, PGT-A has been suggested as a treatment option for RPL. However it is questionable on the cost effectiveness, effect on prognosis and labor-intensive. In addition, currently PGT-A use is not recommended by any society guidelines in RPL patients. ESHRE guideline on RPL stated ‘All couples with results of an abnormal fetal or parental karyotype may be informed about the possible treatment options available including their advantages and disadvantages.’ Study design, size, duration This retrospective cohort study from January 2018 to January 2023 was conducted at Bahçeci Health Group included patients underwent PGT-A/ICSI cycles. Patients with RPL (2 or more clinical pregnancy loss) had a complete RPL workup recommended by ASRM and reported no etiology for RPL was included in the study. Patients with uterine synechia, hydrosalpinx, endocrinological disorders and thin endometrium (endometrial thickness <6mm) were excluded Participants/materials, setting, methods A total of 366 single euploid blastocyst FETs employing hormone replacement therapy for endometrial preparation were evaluated. Patients were categorized into 2 group regarding female age (female age< 35 years and female age≥35 years. Demographic, embryological and endocrinological parameters were analyzed. Live birth rate (LBR) was the primary outcome. Clinical pregnancy rate (CPR) and miscarriage rate (MR) were the secondary outcome. Main results and the role of chance The mean female age of the study population was 33.9±4.6 years, the number of previous pregnancy loss was 3.2±1.6 and BMI was 23.8 ±7.9 kg/m2. Overall CPR, MR and LBR were 61.7% (226/366), 24.8% (56/226) and 46.5% (170/366) respectively. Comparison of clinical characteristics of the 195 women <35 years and 171 women ≥35 years revealed no significant deference in BMI, number of previous live birth, and number of previous pregnancy loss. CPR, MR and LBR were similar among two groups [ 63.6% (124/195) vs 59.6% (102/171), p = 0.44; 27.4% (34/124) vs 21.6% (22/102), p = 0.31; 46.2% (90/195) vs 46.8% (80/171), p = 0.9; respectively]. Binary logistic regression evaluating the independent factors that effect live birth revealed that female age and number of previous pregnancy loss were not found to be statistically significant factors. The only independent factor for live birth was day of embryo biopsy, transferring the day 6 biopsied embriyo significantly decreased the live birth [p = 0.003, OR: 0.47, 95% CI (0.29-0.77)]. Limitations, reasons for caution The maim limitation of the study is its retrospective nature. Wider implications of the findings PGT-A has been suggested to be a treatment for RPL. However there is insufficient data to call PGT-A as an efficient treatment for RPL. Investigating the factors that effect live birth after PGT-A may be the first step for selecting the most favorable group Trial registration number Not applicable

P-195 Euploid embryo transfer in a hyaluronic acid-enriched media results in a higher ongoing pregnancy rate than euploid embryo transfer in media not containing hyaluronic acid

Abstract Study question Will using a hyaluronan-rich transfer medium increase the ongoing pregnancy rate and live birth rate when transferring euploid embryos cultivated in a dry incubator? Summary answer Transferring euploid embryos in a media with high concentrations of hyaluronic acid increases the ongoing pregnancy rate compared with media with no hyaluronic acid. What is known already Cochrane Review found that solutions with high concentrations of hyaluronic acid increase the chance of live birth by adding probably 1 additional live birth for every 14 embryos transferred in a high-concentration hyaluronic acid solution for patients aged 27 to 35. A study involving 85 frozen embryo transfer cycles showed a positive effect of high concentrations of hyaluronic acid on the results of transfer of morphologically poor euploid blastocysts. Previous studies were conducted in humidified incubators where hyaluronic acid-rich media was equilibrated without oil overlay. Our clinic uses dry incubators, necessitating oil overlay with all media, including hyaluronic acid-rich media. Study design, size, duration A randomized prospective study involving 255 patients aged 19-44 undergoing a transfer of a single euploid embryo between June 2023 and November 2023. Patients were randomly assigned to the study and control groups. Blastocysts were incubated after thawing for 3 hours either in a hyaluronic acid-enriched medium or in a medium without hyaluronic acid. Participants/materials, setting, methods Embryos underwent PGT-A at the blastocyst stage on either day 5 or day 6, using the EmbryoMap method. Trophectoderm biopsy was performed using the Flicking technique without a laser. 5-7 cells were removed for analysis. Blastocysts were vitrified following biopsy. Hyaluronic acid-enriched media was equilibrated for 16-18 hours in a 5%O2 6%CO2 atmosphere in a well of 4-well culture dish under oil overlay (600 microliters of media and 500 microliters of oil). Main results and the role of chance In this ongoing study, 127 transfers of euploid blastocysts in a hyaluronic acid-rich media resulted in 70 ongoing pregnancies, with a 55.1% ongoing pregnancy rate. In comparison, 128 transfers of euploid blastocysts in a media with no hyaluronic acid resulted in 65 ongoing pregnancies, with a 50.8% ongoing pregnancy rate. The relative improvement in the ongoing pregnancy rate is 8.5%. Our data suggests that using a hyaluronic acid-rich medium for the transfer of euploid blastocysts of good morphology can improve clinical pregnancy rates, although the effect is less dramatic than was shown in a 2023 study by Nakagawa et al. for morphologically poor euploid blastocysts, where more than twofold increase in clinical pregnancy rates was detected. Limitations, reasons for caution Specific media equilibration protocol is required for the incubation and transfer of embryos in a hyaluronan-rich transfer medium for IVF clinics utilizing dry incubators. Further research is required to confirm results and to establish whether transferring embryos in hyaluronic acid-enriched media increases the live birth rate after euploid embryo transfer. Wider implications of the findings Our results show that it is feasible to use a hyaluronic acid-enriched medium in a dry incubator with an oil overlay and that using it according to described protocol could increase the ongoing pregnancy rate after a single euploid blastocyst transfer. Trial registration number not applicable

O-128 Can embryo re-expansion pattern post-biopsy for PGT-A have a predictive value on the outcome?

Abstract Study question Does re-expansion grade at 1 hour post-biopsy correlate with post-thawing re-expansion rate and live birth rate? Summary answer Embryo re-expansion 1-hour post-biopsy is associated with post-warming re-expansion and increased live birth. What is known already Blastocyst cryopreservation is a key component of PGT-A procedures. In non-PGT-A cycles, collapsing blastocysts before vitrification is commonly recommended to improve blastocyst viability. However, the procedure becomes more complex in PGT-A cycles.The European Society of Human Reproduction and Embryology (ESHRE) recommends an immediate freeze post-biopsy, although a definitive timeframe remains elusive. To date, no conclusive timing has been agreed upon. Some studies support prolonged culture post-biopsy (> 3hrs), citing improved implantation and pregnancy rates. Conversely, others advocate for rapid cryopreservation, endorsing an immediate approach within an hour, while mostly blastocysts are still collapsed due to the biopsy procedure. Study design, size, duration Retrospective study of 1141 single euploid blastocyst transfers (seFET) conducted between April 2021 and February 2023. Analysed factors included patients' age, BMI, embryo quality at biopsy and 1-hour post-biopsy (at vitrification), and the biopsy day. Live birth and Ongoing pregnancy (OPR), defined as viable pregnancy beyond 24 weeks, were assessed. Blastocysts were scored at a standardized time (1hr post TE-biopsy): 0 for complete collapse, 1 for starting re-expansion, and 2 for clear re-expanded cavity. Participants/materials, setting, methods PGT-A patients undergoing a seFET. Only cycles with embryos being biopsied on Days 5,6 or 7 and with a known pregnancy outcome were included. Main results and the role of chance Out of 1141 transfers, 461 transfers resulted in live birth (40.4%) and 108 were still ongoing above 24 weeks’ gestation at the time of last contact (9.4%) for a total OPR/LB rate of of 49.8%. One-hour post-biopsy 64.0% of blastocysts re-expanded to Grade 2, 29.3% to Grade 1, and 6.7% showed no re-expansion (Grade 0). The OPR was significantly different between embryos that re-expanded to grade 2, 1 or 0 (54.2%, 44.3% and 32.5%, respectively, P < 0.001). Regression analysis showed re-expanding to Grade 2 at 1-hour post-biopsy was significantly and independently associated with OPR/LB compared to embryos that did not re-expand (i.e., Grade 0) (aOR: 1.77, 95% CI: 1.01–3.12, P = 0.047), after adjusting for embryo quality metrics (ICM, TE grade, day of biopsy), body-mass index and endometrial preparation type. . Embryos with greater re-expansion 1-hour post-biopsy had higher re-expansion slopes post-thawing, indicating faster and greater re-expansion to the point of transfer. Results were significant for all trophectoderm categories (Grade A, B, and C; P = 0.016, <0.001 and <0.001, respectively). Moreover, re-expansion rate 1-hour post-biopsy was significantly and independently associated with post-thawing re-expansion slope after adjusting for ICM, TE, expansion grade and biopsy day (P < 0.001). Limitations, reasons for caution The re-expansion grading remains an in-house system that involves subjective assessment with a possible inter-observer variation. Wider implications of the findings This study clearly demonstrates that post-biopsy re-expansion pattern, obtained in a standardized approach, can be helpful for predicting live birth. It is a novel independent marker that is not reflected by ICM, TE quality or day of biopsy for selecting embryos for thawing and transfer. Trial registration number not applicable

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

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

P-588 Serum Luteinizing Hormone levels before progesterone supplementation are significantly associated with live birth rate in single euploid frozen embryo transfers performed in hormonal replacement cycles

Abstract Study question Do luteinizing hormone (LH) levels measured prior to initiating progesterone supplementation influence live birth rate (LBR) in artificial frozen-thawed single euploid blastocyst transfer cycles? Summary answer Serum LH levels, measured prior to progesterone initiation in artificial frozen-thawed single euploid blastocyst transfer cycles, are significantly associated with LBR. What is known already In the absence of a dominant follicle, estrogen supplementation in artificial endometrial preparation for frozen embryo transfer (FRET) can lead to a rise in endogenous LH levels, as observed in a natural cycle. Since the LH rise is a key feature in natural conception and the embryo, the endometrium, and the myometrium display receptors for LH, the rise observed during FRET preparation may have an impact on the clinical outcomes of the treatment. Previous studies found contradictory results hence none of them excluded embryo aneuploidy by implementation of preimplantation genetic testing for aneuploidies and the transfer of euploid embryos. Study design, size, duration Retrospective analysis of 639 frozen-thawed single euploid blastocyst transfer cycles performed in a tertiary referral IVF center from January 2018 to August 2023. Cycles with hormonal pre-treatment or pituitary suppression were excluded. Participants/materials, setting, methods Single euploid FRET cycles were included. Oral estradiol administration commenced on cycle-day 2/3. Estradiol, progesterone, and LH were measured at the start of endometrial preparation and up to 3 days before progesterone initiation, and the slope of LH was calculated. Patients were stratified in groups according to their LH values before progesterone administration, and the impact of LH on LBR and implantation rate was assessed. Logistic regression analysis was performed to adjust for potential confounders. Main results and the role of chance In total 639 cycles were included. Patients’ characteristics (mean+/-SD) were: age 33.2±5.9 years; BMI 27.5±4.9 kg/m2. The median duration of estrogen administration was 15 days (range: 11-27 days). The overall implantation rate and LBR were 65.9% (421/639) and 46.9% (300/639), respectively. Patients were stratified into six groups according to their LH levels prior to progesterone supplementation: <10th percentile (≤6mIU/ml, n = 57), p10th-p25th (>6 to ≤ 8.5mIU/ml, n = 81), p25th-p50th (>8.5 to ≤ 12.4mIU/ml, n = 176), p50th-p75th (>12.4 to ≤ 18.1mIU/ml, n = 180), p75th-p90th (>18.1 to ≤ 25.0mIU/ml, n = 91) and >p90th (>25.0mIU/ml, n = 54). A significant trend for higher LBR with increasing serum LH levels was observed (Cochran-Armitage Test, P = 0.041) but not for implantation rate (P = 0.158). Those with higher basal serum estradiol levels before estrogen supplementation had a steeper LH increase from basal to progesterone-supplementation day (0.40 mIU/mL higher per 10pmol/L increase in basal estradiol, P = 0.027). Regression analysis identified as independent predictors of LBR BMI (OR 0.96, 95%CI: 0.93-0.99) and embryo quality (CC vs. AA p < 0.001, CB vs. AA p = 0.027, BC vs. AA p = 0.013). Patients with steeper LH increases from estrogen to progesterone-supplementation day had higher LBR in both univariable (OR:1.27, 95%CI: 1.00–1.63, P = 0.052) and multivariable analyses (OR:1.26, 95%CI: 0.97–1.64, P = 0.085) but the difference didn’t reach statistical significance. Limitations, reasons for caution The retrospective design of the study is a limitation. Results should be interpreted with caution due to the small number of patients in the lowest and highest LH percentile groups. Wider implications of the findings The results of this study suggest that serum LH levels measured before progesterone supplementation may have an impact on the clinical outcome of artificial frozen-thawed blastocyst transfer cycles. Further studies with a higher sample size are necessary to confirm or refute our findings. Trial registration number not applicable

The Impact of Embryo Quality on Pregnancy Outcomes in Single Day 5 versus Day 6 Euploid Blastocyst Transfer: A Retrospective Cohort Study.

Selecting embryos with the highest implantation potential is crucial for in vitro fertilization (IVF) success. Both the timing of blastulation, day 5 (D5) or D6, and the embryo quality have been suggested as influential factors in determining the clinical outcome of single euploid blastocyst transfers. However, evidence supporting the superiority of D5 over D6 blastocysts remains inconclusive. The aim of this study was to compare clinical outcomes following the transfer of euploid blastocysts with different quality and timing of blastulation. A retrospective cohort study was conducted at our Assisted Reproductive Center, analyzing the outcome of 774 transfers with D5 euploids and 155 transfers with D6 euploids performed between January 2019 and February 2022. The live birth rate was significantly lower in the euploid D6 group compared to the euploid D5 group (38.71vs. 55.04%, P=0.001). The outcome was significantly influenced by the quality of the embryos. Live birth rates were 62.14 and 53.61% following transfers of D5 and D6 excellent embryos respectively, 45.18 and 32.21% following transfer of D5 and D6 good embryos but only 28.64 and 19.32% following transfer of D5 and D6 fair embryos. The outcome difference was statistically significant across embryo quality categories (P=0.001). The adjusted risk ratios (RR) of clinical outcomes indicated that excellent euploid D5 embryos consistently outperformed other types of embryo quality. The timing of blastulation and embryo quality are crucial factors in determining the success of single euploid blastocyst transfers. Excellent euploid D5 transfers yielded superior clinical outcomes, providing valuable insights for IVF teams and patients when selecting embryos to be transferred.

Impact of double trophectoderm biopsy on reproductive outcomes following single euploid blastocyst transfer

ObjectivesTo study the effect of double trophectoderm biopsy on clinical outcomes following single euploid blastocyst transfer. Study designRetrospective cohort study of 2046 single euploid frozen-thawed blastocyst transfers from January 2015 to June 2022 in a single centre.All patients undergoing a frozen-thawed embryo transfer (FTET) cycle with euploid blastocysts, biopsied for any indication, were included. The outcomes were compared for blastocysts which were biopsied and vitrified once (Group 1, n = 1684), biopsied once but vitrified twice (Group 2, n = 312) and biopsied and vitrified twice (Group 3n = 50). We adjusted for confounders and performed subgroup analysis for PGT-A, PGT-M and PGT-SR cycles. The primary outcome was live birth rate. Secondary outcomes included pregnancy, clinical pregnancy, birthweight and sex ratio. ResultsAfter adjusting for confounders (previous failed euploid implantations, embryo quality and day of biopsy), embryos which were biopsied twice had lower OR for clinical pregnancy (0.48, CI 0.26–0.88, p = 0.019) and for live birth (0.50 CI 0.27–0.92, p = 0.025) compared to controls. Embryos which were biopsied once but vitrified twice had no different ORs for all reproductive outcomes compared to controls. No significant difference was observed for neonatal birthweight or sex ratio amongst the three groups. This is a retrospective single centre study with inherent bias and results may not be transferable to all settings. ConclusionThis study is the largest to date assessing the outcomes of FTET cycles following double trophectoderm biopsy. The results are in keeping with the existing literature and can be incorporated into patient counselling. Whilst double biopsy seems to adversely impact LBR, it is only one of the many factors that can affect success rates. The subfertility background and embryo characteristics should not be overlooked. This study provides reassuring evidence since double biopsied embryos still result in live births with no difference in sex ratio or birthweight. However, long term follow up of the off-springs is lacking and should be reported in future studies.

Female obesity increases the risk of preterm birth of single frozen-thawed euploid embryos: a retrospective cohort study.

Obesity has been associated with an increased risk of reproductive failure, especially preterm birth. As preimplantation genetic testing for aneuploidies (PGT-A) is increasingly used worldwide, however, it is still unclear whether body mass index (BMI) has an effect on the preterm birth rate in patients undergoing in vitro fertilization (IVF) with PGT-A when transferring a single euploid blastocyst. This retrospective, single-center cohort study included 851 women who underwent the first cycle of frozen-thawed single euploid blastocyst transfer with PGT-A between 2015 and 2020. The primary outcome was the preterm birth rate. Secondary outcomes were clinical pregnancy, miscarriage, ectopic pregnancy, pregnancy complications, and live birth. Patients were grouped by World Health Organization (WHO) BMI class: underweight (<18.5, n = 81), normal weight (18.5-24.9, n = 637), overweight (25-30, n = 108), and obese (≥30, n = 25). There was no difference in the clinical pregnancy, miscarriage, ectopic pregnancy, pregnancy complication, and live birth by BMI category. In multivariate logistic regression analysis, preterm birth rates were significantly higher in women with overweight (adjusted odds ratio [aOR] 3.18; 95% confidence interval [CI], 1.29-7.80, p = .012) and obese (aOR 1.49; 95% CI, 1.03-12.78, p = .027) compared with the normal weight reference group. Women with obesity experience a higher rate of preterm birth after euploid embryo transfer than women with a normal weight, suggesting that the negative impact of obesity on IVF and clinical outcomes may be related to other mechanisms than aneuploidy.

Management strategies following implantation failure of euploid embryos.

Euploid blastocyst implantation failure may result from embryonic factors undetectable by preimplantation genetic testing for aneuploidy (PGT-A); however, various nonembryonic factors can also intricately interfere with implantation. This review seeks to clarify evidence-based testing and treatments for implantation failure after euploid embryo transfer. We conducted a review of the literature on implantation failure after euploid embryo transfer or multiple embryo transfer cycles, which mainly included systematic reviews and meta-analyses. The recommended tests for implantation failure include (1) hysteroscopy, (2) endometrial CD138 immunohistochemistry and bacterial culture, (3) serum 25-hydroxyvitamin D3, and (4) thrombophilia screening. Based on diagnostic findings, the following treatments have been recommended: (1) antibiotics for chronic endometritis, (2) vitamin D replacement, (3) lifestyle modification, and (4) low-dose aspirin starting from the postimplantation period for thrombophilia. Moreover, frozen-thawed single euploid blastocyst transfer using assisted hatching and hyaluronan-enriched transfer medium may support embryo implantation. To ensure a successful pregnancy in subsequent embryo transfers, simple, inexpensive, and evidence-based tests and treatments should be selected.

#105 : Single Day 5 Versus Day 6 Euploid Blastocyst Transfer: The Impact of Embryo Quality on Pregnancy Outcomes

Background and Aims: Selecting embryos with the highest implantation potential is crucial to in vitro fertilization (IVF) success. The day of biopsy, Day 5 (D5) or Day 6 (D6), and embryo quality have been suggested as influential factors in determining the clinical outcomes of single euploid blastocyst transfers. However, evidence supporting the superiority of D5 over D6 blastocysts remains inconclusive. This study aims to compare clinical outcomes following transfer of euploid blastocysts of varying quality biopsied on day 5 versus day 6. Methods: A retrospective cohort study was conducted at an Assisted Reproductive Center, analyzing 774 patients with Day 5 biopsies and 155 patients with Day 6 biopsies who underwent embryo transfer between January 2019 and February 2022. Results: The live birth rate was significantly lower in the euploid D6 group than in the euploid D5 group (38.71% vs. 55.04%, RR=0.70, 95% CI=0.57-0.87). The outcomes were significantly influenced by the quality of the embryos. Excellent embryos yielded live birth rates of 62.14% and 53.61% for D5 and D6 biopsies, respectively, while good embryos demonstrated live birth rates of 45.18% (D5) and 32.21% (D6). For fair embryos, the live birth rates were 28.64% (D5) and 19.32% (D6). The outcome difference was statistically significant across embryo quality categories (p&lt;0.05). The adjusted risk ratios of clinical outcomes indicated that excellent euploid Day 5 embryos consistently outperformed other types of embryos. Conclusions: Our study supports that the biopsy day and embryo quality are crucial factors in determining the success of single euploid blastocyst transfers. Excellent euploid Day 5 transfers yielded superior outcomes, providing valuable insights for clinicians and patients when selecting embryos for transfer in IVF cycles.

Effects of embryo characteristics in frozen-thawed single euploid blastocyst transfers on pregnancy outcomes: a retrospective cohort study

Objectives: Our study examined the effects of the trophectoderm biopsy (TB) day and the presence of necrotic foci (NF) or separate blastomeres (SB) within euploid embryos on in vitro fertilization (IVF) pregnancy outcomes. Methods: This retrospective cohort study was conducted from January 2017 to September 2021 at Memorial Sisli Hospital, Istanbul, Turkey. The study comprised a total of 2758 frozen-thawed euploid embryo transfer cycles. After thawing, blastocysts were graded using Gardner's classification Top-Quality (TQ), Good-Quality (GQ), Moderate-Quality (MQ), Poor-Quality (PQ) and further divided into groups according to the presence of NF and/or SB and evaluated for pregnancy outcomes. Results: There were significant correlations between pregnancy outcomes and the degree of blastocoele expansion, as well as the presence of NF or SB in the euploid embryo. Ongoing pregnancy rates were lower in the group with NF in the inner cell mass (ICM) or trophectoderm (TE) than in the group without NF. The presence of SB decreased the rates of ongoing pregnancy and increased the rates of miscarriage. Embryos with expansion grades ≤ 3 had lower rates of ongoing pregnancy and higher rates of miscarriage compared to embryos with expansion grades&amp;gt; 3. TQ and GQ embryos had a higher rate of ongoing pregnancy and a lower rate of miscarriage than MQ and PQ embryos. Conclusions: When selecting the embryo to be transferred to a patient, careful consideration should be given to the morphological grade of the embryo as well as whether or not it contains NF and SB.

Effect of the Endometrial Thickness on the Live Birth Rate: Insights From 959 Single Euploid Frozen Embryo Transfers Without a Cutoff For Thickness

ABSTRACT Although numerous studies have examined whether endometrial thickness (ET) independently affects the live birth rate (LBR) after an embryo transfer, contradictory conclusions have resulted in an unclear answer. It has been hypothesized that a thin endometrium decreases implantation rates possibly due to elevated oxygen concentration from spiral arteries; however, this mechanism has not been unequivocally demonstrated. This retrospective analysis aimed to investigate whether ET independently affects the LBR after a frozen embryo transfer cycle. All patients who underwent a single euploid blastocyst transfer between March 2017 and March 2020 at a single-assisted reproductive technology clinic were included. Live birth was defined as the delivery of a live fetus after 22 weeks of gestation. Programmed cycle (PC) or natural cycle (NC) for endometrial preparation was conducted at physician’s and patient’s discretion. The presence of a linear relationship between ET and LBR was assessed by conditional density plots (CDPs), and receiver operating characteristics (ROC) curve analyses were used to identify whether a threshold of the ET existed to predict the occurrence of live birth. The CDPs were analyzed for an optimal range of the ET that could be associated with a higher LBR, and the distribution of cycle characteristics and embryo quality in that range were compared with cycles outside of that range to identify confounders. Logistic regression models were constructed separately for the PC and NC. A total of 959 single euploid FETs were included in this analysis, of which 33% (n = 315) were NC and 67% (n = 644) were PC with an overall LBR of 47.1% (452/959). No linear relationship between the ET and LBR or threshold below which the LBR decreased perceivably was identified. In addition, ROC analysis did not suggest a predictive value of the ET for occurrence of live birth based on endometrial preparation or in the overall cohort. Similarly, logistic regression analyses showed no independent effect of the ET on LBR. The results of this study suggest that there is no linear relationship between the ET and LBR or a clinically significant cutoff for ET below which the LBR decreases in FET cycles. Significantly, the common practice of canceling embryo transfers when the ET is &lt;7 mm is not supported by these data.

P-244 Nucleation errors at four-cell stage reduces chances of live births in single euploid-blastocyst transfers

Abstract Study question What is the impact of nucleation errors (NE) and morphokinetic parameters on live birth rates in single euploid blastocyst transfers? Summary answer Nucleation errors (NE) at the four-cell stage was the only parameter associated with decreased live birth (LB) rates. What is known already Morphokinetic studies using time-lapse monitoring (TLM) systems have proposed algorithms to select embryos with the highest implantation potential. However, no single biomarker can reliably predict live-birth so far. Morphokinetics can deselect embryos for Preimplantation-Genetic-Testing for Aneuploidies (PGT-A). However, it is unclear whether morphokinetic markers can be used for selecting embryos with highest chance of LB among the euploid blastocysts. Performing morphokinetics enable detection of features such as nuclear errors (NE) which could be underdiagnosed with intermittent evaluation. It remains to be determined to which extend NE, next to morphokinetic parameters, are associated with LB potential of euploid blastocysts. Study design, size, duration Retrospective observational study including 383 single euploid blastocyst transfers in FET cycles between October 2017 to June 2021. These blastocysts were graded ≥BL3CC (Gardner criteria) and underwent TE biopsy for PGT-A by Next Generation Sequencing (NGS). Only ICSI with fresh autologous cycles using ejaculated sperm were considered. Outcomes of euploid blastocyst transfers were recorded and as pregnant (P): biochemical pregnancy (BP) (n = 17), clinical miscarriage (CM) (n = 35), live birth (LB) (n = 205); or not pregnant (NP) (n = 126). Participants/materials, setting, methods Patient baseline characteristics (Age, AMH, BMI) and infertility duration were considered. Morphokinetics were annotated with time-lapse-imaging (KID-score) for time (t) of PB2 (tPB2), tPNa (PN-appearance), tPNf (PN-fading), t2-t9, CP (compaction), tM (Morula), tSB (start blastulation), tB (blastocyst) and timings between each developmental event. Nuclear errors (NE) such as micronucleation, binucleation, multinucleation were annotated when present in at least one blastomere at two- and four-cell stages between 19.4-51.4 hours and 22.1-63.1 hours, respectively, post-insemination. Main results and the role of chance Median (IQR) values of Age and AMH of female partner, and infertility duration were similar among the P and NP groups. BMI was significantly higher in patients who miscarried with Median (IQR) values of 28.4kg/m2 (26.1-33.4) vs &amp;lt; 27.9kg/m2 in all other groups (P &amp;lt; 0.01). Significantly more blastocysts with type A inner cell mass quality resulted in LB (24.9%) than to CM (14.3%), BP (5.9%) and NP (13.5%) (P &amp;lt; 0.001). Concerning TLM morphokinetics evaluation, none of the timed parameters evaluated, neither the timings between the different morphokinetics events resulted in significant differences among the LB and the other P and NP groups. However, LB group had significantly less euploid embryos presenting NE at four-cell stage (7.8%) compared to CM (17.0%), BP (23.5%) and NP (17.5%) (P = 0.02). In a logistic regression model, NE when present at four-cell stage but not at two-cell stage, had a negative impact in LB rates (OR:0.39, CI: 0.2-0.72; P = 0.003). Limitations, reasons for caution The retrospective nature of the study and the fact that NE evaluation was performed manually based on TLM videos by a single observer. Wider implications of the findings NE-constituted embryos can give rise to euploid blastocysts and have an implantation potential. Since these embryos are associated with lower LB, it is relevant to include NE as an evaluation parameter to deprioritize multinucleated four-cell embryos for transfer, even when they result in euploid blastocysts. Trial registration number not applicable

P-725 Role of Non-Invasive Preimplantation Genetic Testing- Aneuploidy (NIPGT-A) using spent culture media (SCM) and its concordance with Trophoectoderm (TE) biopsy: A Prospective Cohort Study

Abstract Study question What is the ploidy concordance rate between spent culture media (SCM) and trophectoderm biopsy (TE) using Next Generation Sequencing (NGS) and correlation with clinical outcome? Summary answer DNA could be isolated from SCM with successful NGS library sequencing. Ploidy and per-chromosome concordance rate of TE biopsy vs SCM was 68.38% and 85.13%. What is known already Many challenges are associated with TE biopsy, and the possibility of NiPGT-A using cell free DNA (cfDNA) from SCM is very intriguing, as it will be simpler, safer and cheaper alternative to the gold standard TE biopsy. The bottlenecks associated with TE biopsy like additional equipment and trained embryologist, can be substantially reduced. The disparities in reported results in various studies comparing SCM vs TE, could be attributed to culture media contamination (maternal, paternal or environment), different laboratory workflow methodologies used for embryo culture, different whole genome amplification methods, library sequencing methods and different algorithms for analysis. Study design, size, duration Prospective cohort study was conducted in tertiary care centre from August 2021-June 2022. Ethical Approval was taken from Institute Ethics Committee. Couples with female age ≥ 35 years, one or more implantation failures, male factor infertility requiring ICSI, opting for elective single euploid blastocyst transfer, were included in study. Forty four blastocysts were obtained from 14 patients who underwent IVF/ICSI cycles, gave consent for TE biopsy and SCM collection for PGT-A and NiPGT-A respectively. Participants/materials, setting, methods Individualised ovarian stimulation was carried out with GnRH antagonist protocol. ICSI was done for fertilization. Embryos were cultured in sequential medium. On day 3, after thorough washing embryo was cultured separately in 10μl micro-droplets. SCM was collected on day-5, before biopsy. DNA extraction, amplification and library preparation from TE biopsy and SCM was done using Veriseq PGS kit (Illumina) and sequencing was done on Illumina MiSeq system. Euploid embryo transfer was done in FET cycle Main results and the role of chance Out of 44 blastocysts, 31 were day 5 and 13 were day 6. WGA-DNA from TE biopsy and SCM was 100% successful. Average concentration of amplified DNA obtained from TE biopsy and SCM was 34.88 ± 9.56 ng/µl, 32.3 ± 6.84 ng/µl respectively. Quality control parameters for library preparation and sequencing for samples were as per recommended standards. CNV visualization and analysis was done using BlueFuse Multi-Software (Illumina). Ploidy concordance rate could be analysed in 26 embryos, which was 68.38%, per chromosome concordance was 85.13% and sex-chromosome concordance was 73.0%. The sensitivity, specificity, PPV, NPV and diagnostic accuracy of NiPGT-A was 66.6%, 60%, 87.5%, 30% and 65.38% respectively. On comparing day 5 and day 6 blastocysts, day 6 had better concordance rate 72.7% vs 60%, p &amp;gt; 0.05. On comparing good (&amp;gt;BB) and poor morphology (&amp;lt;BB)embryos, &amp;lt;BB had better concordance rate 83.33% vs 50%, p = 0.16. Per chromosome concordance rate was higher for &amp;lt;BB embryos, 90.5% vs 80.5%,p= 0.001. Full maternal cell contamination was suspected when TE was aneuploid/mosaic, XY and SCM was euploid, XX (n = 4/14), assessed only in XY embryos. Single euploid blastocyst transfer had 66.6%clinical pregnancy rate. One resulted in healthy live-birth, two on-going pregnancies, 30weeks and 12weeks period of gestation. Limitations, reasons for caution Before considering NiPGT-A in routine clinical practice, each embryology lab needs standardization, to implement necessary modifications in routine embryology protocol. Labs should ensure that embryo viability is not affected and should try to minimize chances of maternal or external contamination in SCM to reliably predict concordance rates with high confidence. Wider implications of the findings NiPGT-A is useful technique to assess ploidy but presently, NiPGT-A in combination with PGT-A, can help in prioritizing embryos according to their implantation potential. Further studies are required to find out whether NiPGT-A may serve as an alternative to PGT-A and implemented alone in routine clinical practice in future. Trial registration number CTRI/2021/04/033121