Blastocyst Day Research Articles

Comparison of live birth rates following the transfer of day-6 blastocysts on the 6th versus 7th day of progesterone exposure in hormone replacement treatment–frozen embryo transfer cycles

Objective: The duration of progesterone exposure is critical for establishing and maintaining a pregnancy in hormone replacement treatment (HRT)–frozen embryo transfer (FET) cycles. This study compared the live birth rates (LBRs) of patients undergoing day-6 warmed blastocyst transfer on either the 6th or 7th day of progesterone administration in HRT-FET cycles.Methods: A retrospective cohort study was conducted using data from the central registry of Indira IVF Hospital Private Limited. In total, 2,633 patients who underwent day-6 blastocyst transfer cycles with HRT-FET were identified. The cycles were categorized based on the timing of the day-6 blastocysts transfer: P+6 vs. P+7 (initiation date: P+1). Propensity scores were calculated. The primary outcome measure was the LBR.Results: Following propensity score matching, a total of 1,401 patients were included in the final analysis (P+6: n=1,212; P+7: n=189). The number of previous attempts was significantly higher in the P+6 group, whereas the rate of preimplantation genetic testing for aneuploidy was significantly higher in the P+7 group. The LBRs were 59.2% and 54.5% in the P+6 and P+7 groups, respectively (p=0.21). Multivariate regression analysis revealed that the number of previous in vitro fertilization attempts (odds ratio [OR], 3.246; 95% confidence interval [CI], 2.429 to 4.337; p<0.001), the number of blastocysts transferred (OR, 2.011; 95% CI, 1.461 to 2.768; p<0.01), and endometrial thickness on the day of progesterone initiation (OR, 1.143; 95% CI, 1.022 to 1.28; p=0.02) were independent predictors of live birth.Conclusions: The LBRs were comparable in patients who underwent day-6 warmed blastocyst transfer on day 6 or 7 post-progesterone initiation in HRT-FET cycles.

P-131 Let’s not forget the day-6 blastocysts: what is the clinical pregnancy prediction of day-6 blastocysts from a commercial software for day-5 embryo selection?

Abstract Study question How is the performance of KIDScoreTM D5 version 3 for clinical pregnancy prediction in day 6 blastocysts? Summary answer Differing on Day 5 performance, KIDScoreTM D5 algorithm does not predict clinical pregnancy for day 6 blastocysts according to score subgroup grades. What is known already Embryo selection is a crucial step for a successful outcome in assisted reproductive technologies (ART). In the past years, the use of software and algorithms developed with artificial intelligence (A.I.) tools brought new parameters to be considered by the embryologists in embryo decision at the ART lab. KIDScoreTM is a commercial software (Vitrolife®, Sweden) developed and validated with a large database of known clinical outcome embryos transferred on day 3 or day 5 of development. Yet, it is unknown the performance of KIDScore D5 v3 on day 6 blastocysts, which are widely used for embryo transfer. Study design, size, duration Large retrospective cohort study including sequential single embryo transfers with blastocysts submitted or not to trophoectoderm biopsy for PGT-A, cultured in the Embryoscope® Plus incubator (Vitrolife®) in a single private IVF center between Jan/2020 and Jul/2023. Positive or negative clinical pregnancy, CP (presence/absence fetal heartbeat and gestational sac) from 738 patients/771 blastocysts developed on day 5 or day 6 were considered. Biochemical pregnancy and miscarriage were excluded from this analysis. Participants/materials, setting, methods Day 5 and day 6 embryo transfer outcomes data were compared considering three KIDScoreTM subgroups, according to the following score intervals: subgroup 1: 1.0-3.9 (n = 102), subgroup 2: 4.0-6.9 (n = 273) and subgroup 3: 7.0-9.9 (n = 396 blastocysts). Euploid (n = 517) and non-biopsied embryos (n = 254) were also analyzed in the described subgroups. For the analysis, Mann-Whitney, Chi-square and Fisher tests were used properly for statistical analysis, values of p < 0.05 were considered significant. Main results and the role of chance Maternal age were similar between positive and negative pregnancies in D5 and D6 transfers (D5:39,32±4,34 vs 39,02±4,47 and D6:38,75±3,99 vs 38,18±3,76, p = 0,35 and 0,28 respectively). In D5 transfers, CP rates significantly increased in higher score subgroups [subgroup 1:42.9% (6/14); subgroup 2:48.9% (86/176); subgroup 3:61.6% (236/383),p=0.01]. As expected, subgroup 3 showed a higher CP rate when compared to subgroup 1 + 2 (scores from 1.0 to 6.9,p=0,003). However, in D6 transfers, CP rates were similar between score subgroups 2 and 3 and lower in group 1 [subgroup 1:28.4% (25/88); subgroup 2:50.5% (49/87); subgroup 3:53.8% (7/13),p=0.005 and p = 0,001 between subgroup 1 and subgroups 2 + 3]. Similar results were seen in euploid and non-biopsied embryo transfers; in D5, CP rates were increased in higher scores subgroup (7.0-9.9) when compared to subgroups 1 + 2 (euploid:61,6% vs 42,9% p = 0,05, and non-biopsied:61,3% vs 42,6%,p=0,01 for positive and negative CP respectively in subgroup 3 vs subgroup 1 + 2). And in D6 embryo transfers CP rates were lower in subgroup 1 (1.0-3.9) and similar in subgroups 2 and 3 for both euploid and non-biopsied embryos (euploid:31,5% vs 49,4% p = 0,02, and non-biopsied:13,6% vs 56,5%,p=0,007 for positive and negative CP respectively in subgroup 1 vs subgroup 2 + 3, respectively). Limitations, reasons for caution The KIDScoreTM D5 v3 algorithm is validated for blastocyst on day 5 of development; the results showed here recognizes the limitations of evaluate the potential of CP prediction on Day 6 blastocysts and the score were not considered for embryo selection decision during the time of study. Wider implications of the findings The KIDScoreTM is effective to predict the potential of clinical pregnancy in blastocysts on day 5 of development, with higher rates on higher score subgroups, but not for blastocysts on day 6 of development, where the embryos had similar rates with score greater than 4.0. 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 (<120, 120-132, 132-144, 144-156, 156-168, and >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.

P-243 Effect of delayed development of embryos 7 days on embryo quality, ploidy and reproductive cycle outcomes

Abstract Study question How does delay in embryo development before Day7 affect blastocyst quality, ploidy and reproductive cycle outcomes? Summary answer Embryo quality and ploidy are statistically significantly lower in the Day7 group, no difference in implantation rate was found. What is known already In clinics that allow extended culture up to Day7, on average 5% of embryos have a 48-hour delay in development, blastulation occurs on the 7th Day (Hammond et al., 2018). Day7 blastocysts may have good morphology and be euploid, give a pregnancy and birth of a healthy baby. Back in 2013 it has been proven that embryos from patients over 35 years of age take longer to form a blastocyst (Shapiro et al., 2013). Cultivation until Day 7 is justified for couples with a poor prognosis in cycles. Study design, size, duration This was a retrospective cohort study in a private Delta Fertility Clinic of consecutive female infertility patients (n = 422) between Sept 2021 and Sept 2022. Average age of the patients was 39,7 years who underwent frozen embryo transfer (FET) of a single euploid day 5, 6 or 7 blastocyst. Then we compared the outcomes in the 5, 6 and 7 Day groups. Participants/materials, setting, methods The results of culture and subsequent biopsy of trophoectoderm with PGT-A of 2095 blastocysts (1516 were tested with PGT-A) in 482 cycles were analyzed, of these, embryos Day7 suitable for biopsy were 9.8% (149). Blastocysts were classified as good (AA/AB/BA/BB) or poor (AC/BC/CA/CB) (Gardner D.K. et al., 1999). Cultivation performed in single drops, biopsy was performed using the flicking method. Statistical analysis was performed using the Pearson’s Chi-Squared Test. Main results and the role of chance In 3.4% of cases, patients had only Day7 blastocysts. Poor quality blastocysts were predominant in D7 blastocyst group (good quality: 91.0%, 65.2%, 46.3%; poor quality: 9.0%, 34.8%, 53.7%, P < 0.001 for D5, D6 and D7 blastocysts; respectively). The number of euploid embryos was significantly lower in the Day 6 and Day 7 group compared to Day 5 (51.8% and 50.0% vs 64.4%, p < 0.001 and p < 0.001). We found no differences in cycle outcomes, implantation rate was 52,7%, 49,6% and 38,5% for 5, 6 and 7 Day respectively (P = 0,474). We speculate that this may be due to the small number of day 7 embryo transfers (n = 13), but we have 5 live births in our clinic after euploid Day7 embryo transfer. Limitations, reasons for caution Due to the decrease in the percentage of euploid embryos on day 7, such embryos should be transferred only after PGT-A. There is little information about the outcomes of Day 7 embryo transfers because they are the last embryos to be transferred. Wider implications of the findings Cultivation before day 7 should be introduced into routine practice as an option for patients over 35 years of age or with a known poor prognosis. Having previously assessed the laboratory’s capabilities and financial costs. Trial registration number not applicable

P-511 Euploidy potential of day 7 embryos and the relevance in Assisted Human Reproduction treatments

Abstract Study question Is it worth waiting embryos with late blastulation (Day 7) for preimplantation genetic analysis? Summary answer Embryo biopsy on Day 7 (D7) benefits cases of ovarian insufficiency and severe male factor with a euploidy rate of 15.1%. What is known already New technologies and improvements in IVF laboratories has allowed the extended cultivation of blastocysts to increase the pool of embryos per IVF cycle, resulting in greater chances, especially in cases of low blastulation rate, blastocysts with low quality or with few cells at Day 5(D5) and Day 6(D6). The variation in the time of the compaction stage depends on many factors, which may take more time, leading some embryos to reach the blastocyst stage only on D7 of development. These embryos have a lower euploidy rate, but there is embryonic competence and these embryos can be eligible for embryo transfer. Study design, size, duration Retrospective study that evaluated the chromosomal status (by Next Generation Screening - NGS) of 33 blastocysts biopsied on day 7 of development, between 2016 and 2023. Subsequently, it was compared the rate of euploidy and mosaicism with 295 blastocysts biopsied at D5 and D6, biopsied from January to October 2023. D7 embryos were originated from IVF cycles of couples with female age between 26 and 44 years, and males age between 29 and 56 years. Participants/materials, setting, methods D7 blastocysts from fresh and own oocytes fertilized by ICSI. The euploidy and mosaicism rates was compared, as well as the morphology of the ICM and trophectoderm (A-C; by Gardner Blastocyst Grade), the day which biopsy was performed and the respective infertility factors, in the groups: Group 1: D7 euploid and Group 2: D7 aneuploid. Subsequently, the rate of euploidy and mosaicism was compared between D7 embryos (Group 3) and D5/D6 embryos (Group 4). Main results and the role of chance There was no difference (p > 0.05) between maternal age for group D7 euploid (38.4 years) vs group D7 aneuploid (37.9 years) and group 3 (38.4 years) vs group 4 (38.2 years). Embryonic morphological evaluation of D7 embryos showed that ICM was classified as B (18.2%) or C (81.8%) and trophectoderm as A (6.1%), B (24.2%) or C (69.7%). Comparisons of ICM and trophectoderm classifications between euploid (group 1) and aneuploid (group 2) in D7 blastocysts showed no difference (p > 0.05). There was a predominance of 5CC embryos in both groups (Group 1: 40.0% vs Group 2: 42.4%), with no difference between them (p > 0.05). In most cases, the biopsy was performed on two days (D5+D7 – 39.3%; D6+D7 – 35.7%), while in 10.7% of cases only on Day 7, when the infertility factor was 100% ovarian insufficiency. In cases of biopsy within 3 days (D5+D6+D7), we observed that 100% of cases were related to severe male factor. When comparing the euploidy rate of D7 embryos – group 3 (15.1%) with that of D5/D6 – group 4 (34.2%), we observed a statistical difference (p < 0.05), while, for in mosaicism rates, D7 embryos represented 6.1% and D5/D6 embryos 9.1%, with no statistical difference (p > 0.05). Limitations, reasons for caution The retrospective nature of our study may lead to bias derived from clinical practices and variability in relation to patient age, embryonic quality and infertility factor. Wider implications of the findings Extended culture and biopsy on D7 benefits cases of ovarian insufficiency and severe male factor by optimizing the IVF cycle. Although the euploidy rate in D7 embryos is lower than D5/D6 embryos, 15% of D7 embryos are euploid, demonstrating that the slower development is not caused exclusively by aneuploidy Trial registration number NA

O-298 Embryo long-term storage does not affect ART outcome: analysis of 58,001 vitrified blastocysts

Abstract Study question Does duration of storage affect clinical outcome in frozen embryo transfer (FET) cycles? Summary answer Long-term storage does not affect live birth rate (LBR) in elective and non-elective FET cycles. What is known already Despite the success rates provided by the embryo vitrification programs, some studies found an association between the duration of cryo-storage and decreased clinical outcome, raising concern about the potential negative effect that prolonged storage may have on embryos. Study design, size, duration This was a retrospective multicenter study including a total of 58001 vitrified/warmed day-5 blastocysts from two different populations according to the reason for FET: elective FET which involved patients undergoing freeze all cycles (N = 16,615 blastocysts and 16,615 patients); and non-elective FET including 41,386 embryos from 25,571 patients. FETs were performed in the period between January 2010 and December 2020. Participants/materials, setting, methods Main outcome was live birth rate (LBR) and secondary outcomes were embryo survival, miscarriage, clinical and ongoing pregnancy rates. The impact of storage time and the potential confounding variables was assessed by univariate analyses. Those covariables found to significantly modify the LBR were included in a multivariable logistic regression. GEE (generalized estimated equation) was performed to allow the analysis of repeated measurements. Odds ratios (OR) and 95% confidence intervals (CI) were provided. Main results and the role of chance The blastocysts sample was clustered into deciles which provided specific storage duration categories that ranked from ≤0.67 to ≥ 4.34 and ≤1.8 to ≥ 34.81 months in freeze-all and non-elective FET, respectively. In the freeze-all group, maximum storage time was 34.8 months (2.9 years), while it was 121.2 months (10.1 years) in the non-elective FET group. Embryo survival did not show statistical differences across the categories of storage time in freeze-all and non-elective FET groups. In the freeze-all group, statistical differences were found for LBR across some, but not all the subgroups of storage duration. Similarly, in the non-elective FET group, LBR among the deciles showed statistical differences in some comparisons but not in all. However, after adjusting for confounding variables, the multivariate analysis showed no association between storage time and LBR in both groups. Blastocyst quality, body mass index (BMI), number of retrieved oocytes, endometrial preparation, male factor and uterine factor were related to the drop in the LBR in the freeze-all group (p < 0.05). In the non-elective FET group, the variables that showed significant association with the LBR were age at retrieval and at FET, number of embryos transferred, number of retrieved oocytes, BMI, endometrial preparation, sperm origin and female factor. Limitations, reasons for caution The retrospective design of the study represents its main limitation. Wider implications of the findings Our results demonstrate the safety of long-term cryo-storage. This is reassuring for both IVF practitioners and patients especially considering the exponential increase in FET cycles observed in the last decades. Trial registration number Not applicable

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)

P-138 Effect of biofeedback from the blastocysts on the cumulative pregnancy rate

Abstract Study question The study is to compare cumulative pregnancy rates of embryos transferred with and without spent culture media to confront biofeedback’s role in clinical outcomes Summary answer Transferring the blastocysts with spent culture media improved the pregnancy rates. Biofeedback was observed from the blastocyst stage. The blastocyst freezing and transfer approach improved. What is known already Biofeedback is often encountered in regulating menstrual cycles and other events in continuing gestations with estrogen, progesterone, beta hCG..etc. A similar biofeedback is possible with growing blastocyst for implantation. Since the in-vitro culture of embryos is devoid of its occurrence, it may lead to asynchrony of subsequent events and implantation. There is no scientific evidence from metabolomics on the characterization of these substances or secretions from spent culture medium that includes cell-free DNA giving feedback and its role in improving clinical outcomes. Study design, size, duration This retrospective multi-centric study included the 3591 frozen embryo transfers from 1st Apr 2022 to 31st Nov 2023 out of 4623 women who underwent oocyte retrieval from 1st Jan 2022. Patients were divided into study (group A) and control (group B). In group A, 1035 women received the transfers from spent culture media; in group B, 2556 women received transfers after shifting the blastocysts to fresh culture media before loading into the ET catheter. Participants/materials, setting, methods All the transfers considered in this study were day-5 blastocysts, irrespective of their frozen state. Day-5 frozen-thawed were incubated short-time (∼1 hour) before the transfer. Whereas, the frozen embryos and morulas were cultured till day 5. In the study group, all cultures were conducted in 100ul of culture media under oil per embryo to maintain consistency amongst the group. The rest of the laboratory and clinical procedures were consistent between the two groups. Main results and the role of chance The cumulative pregnancy rate was significantly higher in group A than in group B (60.9% Vs 55.6%; OR = 1.23; 95% CI, 1.07 -1.44, Z-score 2.901, P < 0.003). The effect of spent culture media was further observed by regrouping the day-3 or 4 frozen (group A1) and day-5 frozen patients (group A2). Day-5 thawed blastocysts transferred after short incubation to allow the secretions to directly deposit on the endometrium. Group A1 might have a high concentration of secretions due to its accumulation from the embryo or morula stages. Pregnancy rates in group A1 were higher than A2 but not statistically significant (61.7% Vs 59.7%; OR = 1.08, 95% CI, 0.84-1.4, Z-score 0.654, P = 0.51). The less impact indicates that the feedback came from blastocysts rather than embryos. Limitations, reasons for caution It is not evident how these secretions contribute to biofeedback. Quantification, identifying their time of release and recovery from the qualitative tests for their therapeutic use are the limitations. Nevertheless, spent media can be employed if found beneficial. Wider implications of the findings We have taken a large possible data set for control against the test group and took cumulative pregnancy rate as the primary outcome indicator to prove biofeedback from blastocysts can improve the clinical outcomes irrespective of the age and other factors of infertility. Trial registration number Not applicable

P-233 Embryo transfer timing and clinical pregnancy success: a retrospective study

Abstract Study question The aim of this study is to compare the clinical outcome of day5 versus day6 genetically normal blastocyst transfers. Summary answer 1000 oocytes were analyzed showing a highly significant difference in the number of normal embryos and clinical pregnancy rates in day5 versus day6 blastocysts What is known already studies have shown that day 5 embryos have a higher pregnancy rate compared to day 6 embryos, some studies had only euploid embryos transferred and others did not have any genetic testing done on embryos. No study had a sample size as large as 1760 and the number of embryos studied was a limiting factor in their results. Study design, size, duration The study is a retrospective cross-sectional chart review with multivariate analysis, data from January 2019 up to February 2023 were extracted. 1760 patients were recruited, 1000 in the day5 group and 760 in the day6 group. The data recorded for each patient included maternal age, body mass index (BMI), anti-Müllerian hormone (AMH) level, number, prenatal genetic diagnostic testing, frozen embryo transfer, sperm parameters, and pregnancy status. Participants/materials, setting, methods All embryos were genetically tested for aneuploidy before transfer, using next-generation sequencing technology to rule out 24-chromosome aneuploidies (1-22, X, Y). All patients who have single embryo transfer, frozen embryos and pre-implantation genetic diagnosis (PGD) were included in the study. Continuous variables were expressed as mean with standard deviation and categorical variables as numbers and frequencies (percentages). Statistical differences between groups were estimated using the chi-square or Fisher’s test. All analyses were performed using SAS studio. Main results and the role of chance 1,000 cycles were analyzed for Day 5 and 760 for Day 6 embryo. The number of blastocysts formed, and genetically diagnosed embryos significantly favor the Day 5 group. There’s a significant difference in the number of normal embryos, with the Day 5 group having more. There is a highly significant difference in clinical pregnancy rates. On the other hand, the differences in biochemical pregnancies are not statistically significant. The occurrence of ectopic pregnancies shows no significant difference. Additionally, there is no statistically significant difference in the rates of miscarriage. There is a highly significant difference in blastocyst formation rates, with Day 5 embryos. This indicates that Day 5 embryos have a significantly higher likelihood of forming blastocysts compared to Day 6 embryos. Implantation: Day 5 embryos have an implantation rate of 62.0% compared to 49.7% for Day 6 embryos. Day 5 embryos exhibit a significantly higher likelihood of successful implantation. Euploid: The difference in euploid rates is statistically significant, albeit with a lower p-value of 0.0236. Day 5 embryos have a rate of 59.6%, while Day 6 embryos have a rate of 56.0%. Day 5 embryos are associated with a lower likelihood of being aneuploid Limitations, reasons for caution Limitation of this study is that we do not have data available regarding complications during pregnancy in the day 5 versus day 6 groups and we had no data regarding the the live birth rate between the two groups. Wider implications of the findings This study has the potential to revolutionize worldwide discussions during IVF consultations. With the large sample size we can provide for the first time a statistically significant difference in the percentage of success when transferring a day5 (61%) versus day6 (49%) euploid embryo. Trial registration number not applicable

Comparison of pregnancy outcomes between 4th day morula and 5th day blastocyst after embryo transfer: a retrospective cohort study

BackgroundThis study was designed to evaluate pregnancy outcomes between morulae transferred on day 4 (D4) and blastocysts transferred on day 5 (D5).MethodsFrom September 2017 to September 2020, 1963 fresh transfer cycles underwent early follicular phase extra-long protocol for assisted conception in our fertility center were divided into D4 (324 cases) and D5 (1639 cases) groups, and the general situation and other differences of patients in both groups were compared. To compare the differences in pregnancy outcomes, the D4 and D5 groups were further divided into groups A and B based on single and double embryo transfers. Furthermore, the cohort was divided into two groups: those with live births (1116 cases) and those without (847 cases), enabling a deeper evaluation of the effects of D4 or D5 transplantation on assisted reproductive outcomes.ResultsIn single embryo transfer, there was no significant difference between groups D4A and D5A (P > 0.05). In double embryo transfer, group D4B had a lower newborn birthweight and a larger proportion of low birthweight infants (P < 0.05). The preterm delivery rate, twin delivery rate, cesarean delivery rate, and percentage of low birthweight infants were lower in the D5A group than in the D5B group (P < 0.05). Analysis of factors influencing live birth outcomes further confirmed the absence of a significant difference between D4 and D5 transplantation in achieving live birth (P > 0.05).ConclusionWhen factors such as working life and hospital holidays are being considered, D4 morula transfer may be a good alternative to D5 blastocyst transfer. Given the in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) success rate and risk of twin pregnancy, D4 morula transfer requires an adapted decision between single and double embryo transfer, although a single blastocyst transfer is recommended for the D5 transfer in order to decrease the twin pregnancy rate. In addition, age, endometrial thickness and other factors need to be taken into account to personalize the IVF program and optimize pregnancy outcomes.

P-444 The battle between a machine learning model, a gynecologist and an embryologist to predict pregnancy outcomes after single day-5 embryo transfer

Abstract Study question Are algorithms more effective than clinicians/embryologists at predicting pregnancy outcomes in the context of Assisted Reproductive Technology (ART) following the transfer of a single blastocyst? Summary answer Evaluation of a pregnancy prediction algorithm, trained on local data and assessed by two ART professionals, yielded an accuracy comparable to a coin toss. What is known already Artificial Intelligence’s ability to synthesize multifaceted embryological and clinical data and process extensive datasets holds promise for elevating IVF success rates through data-driven, individualized treatments. Refining embryo selection through deep learning and computer vision, individualizing treatment protocols, predicting oocyte retrieval after Controlled Ovarian Hyperstimulation (COH) and ultimately predicting Live Birth (LB) following embryo transfer are one of the early promises of this technology. Study design, size, duration A retrospective analysis was conducted on 475 stimulation cycles that resulted in a single day-5 blastocyst transfer (fresh or frozen, classical IVF or ICSI) from January 2015 to January 2022 at the Poissy/Saint-Germain-En-Laye hospital, France. The study encompassed 32 clinical and biological parameters, ranging from general characteristics of patients to controlled ovarian hyperstimulation (COH) details and embryo grading. Exclusively, embryos fertilized with autologous oocytes and sperm were considered, excluding any donor-fertilized embryos. Participants/materials, setting, methods Preliminary testing with Machine Learning algorithms (XGboost, LightGBM, Random Forest (RF), Support Vector Machine (SVM), Multi-layer Perceptron (MLP)) were conducted before selecting XGboost model for this study. Data was split into a training (n = 331) and testing (n = 144) set. Pregnancy outcome predictions (binary) were compared between the model, a clinician, and an embryologist. Feature extraction was conducted, and both professionals were asked to give the top 5 most relevant parameters used to make the pregnancy predictions. Main results and the role of chance The study analyzed 475 stimulation cycles, including 362 frozen and 114 fresh transfers. Participants’ mean age was 33, with a Body Mass Index (BMI) of 24.9. The average Antral Follicle Count (AFC) was 16.8, and the mean starting gonadotropin dose was 225 UI, yielding 8.3 MII oocytes on average. 36 patients were active smokers. Preliminary AUC results showed XGboost leading (AUC = 0.57) compared to LightGBM (AUC = 0.56), Random Forest (AUC = 0.56), SVM (AUC = 0.49), and MLP (AUC = 0.47) in predicting pregnancy outcomes. After hyperparameter optimization via GridSearch, the XGboost model enhanced its AUC to 0.61, outperforming the embryologist (AUC = 0.58) and the gynecologist (AUC = 0.44). Top predictive features identified after feature extraction included endometrial thickness at or before ovulation trigger, AMH levels at patient admission, AFC, BMI, and age. The gynecologist primarily prioritized age, BMI, embryo grading (TE and ICM), and the number of mature oocytes retrieved. The embryologist, valued embryo grading (TE and ICM) foremost, followed by age, smoking status, BMI, and COH duration. Limitations, reasons for caution The study’s limited dataset due to dynamic practices and difficulty maintaining curated data over the years, necessitates caution. The selected 32 parameters from 762 available reflect typical incomplete datasets in such settings, highlighting the importance of careful interpretation and potential need for broader data integration to enhance representativeness and reliability. Wider implications of the findings The findings advocate for developing ART-specific AI, integrating multimodal data and novel parameters, to enhance live birth predictions. This approach underscores the complex synergy between algorithmic precision and clinical expertise in ART, highlighting the stochastic nature of pregnancy prediction and the potential of tailored, data-driven strategies to improve outcomes. Trial registration number not applicable

Relationship between fresh single embryo morphology scores and serum HCG values at 14 days and 14–18-day doubling values

Research questionWhat is the effect of embryo morphology score on 14-day β-HCG levels and 14–18-day β-HCG doubling values, and do they have differences in day-3 embryo or day-5 blastocyst transfers? DesignRetrospective analysis of 4434 fresh cycles of single embryo transfers (SET) with β-HCG ≥15 mIU/ml on day 14 after transfer via IVF and ICSI. The correlation between embryo morphology score and 14-day β-HCG was examined. Doubling of 14–18 day β-HCG was analysed in 2628 cycles to determine correlations with embryo morphology score. ResultsIn day-3 SET, number of embryonic cells was positively correlated with 14-day post-transfer β-HCG values (R = 0.076; P = 0.013). No significant correlation was observed between the grade of the transferred embryos and the 14–18-day serum β-HCG doubling values. In day-5 single blastocyst transfers, the degree of blastocyst expansion, trophoblast cell and inner cell mass (ICM) grades demonstrated a significant positive correlation with 14-day post-transfer β-HCG (P < 0.001, P = 0.014, P = 0.003). Degree of blastocyst expansion was significantly correlated with 14–18-day β-HCG doubling values (R = –0.051, P = 0.027). Grades of the ICM and trophoblast cells showed no significant correlation with 14–18-day β-HCG doubling values. ConclusionIn fresh SET, embryo morphology score influences 14-day β-HCG values in day-3 embryos and day-5 blastocyst transfers. Embryo morphology score in day-3 SET does not affect 14–18-day β-HCG doubling values. Degree of blastocyst expansion significantly affects 14–18-day β-HCG doubling values in day-5 blastocyst transfers.

The effects of a glyphosate-based herbicide on the bovine gametes during an in vitro embryo production model

Roundup® (R), while it is the most used herbicide globally, and its residues are ubiquitous in urban and suburban areas, its impact on vertebrates' safety remains highly debated. Here, in three in vitro experiments, we investigated the effects of a very low dose (1 ppm) of R on the fertilization capacity and embryo development in cattle. In the first experiment, frozen-thawed bull semen exposed to R for 1 h exhibited reduced motility parameters but unaffected fertilization ability. However, after in vitro fertilization, the rates of embryo formation were significantly lower compared to the untreated controls. In the second experiment, oocytes exposed to R during in vitro maturation showed reduced cleavage rates, and the embryo yield on days 7, 8, and 9 of embryo culture was significantly lower than that of the controls. In the third experiment, oocytes were matured in the presence of R and in a medium containing both R and Zinc, chosen to offer antioxidant protection to the oocytes. Day-7 blastocysts were analyzed for the expression of genes associated with oxidative stress, apoptosis, and epigenetic reprogramming. Exposure to R markedly suppressed embryo formation rates compared to the controls. The combination of R with Zinc restored the blastocyst yield, which on days 8 and 9 was comparable to that of the controls and higher than the groups exposed only to R on all days. The gene expression analysis revealed that R promotes oxidative stress development, triggers apoptosis, and induces epigenetic changes in developing embryos, while zinc presence alleviates these adverse effects of R. These findings imply that even at very low doses, R could be highly toxic, leading to functional abnormalities in both gametes, potentially affecting fertility in both genders.

Embryo long-term storage does not affect assisted reproductive technologies outcome: analysis of 58,001 vitrified blastocysts over 11 years

BackgroundRecently the potential detrimental effect that the duration of storage time may have on vitrified samples has raised some concern, especially when some studies found an association between cryo-storage length and decreased clinical results. ObjectiveThis study aimed to evaluate the effects of the storage time length of day-5 vitrified blastocysts in two study groups: freeze-all cycles and non elective frozen embryo transfers (FET). Study designThis was a retrospective study including 58001 vitrified/warmed day-5 blastocysts from two different populations according to the reason for FET. Elective FET comprised freeze-all cycles (N=16615 blastocysts and 16615 patients) where only single embryo transfers (SET) and only the first FET were included. The non elective FET group included 41386 embryos from 25571 patients where FET took place using supernumerary embryos after fresh embryo transfer. All the possible FETs were included. Both SET and double embryo transfers (DET) were included. Donor and autologous oocytes were used. The period covered by this study was 11 years. The blastocysts sample was clustered into deciles which provided specific storage duration categories. Main outcome was live birth rate (LBR) and secondary outcomes were embryo survival, miscarriage, clinical and ongoing pregnancy rates according to storage duration. The impact of storage time was assessed by univariable analyses in both groups. The comparison was made between each decile to the last one. A multivariable logistic regression analysis was conducted including the variables with significant association found in the univariate analysis. Student's t-test and Chi squared tests, or an analysis of variance were used wherever appropriate. P < 0.05 was considered statistically significant. ResultsThere were statistical differences in baseline characteristics of patients included in the study groups. Storage durations ranged from ≤ 0.67 to ≥ 4.34 and from ≤ 1.8 to ≥ 34.81 months in freeze-all and non elective FET respectively. Embryo survival did not show statistical differences across the categories of storage time in freeze-all and non elective FET groups. Statistical differences were found for the LBR across some, but not all the subgroups of storage duration. The multivariable analysis showed no association between storage time and the LBR in both groups (NS). Blastocyst quality, body mass index (BMI), number of retrieved oocytes, endometrial preparation, male factor and uterine factor were related to the drop in the LBR in the freeze-all group (p<0.05). In the non elective FET group the variables that showed significant association with the LBR were, age at retrieval and at FET, type of FET (SET or DET), number of retrieved oocytes, BMI, endometrial preparation, origin of sperm sample and female factor. ConclusiónThis large study demonstrated no association between storage time and clinical outcome. Other variables such as patient's age, embryo quality, BMI and etiology, are somewhat responsible for impacting outcome. This provides evidence for the safety of embryo vitrification, even after long storage periods. This is reassuring for both IVF practitioners and the patients undergoing FET of either elective or non elective embryos.

Blastocyst formation in vitrified-warmed preimplantation embryos derived from vitrified-warmed oocytes in a mouse model.

The purpose of this study was to use a mouse model to investigate the blastocyst formation rate in vitrified-warmed embryos derived from vitrified-warmed oocytes. Metaphase II oocytes obtained from BDF1 mice were vitrified and warmed, followed by fertilization with epididymal sperm. On day 3, a total of 176 embryos, at either the eight-cell or the morula stage, were vitrified-warmed (representing group 1). For group 2, 155 embryos at the same developmental stages were not vitrified, but rather were directly cultured until day 5. Finally, group 3 included day-5 blastocysts derived from fresh oocytes, which served as fresh controls. The primary outcome measured was the rate of blastocyst formation per day-3 embryo at the eight-cell or morula stage. The rates of blastocyst formation per day-3 embryo were comparable between groups 1 and 2, at 64.5% and 69.7%, respectively (p>0.05). The formation rates of good-quality blastocysts (expanded, hatching, or hatched) were also similar for groups 1 and 2, at 35.5% and 43.2%, respectively (p>0.05). For the fresh oocytes (group 3), the blastocyst formation rate was 75.5%, which was similar to groups 1 and 2. However, the rate of good-quality blastocyst formation in group 3 was 57.3%, significantly exceeding those of group 1 (p=0.001) and group 2 (p=0.023). Regarding developmental potential to the blastocyst stage, vitrified-warmed day-3 embryos originating from vitrified-warmed oocytes demonstrated comparable results to non-vitrified embryos from similar oocytes. These findings indicate that day-3 embryos derived from vitrified-warmed oocytes can be effectively cryopreserved without incurring cellular damage.

The Association between Embryo Development and Chromosomal Results from PGT-A in Women of Advanced Age: A Prospective Cohort Study.

Embryo morphology and morphokinetics have been studied for their association with euploid embryos. However, the results are controversial, especially in the advanced-aged women group, when the risk of aneuploidy increases significantly. This prospective cohort study evaluated the association between embryo development between day-3 cleavage and day-5 blastocyst stages and euploidy rates, determined using preimplantation genetic testing for aneuploidy (PGT-A). Embryos from women aged 35 years and above who underwent intracytoplasmic sperm injections and PGT-A were studied. Day-3 cleavage-stage embryos were evaluated for their cell number, and day-5 blastocyst-stage embryos were evaluated for their morphological grade. Embryo development from day 3 to day 5 was categorized as either good or poor development and evaluated for its association with the PGT-A results. We evaluated 325 embryos from 101 infertile couples. It was found that 55.17% of blastocysts with good development and 29.83% with poor development were euploid. A significant association was found between embryo development and euploidy rates in advanced-aged women (p < 0.001). Also, there were significantly higher rates of euploid embryos with good blastocyst morphological grades, especially blastocyst expansion grades and trophectoderm grades. In conclusion, embryo morphokinetics shows promising results in predicting euploidy in advanced female age.

Comparing Day 5 versus Day 6 euploid blastocyst in frozen embryo transfer and developing a predictive model for optimizing outcomes: a retrospective cohort study.

Optimal protocols for frozen-thawed embryo transfer (FET) after preimplantation genetic testing (PGT) remain unclear. This study compared Day 5 (D5) and Day 6 (D6) blastocysts and evaluated predictors of FET success. A total of 870 patients with genetic diseases or chromosomal translocations who received PGT at the First Affiliated Hospital of Zhengzhou University from January 2015 to December 2019 were recruited. All patients underwent at least one year of follow-up. Patients were divided into groups according to the blastocyst development days and quality. Univariate and multivariate logistic regression were applied to identify risk factors that affect clinical outcomes and to construct a predictive nomogram model. Area under the curve (AUC) of the subject's operating characteristic curve and GiViTI calibration belt were conducted to determine the discrimination and fit of the model. D5 blastocysts, especially high-quality D5, resulted in significantly higher clinical pregnancy (58.4% vs 49.2%) and live birth rates (52.5% vs 45%) compared to D6. Multivariate regression demonstrated the number of blastocysts, endometrial preparation protocol, days of embryonic development and the quality of blastocysts independently affected live birth rates (P<0.05). A nomogram integrating these factors indicated favorable predictive accuracy (AUC=0.598) and fit (GiViTI, P=0.192). Transferring high-quality D5 euploid blastocysts after PGT maximizes pregnancy outcomes. Blastocyst quality, blastocyst development days, endometrial preparation protocols, and number of blastocysts, independently predicted outcomes. An individualized predictive model integrating these factors displayed favorable accuracy for counseling patients and optimizing clinical management.

#193 : Comparison of the Different Expression of OCT-4, GATA-3 and Clinical Pregnancy Outcome Between the Day 5 and Day 6 Blastocysts

Background and Aims: In common experience of in vitro-fertilization (IVF) cycle, blastocysts formed at Day 5 (D5) are regarded as higher quality and are more desirable for embryo transfer compared with those formed at Day 6 (D6). How those delayed D6 embryo differs from D5 embryo is less discussed. This study aimed to elucidate the cell content distinguished by immunofluorescent (IF) staining between the same grade of D5 and D6 blastocysts that might link to the clinical pregnancy outcome in frozen–thawed embryo from different day of blastocyst formation. Method: The retrospective study included 1327 frozen-thawed single blastocyst transfer (SBT) cycles in the Taipei Fertility Center during March 2020 to December 2022. All D5 (D5-SBT) and D6 (D6-SBT) frozen-thawed SET cycles (n=1327) were separated to non-PGS (n=671) and PGS-euploid (n=656) groups, then assigned to high grade (AA, AB, BA, BB) and low grade (BC, CB, CC). Aneuploidy and mosaicism embryo are excluded. For cell content analysis, pluripotent marker OCT-4 initially expressed in all blastomeres but restricted to the inner cell mass (ICM) of the blastocyst and downregulated in the trophectoderm (TE). GATA-3 is a TE specific marker which increases during pre-implantation embryo development. The cell number, OCT-4 and GATA-3 expression of same grade frozen-thawed D5 (n=14) and D6 (n=14) blastocysts were analyzed with IF staining. D5 extensively cultured to D6 (n=10) were used as the control group of regularly development. Cell numbers of TE and ICM region were examined by OCT-4 and GATA-3 IF detection. The total cell number was visualized with DAPI staining. Results: According to staining results, there were no significant differences in total cell number, TE and ICM cell number between the same grade D5 and D6 blastocysts. However, TE cell number significantly increased in D5 extensively cultured blastocysts, indicating higher proliferative capacity in TE cells from D5 blastocysts. In the clinical data of the non-PGS group, chemical pregnancy rate (HCG+) and clinical pregnancy rate (SAC+) of high grade D5-SBT were significantly increased compared with high grade D6-SBT (HCG+: 69.1% versus 47.0%, P&lt;0.001; SAC+: 63.0% versus 43.4%, P=0.001). In the PGS-euploid group, the chemical pregnancy rate (HCG+) showed significantly increased in high grade D5-SBT compared with high grade D6-SBT (69.5% versus 60.0%, P=0.047). The clinical pregnancy rate (SAC+) was significantly higher in high grade D5-SBT compared with high grade D6-SBT (65.5% versus 52.5%, P=0.009). The ongoing pregnancy rate was significantly higher in high grade D5-SBT compared with high grade D6-SBT (57.6% versus 43.3%, P=0.005). Conclusion: According to the IF data in this study, we demonstrated that the number of GATA3-positive TE cells were significantly decreased in D6 blastocyst compared with D5 extensively cultured blastocysts. Due to decreased proliferative capacity in high grade D6 blastocyst, transfer of high grade D5 blastocyst would exhibit higher ongoing pregnancy rate than D6 blastocyst.

#183 : The Use of Follitropin-Delta in Indonesia: Current Evidence and Development

Background and Aims: The current success of IVF treatments was aided by the fulminant ascent of recombinant DNA technologies. Follitropin delta (FD) is used to promote ovarian follicular proliferation. This research was aimed to investigate the current evidence and development of follitropin delta in its novel in Indonesia. Method: This prospective cohort study was conducted at the Yasmin IVF Center, Dr. Cipto Mangunkusumo General Hospital Jakarta from January 2021 to October 2022. All IVF/ICSI cycles in which follitropin delta (RekovelleⓇ, FE 999049; Ferring Pharmaceuticals, St. Prex, Switzerland) was used for ovarian stimulation. The outcome data were collected, including initial dose, and total dose of follitropin delta, retrieved oocytes, fertilized eggs, blastocyst day 3-5, embryo transfer, clinical pregnancy rates, and clinical implantation rates. The collected patients’ data (n = 20) who underwent IVF and received follitropin delta ovarian stimulation were divided into 2 groups by age (under 35 years and above 35 years). Results: The average oocyte, blastocyst, and embryo day 3 obtained in the study sample were higher in women under 35 years old. The pregnancy rate was higher in group 1 (33,30% vs. 25,0%) which was assessed based on a positive b-HCG results and a P4 value after receiving FET. Patients who followed follitropin delta ovarian stimulation had a pregnancy rate of 30%. Follitropin delta can be used in patients to successfully obtain a pregnancy in both groups, as it achieves higher result in women under 35 years old (33.3% vs. 25%). Conclusion: Optimizing ovarian response in IVF by using a program of follitropin delta dose achieved considerable rise in the quantity of good-quality, usable blastocysts. Assessing follitropin delta in individualized protocols and improving understanding of the dosages can help clinicians in evaluating follitropin delta in their conventional protocols.

#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.