Blastocyst Day Research Articles

Comparison of the effects of soft and firm embryo transfer catheters on clinical pregnancy rates in a homogeneous group of thawed day-5 blastocyst embryo transfers

Comparison of the effects of soft and firm embryo transfer catheters on clinical pregnancy rates in a homogeneous group of thawed day-5 blastocyst embryo transfers

P-759 Discard, or not discard, that is the question. An international survey among 117 embryologists about their decision on 50 poor-quality/day7 blastocysts cultured in time-lapse incubators.

Abstract Study question Are decisions on day7 and/or poor-quality blastocysts’ (PQB; <Gardner’s BB) use consistent among embryologists of diverse European countries and what factors influence them? Summary answer “Discard” choice prevalence was ≈40%, however inter-rater agreement was just fair (Fleiss-k=0.26). Country of practice, maternal age and number of sibling embryos influence each decision. What is known already Blastocyst morphokinetics and morphological quality are associated with competence and used to make clinical decisions, even though evaluations are poorly reproducible across embryologists. Moreover, the increasing utilization of PQB and adoption of culture beyond 144 hours-post-insemination is further challenging the decision-making process. Previous surveys reported limited concordance among embryologists in the “discard”/“use” choices made for day7 and/or PQB based on single 2D-pictures without cycle/patient information. However, for instance whole embryo preimplantation development, reproductive history, number of sibling embryos, can influence each decision. Different regulations and mindsets across countries may further influence these choices and require investigation. Study design, size, duration Anonymous survey in Microsoft-Forms run within a large IVF-network. Each laboratory-director shared a link to the survey with his/her colleagues via 3 e-mails sent weekly between 22/12/2023 and 12/1/2023. 117 embryologists participated representing 6 European Countries, 9 IVF-groups, and 28 centers. Raters inspected EmbryoScope videos of 50 PQBs and/or day7 blastocysts’ whole preimplantation development and decided whether to “use” (“transfer-fresh”/“cryopreserve”/“biopsy and cryopreserve”) or “discard” each of them. Inter-rater agreement was measured with Fleiss-k. Participants/materials, setting, methods Couples’ information (age, infertility diagnosis, previous attempts, sperm analysis, number of sibling blastocysts obtained) were provided, and participants were asked their years of experience, center location, average number of cycles and average maternal age, number of colleagues, presence of time-lapse-incubators. The AI-score (CHLOE-EQ, Fairtility and iDAScore v1.0, Vitrolife), chromosomal diagnosis and clinical outcomes were known for all embryos by one embryologist not involved in the survey, but the participants were blinded to them. Main results and the role of chance Participants were Italian (40%,N=47), Spanish (24%,N=28), Portuguese (5%,N=6), Czech (5%,N=6), Swedish (23%,N=27) and Icelandic (3%,N=3). 2263(38.7%) “discard” and 3587(61.3%) “use” decisions were recorded. Czech, Portuguese, and Italian embryologists expressed lower (17±7%,23±14% and 27±18%), Spanish intermediate (37±16%), and Nordic higher (67±11%) “discard” decision rates.The prevalence of “discard” responses per embryo was 37.4±24.2%(2-87). The larger the number of sibling blastocysts the higher the embryologists’ propensity to “discard” PQBs/day7 (unstandardized coefficient-B:+4.4%,95%CI:+0.9 to + 7.9,p=0.015). The most prevalent “use” choice was “biopsy and cryopreserve” (33.1±21.7%,2-78). Maternal age (unstandardized coefficient-B:+2.2%,95%CI:+1.3 to + 3.8,p<0.01) and number of sibling blastocysts (unstandardized coefficient-B:-9.2%,95%CI:-15.2% to -3.3%,p<0.01) influenced this rate.Overall, inter-rater agreement was 0.26 (standard-error:0.003;“fair”), with “moderate” agreement for Czech and Portuguese (0.41,standard-error:0.037), “fair” for Spanish (0.38,standard-error:0.007), Italian and Nordic embryologists (0.28,standard-error:0.006 and 0.009).CHLOE-EQ and iDAScore v1.0 were not associated with embryologists’ decisions. No difference was shown for AI-scores in euploid versus aneuploid blastocysts (CHLOE-EQ: 0.2±0.32,0-0.9 versus 0.2±0.29,0-0.93; iDAScore v1.0: 4.2±1.6,2.2-6.7 versus 4.3±1.5,2.1-9), while the embryologists more often chose “discard” among the latter group (28.3±21%,9-71 versus 41.6±24.8%,2-87). Five blastocysts were transferred and 3 implanted (CHLOE-EQ: 0.001,0.416 and 0.003; iDAScore v1.0: 2.6,5.0 and 6.6; “discard” choice rates: 19%,15% and 10%). Limitations, reasons for caution All embryologists were asked to decide regardless of the regulation enforced in their countries (e.g., PGT-A forbidden, discarding viable embryos not allowed). Nonetheless, the legislative, social, and clinical background might still have affected their choice. The survey included only private IVF clinics located in Europe. Wider implications of the findings European embryologists’ decision-making regarding PQB/day-7 embryos is inconsistent. Mostly, legislative/clinical background and cycles’ characteristics influence each decision. However, this practice has clinical implications, especially in poor-prognosis patients. Artificial-Intelligence may improve the reproducibility by removing intrinsic human subjectivity. However, it is still currently underpowered to assess these outlier embryos Trial registration number Not applicable

O-162 Fresh or frozen Day 6 blastocyst transfer : Is there still a question?

Abstract Study question Whether slow developing expanded day 6 (D6) blastocysts should be either transferred during fresh cycle or systematically vitrified at D6 Summary answer Fresh D6 blastocyst transfer is independently associated with reduced live birth rate compared to frozen thawed D6 transfer What is known already Live birth rate (LBR) after D5 blastocyst transfers are significantly higher when compared with D6 embryos in both fresh and frozen-vitrified embryo transfer cycles according to the last published meta-analyses. Therefore, for women obtaining only D6 blastocysts, chances of pregnancy may be lower but still persist and these embryos should be transferred. The question of the best strategy for transfer (i.e. in fresh or frozen cycles) remains unclear while data on this subject are scarce. Study design, size, duration Retrospective observational cohort study. Patients having only embryos reaching blastocyst stage at D6 were included between January 2018 and May 2021. Two groups were compared : patients having a fresh D6 transfer (D6 fresh transfer group) and patients having a frozen thawed D6 transfer ( D6 frozen transfer group). Participants/materials, setting, methods 830 D6 single blastocyst transfers (736 frozen blastocysts and 94 fresh blastocysts) were analyzed. LBR and neonatal outcome were compared between groups, as well as the influence of clinical characteristics and data related to COS protocols. Correlation between D6 blastocyst morphology according to Gardner’s classification and livebirth occurrence was also evaluated. Statistical analysis of the data was carried out using univariate and multivariate logistic regression models. Main results and the role of chance LBR was significantly lower after D6 fresh blastocyst transfer compared to D6 frozen thawed blastocyst transfer [5.3% (5/96) vs 12.2% (90/736), p < 0.05]. Moreover, when comparing a subgroup of 68 first D6 frozen embryo transfers (1st D6 frozen transfer group) to the 94 fresh D6 blastocyst transfers (D6 fresh elective group), the superiority of D6 frozen blastocyst transfers regarding LBR was also confirmed (17.6% vs 5.3% p < 0.001, respectively). Concerning neonatal outcomes, the mean birth weight was comparable between the two groups. Univariate logistic regression analysis taking into account blastocyst morphology parameters showed that TE grade was the only parameter significantly associated with LBR after D6 embryo transfer (p < 0.001). Multiple logistic regression revealed that D6 embryo fresh transfer was independently associated with reduced LBR compared to frozen thawed D6 transfer (OR 0.367; 95%IC 0.143-0.945; p = 0.038). Moreover, our results showed that transferring a good or top quality D6 blastocyst increases 3 fold chances of live birth. Limitations, reasons for caution The number of fresh cycles seems relatively small, but the consistency of our results was guaranteed by the homogeneity of the 2 subgroups, standardization of all biological and clinical procedures and the robust statistical analysis methodology used in this study. Wider implications of the findings Despite their lower potential, D6 blastocysts have to be transferred with the objective to improve LBR, especially in women obtaining only these type of embryos. Our results recommend to transfer D6 blastocysts in frozen cycles. However, these findings have to be confirmed on larger series in prospective randomized trials. Trial registration number not applicable

P-303 Outcomes of day 5 versus day 6 blastocysts: correlation of euploidy, implantation and embryo quality

Abstract Study question Are outcomes of day 5 (D5) blastocysts better than day 6 (D6) blastocysts in a PGT-A program? Summary answer Outcomes of day 5 embryos show more blastocysts, better quality, more euploidy due to better quality and higher implantation on same quality than day 6. What is known already Human embryos optimally reach the blastocyst stage after five days of culture, but some have a slower development. The superiority of day 5 blastocysts compared to day 6 blastocysts in fresh cycle transfers was previously demonstrated and attributed mainly to endometrial asynchrony. Data from frozen blastocysts transfers showed conflicting results, some studies have announced higher pregnancy rates after day 5 transfer compared with day 6 transfer, while others have shown equivalent outcomes. However, none of these studies have compared outcomes between same quality and euploid day 5 (D5) frozen blastocysts versus those frozen on day 6 (D6). Study design, size, duration Retrospective observational study performed in a private centre between May 2017 and December 2022. The study includes the data analysis of 5599 D5 and D6 blastocysts undergoing PGT-A obtained from 819 patients following 1295 PGS cycles. 948 euploid blastocysts with known implantation outcome were transferred in 789 frozen embryo transfers. Blastocyst morphology was scored in 3 groups: A: excellent (AA, AB, BA), B: good (BB), C: average and poor-quality embryos (BC, CB, CC). (Gardner-Schoolcraft classification) Participants/materials, setting, methods PGT-A with NGS technology was offered to patients of advanced maternal age and/or with repeated IVF failures. Trophectoderm biopsies were performed on D5 and/or D6 embryos, with laser assistance. (Navilase, OCTAX) We compared both populations (D5 and D6) in terms of number of blastocysts achieved and distribution of embryo quality of each population. As primary outcome, same-quality group results of euploid rate and implantation were compared according to the day of blastocyst development (D5 and D6). Main results and the role of chance Vast majority of the blastocysts were biopsied on D5 (71.8%, n = 4017) versus (28.2%, n = 1582) biopsied on D6. The proportion of three quality-categories of blastocysts according to D5/D6 is uneven. Quality A = 18.6% vs 3,5%. Quality B = 72,4% vs 59,6%. Quality C = 9,0% vs 36,9%, respectively, showing significative differences between D5/D6 (Chi^2 p-values of 3 groups< 0.001), concluding that overall D5 quality is better than D6. Overall euploidy rate on D5 population versus D6 was 31,3% vs 23,3% (p value <0.001), showing significant difference in favour of D5. But layering on 3 quality categories on D5/D6, the euploidy rate was 44,7% vs 51,8% for quality A, 29,6% vs 26,6% for quality B and 17,6% vs 14,9% for quality C, respectively, showing no significative differences D5/D6 (Chi^2 p-values: A = 0,331; B = 0,067; C = 0,280). Euploidy is not D5/D6 dependant but quality dependant. Overall implantation on D5 (65,9%) is higher than D6 (40,4%) (p value <0.001). Layering again, on D5 the implantation was 75,0% vs 66,7% for quality A, 63,8% vs 46,4% for quality B and 35,3% vs 26,3% for quality C, respectively. Despite implantation being higher on D5 on all three groups only group B shows D5/D6 significative differences. (Chi^2 p-values: A = 0,651; B < 0.001; C = 0,360). Implantation is embryo quality and D5/D6 dependant. Limitations, reasons for caution The study is limited by its retrospective nature and the low number of grade A D6-euploid blastocysts available to transfer. Additionally, it is common to transfer more than one grade C quality embryo to increase the chances of pregnancy, losing implantation track of these type. Wider implications of the findings It was uncontested that D5 blastocysts had better reproductive potential, but we managed to quantify this potential according to euploidy and implantation rates based on embryo quality, which remains the most important predictive biomarker for selecting the best embryo to transfer and reducing the time to achieve a pregnancy. Trial registration number not applicable

P-722 Impact of blastocyst quality and post-thaw embryo culture factors in non-invasive PGT-A informativity on vitrified blastocysts.

Abstract Study question What are the most important factor/s involved in the informativity of non-invasive PGT-A (niPGT-A) of vitrified blastocysts? Summary answer The day of vitrification and post-thaw time in culture of vitrified blastocysts are the most important factors for niPGT-A informativity, without correlation with blastocyst quality. What is known already niPGT-A is a technique that allows the non-invasive study of the chromosomal content of blastocysts. The informativity and concordance rates for fresh (Rubio, 2020) and frozen-thawed embryos (Garcia-Pascual, 2022) have been related with the time in culture and the day of media collection. However, there is a need to understand the optimal conditions needed to obtain optimal informativity for frozen-thawed vitrified blastocyst undergoing niPGT-A. Study design, size, duration Prospective study including 135 spent blastocyst media (SBM) and the corresponding blastocyst samples from thawed day-5 and day-6 blastocysts from PGT-A (73) and niPGT-A (62) cycles collected from January 2021 until March 2022. Blastocysts were discarded under IRB approval. The embryos were divided into 3 groups depending on the day of development and the time in culture after thawing: group-1, day-5 blastocysts cultured 8h; group-2, day-5 blastocysts cultured 24h and group-3, day-6 blastocysts cultured 8h. Participants/materials, setting, methods Media and blastocysts were obtained from IVF patients (29-42 years-old). After thawing, each blastocyst was washed to remove remaining cumulus cells and individually transferred to a 10 μL droplet of fresh media or to an EmbryoScope dish with 20 μL per well. SBM and blastocysts were collected, frozen and analysed by Next Generation Sequencing. Correlation between concordance rates (overall agreement between SBM and blastocyst samples), informativity, embryo quality and time in culture were evaluated. Main results and the role of chance All the embryos had a good embryo quality (BB or greater using Gardner Criteria) but different expansion degree. Informativity was significantly lower when day-5 embryos were cultured for only 8 hours (group 1), regardless of the degree of expansion (expansion degree 3, 28.57%; degree 4, 64.29% and degree 5-6, 57.14%, p < 0.0005). The concordance rates between SBM and whole blastocysts were very high and no significant different in relation to the expansion degree when comparing day-5 embryos cultured for 8 hours (group 1, 90.5%) with 24 hours (group 2, 93.6%) or with day-6 embryos cultured for 8 hours (group 3, 92.3%). The data suggest that the important factor impacting the informativity rate is the day of vitrification, and the time in culture after thawing, but not the expansion degree of the embryos. Regarding the concordance rates, there is no impact of the time in culture nor the expansion degree. Limitations, reasons for caution The main limitation of this study is that all the embryos analysed had fair or good quality but different expansion degrees. Wider implications of the findings The high concordance rates observed in frozen blastocysts opens the possibility of expanding the use of niPGT-A to frozen blastocyst without an impact of the expansion degree in the results. Trial registration number Not applicable

P-240 High mitochondrial DNA content in Day6 blastocyst may impair ongoing pregnancy rate

Abstract Study question Is there an association between mitochondrial DNA (mtDNA) content and Ongoing Pregnancy Rate (OPR) in D5 or D6 euploid blastocyst? Summary answer Day6 OPR is decreased in high mtDNA content blastocyst. Day5 and Day6 mtDNA levels are independent of maternal age or standard morphology. What is known already Preimplantation development is an energy-demanding process and mitochondrial ATP production is crucial for cellular activity in fast replicating cells. Mitochondrial content in preimplantation embryo has been proposed as a marker of embryo potential in term of viability and implantation success though the dynamics and distribution of mitochondria in human embryo is still debated. In recent years, it has been suggested that low mtDNA levels were associated with euploid chromosomal complement and higher implantation rate while other studies failed to find a correlation between mtDNA content and reproductive outcome. Study design, size, duration This study is a retrospective cohort analysis from 2020 to 2022 including 343 Day5 and 187 D6 single euploid blastocyst transfer. Primary endpoint was OPR beyond pregnancy week 16. Day5 and Day6 frozen-thawed transfer were divided based on blastocyst mtDNA content in four groups: Day5-high, Day5-low, D6-high, D6-low. Secondary endpoints were relationship between mtDNA content, maternal age and standard morphology in all obtained blastocysts. Statistical differences were compared by Chi-Square test. Participants/materials, setting, methods 771 couples performed IVF cycles with next-generation sequencing (NGS)-preimplantation genetic testing of aneuploidy (PGT-A) (age 19-47yrs; Mean age 37.8yrs). Following culture in sequential media 700 euploid blastocyst were obtained and mitochondrial and chromosomal DNA copy number variation were examined simultaneously with next generation sequencing (NGS) methodology. mtDNA copy numbers was based on the observed ratios of sequence coverages between mtDNA and nuclear DNA. Main results and the role of chance In our clinical setting OPR is similar in Day5 vs. Day6 transfers (55% vs. 49% n.s.). However when mtDNA content is considered D6-low OPR is comparable to D5-high or low (57.3%; 53.3%; 57,3% respectively n.s.) while D6-high transfers yield significantly decreased OPR (31,2% p < 0.05). When stratified by age only Advanced Maternal Age (AMA) patients ( > = 38yrs) showed this decrease at a significant level (Day6 high OPR 20% p < 0,05).This result in transferred blastocyst is obtained despite high:low ratio of all biopsied blastocyst is constant in Day5 (1:1) (47%-53%) and markedly decreased in Day6 (1:4) (25%-75%) either overall or in younger (n.s.) and AMA patients (n.s.). Moreover when standard morphology distribution is evaluated according to Gardner classification criteria top, fair and poor quality blastocysts display the same 1:1 high:low blastocysts mtDNA ratio in Day5 and 1:4 in Day 6 overall and in younger and AMA patients. Hence, the mtDNA blastocyst content is independent of patients’ age and blastocyst quality. The origin of Day6 OPR decrease could therefore depend on the number of Day6-high blastocyst transferred in AMA patients and ultimately in the number of blastocyst available to transfer for each patient. Limitations, reasons for caution Due to the small numbers of Day6-high mtDNA blastocyst, a larger sample size is required to confirm these preliminary findings. Moreover, the retrospective nature of the study may introduce some bias mainly in patients’ characteristics and the strategy of embryo selected for transfer. Wider implications of the findings While Day5-high blastocyst yield higher OPR the persistence of higher mtDNA levels in Day6 blastocyst may hamper their implantation potential. Our findings may help to select the embryo to transfer to maximize transfer success. D6 blastocysts with low mtDNA content should be preferred whenever a choice is possible. Trial registration number Not Applicable

O-225 May embryonic cfDNA in the culture media play a role as a non-invasive assessment of aneuploidy in PGT

Abstract Non-invasive preimplantation genetic testing for aneuploidies (niPGT-A) in spent blastocyst media (SBM) is a promising technique that has the potential to revolutionize current preimplantation genetic testing procedures. This technique is based in the analysis of the cell-free DNA (cfDNA) released by the embryo during the latest stages of preimplantation development. It allows for the detection of chromosomal abnormalities in embryos without the need for invasive procedures that can harm the embryo (reviewed in Navarro-Sánchez et al., 2022). Our group among others has conducted different studies in this field, and the early clinical application of the cfDNA analysis in the culture medium has been proposed as a screening tool, giving a priority score for embryo transfer based on the media results (Rubio et al., 2019, 2020). In the final analysis of our multicenter study in day-6 and day-7 blastocysts, we have been able to compare the chromosomal results of the SBM with trophectoderm (TE) biopsies in 2,187 samples, and with the inner cell mass ICM in 230 blastocysts. In this study, the analysis of the cfDNA showed a concordance rate of 87% with the ICM biopsy, which is currently considered the gold standard for PGT-A. However, it is important to continue evaluating the factors that may affect the informativity and concordance rates of cfDNA-based assays, such as the culture day when medium is collected, contamination with external and/or cumulus cell DNA, and previous manipulation of the embryos. Multivariate analysis has shown that the longer the time in culture, the higher the informativity rate, and female age had the greatest impact on concordance rates with TE biopsies. Regarding technical aspects, the SBMs with higher number of reads after sequencing resulted in higher concordance rates, reflecting the importance of analysing samples with sufficient DNA quantity. Finally, the predictive value in terms of concordance with TE biopsies was higher for aneuploid blastocyst. No significant independent correlation was observed for embryo quality. The improvements in IVF lab protocols and the use of time-lapse and short incubation protocols for vitrified-thawed blastocysts are also noteworthy, as they may further increase the concordance rates of cfDNA-based assays with blastocysts. In fact, concordance in previously vitrified-thawed blastocysts as result as high as 90%. Regarding clinical outcomes, several groups have evaluated non-invasive PGT-A with transfer of euploid embryos showing good results. In a pilot study with only 7 SETs in couples with PGT-A or PGT-SR indication, 5 pregnancies were achieved resulting in 5 livebirths (Xu et al., 2016). In a second study, in couples with recurrent spontaneous abortion or repeated implantation failure, 50 transfers of euploid media were performed resulting in a 58% clinical pregnancy rate, with 27 healthy babies born (Fang et al., 2019). Similar results were described in good prognosis patients <38 years of age with ongoing pregnancy rates comparable to PGT-A (61.5%), and higher than conventional IVF or ICSI (48.5%) (Franco et al., 2021). Overall, the high concordance rates with trophectoderm biopsies and inner cell mass biopsies are encouraging, and the clinical outcomes with transfer of euploid embryos are promising. It is exciting to see the progress in non-invasive preimplantation genetic testing using cfDNA in culture medium, and it will be interesting to see how this technology evolves and its potential role as a screening or diagnostic tool in the future.

P-273 Heavy oil and conventional mineral oil produce similar Day 5 embryological outcomes. A prospective, single centre case study on 546 sibling 2PN zygotes

Abstract Study question Does heavy oil improve embryological outcomes compared to conventional mineral oil? Summary answer Heavy oil produces no improvement in embryological outcomes. What is known already Oil overlays are integral part of the culture system as they buffer gas and temperature exchanges in the culture media and reduce the risk of contamination and evaporation. However, slight variations in their composition, oxidative status, storage and utilisation can have a direct effect on culture system performance, eventually resulting in reduced embryo development and subsequent decrease in clinical outcomes. In an attempt to improve the performance of oil overlays, new formulations have recently reached the market. Nonetheless, their positive impact on embryological outcomes, as well as best practice in handling them, are yet to be broadly recognised. Study design, size, duration This is a prospective case-study conducted as part of routine consumable validation procedures undertaken when evaluating the use of new products in the IVF laboratory of a public hospital. A total of 546 sibling 2PN zygotes derived from 52 treatments were cultured to Day5 of development. All cases attending the clinic between March 2022 and January 2023 and leading to the collection of more than 10 oocytes were enrolled in this validation exercise. Participants/materials, setting, methods Sibling oocytes from IVF and ICSI treatments were cultured under the same conditions until fertilisation check (IVF) or after ICSI. Oocytes from each treatment were then equally split between conventional mineral oil (CONTROLS) and heavy oil (CASES) study groups. All embryos were individually cultured in 25µL drops of single step media at 37 °C, 5% CO2 and 5% O2. Embryological outcomes on Day3 and Day5 were compared across the study groups using Chi-square test. Main results and the role of chance CONTROLS and CASES showed similar embryological outcomes, including fertilisation (for ICSI only; 77.2% vs. 79.77%, respectively p = NS), good quality Day3 (69.5% vs. 75.6%, p = NS), good quality Day5 (30.2% vs. 24.0%, p = NS), blastulation (58.9% vs. 53.9%, p = NS) and utilisation rates (34.9% vs. 29.5%, p = NS). However, a sub-analysis was performed on the basis of oil handling/storage method. When heavy oil was pre-aliquoted and stored in 15mL tubes prior to usage, negative effects were measured on embryological parameters. In this subset of cases (n = 28 treatments; n = 304 2PN zygotes), although fertilisation and good quality Day3 rates were comparable (80.0% vs 81.6% and 69.1% vs. 73.0% in CONTROLS and CASES, respectively), CASES revealed a significantly lower rate of good quality Day5 blastocysts (Gardner grade ≥B for ICM and TE and expansion ≥grade3) compared to CONTROLS (32.2% vs. 21.7%, p = 0.04). Moreover, CASES showed significant lower embryo utilisation (36.8% vs. 25.7%, CONTROLS and CASES, respectively, p = 0.03) and a trend towards lower blastulation rate (61.2% vs. 50.7%, respectively, p = 0.06). Although the equal split of sibling oocytes between the two groups has minimised population bias, multiple variables may have influenced the performance of the tested product. We note that adequate storage/handling of products is required to avoid suboptimal results. Limitations, reasons for caution Although designed with sibling oocytes/embryos, this study is limited in population size. Moreover, only one product brand has been tested, limiting the universality of the conclusions about heavy oil performance in IVF culture systems. Wider implications of the findings Introducing a new product in the IVF laboratory should undergo a reliable validation exercise. Despite the commercial claims, heavy oil failed to improve our results. Untested variations may have detrimental effects on clinical performance and, if implemented without control, their effects may become impossible to identify at later stages. Trial registration number not applicable

O-292 Does ovulation trigger enhance live birth in a natural cycle for frozen embryo transfer: A randomized controlled trial

Abstract Study question Does ovulation trigger enhance pregnancy rate in a natural cycle (NC) for frozen embryo transfer (FET)? Summary answer Pregnancy rates after a FET are similar in a natural cycle with detection of a spontaneous LH surge or with a triggered ovulation. What is known already Several studies have explored whether one approach might appear to be more beneficial in terms of pregnancy or live birth rates, but with inconsistent results. Thus, the best protocol to identify the time of ovulation in NC and to plan the FET is still undefined. Study design, size, duration This was a bicentric, two-arm, randomized, controlled and open-label trial, including 108 patients for 18 months. In the spontaneous-NC group, the LH surge was defined as a rise of the serum LH level at least three times above the basal LH serum value with a serum progesterone level three days after the LH surge above 3 ng/mL. Participants/materials, setting, methods Patients included were aged 18- to 39-year-old, had regular cycles and were planned for an autologous frozen day-5 blastocyst transfer in a NC. They did not have endometriosis stage AFSr 3 nor 4 nor adenomyosis. Fifty-four patients per group were included. In the modified-NC group, a spontaneous LH surge was observed in 16 women before the hCG-trigger could be administrated: they were re-allocated to the spontaneous-NC for the per-protocol (PP) analysis. Main results and the role of chance Patients characteristics were comparable in both groups. Pregnancy rates were similar in the spontaneous-NC and the modified-NC group in the intention-to-treat (ITT) (41.3 versus 37.8% respectively, p = 0.85) and in the PP analysis (39.3% versus 40% respectively, p = 0.9). Ongoing pregnancy, miscarriage and live birth rates were also similar. Significantly more visits were needed in the spontaneous-NC (3.4 ± 0.81 versus 2.59 ± 1.0, p < 0.0001 in the ITT analysis and 3.18 ± 0.93 versus 2.63 ± 1.03, p = 0.0064 in the PP analysis). Limitations, reasons for caution Due to group redistribution (spontaneous LH surge before hCG trigger), the number of subjects in the modified-NC group is low for the PP analysis. This may reduce the power of the trial which may fail to detect a small difference. Wider implications of the findings The results reinforce previous data that showed similar efficacy of the s-NC and the m-NC. This allows the patient to choose from several options depending on her preference. Trial registration number NCT03428165

P-707 Evaluating DualStim/PGTA in poor prognosis patients: Comparison of double ICSI-PGTA vs. oocyte cryopreservation plus single PGTA in luteal phase through laboratory, genetic and clinical results.

Abstract Study question Which strategy is best for poor-prognosis patients: Two ICSI-PGTA cycles fresh oocytes (follicular plus luteal phase) or one ICSI-PGTA cycle with vitrified and fresh oocytes?" Summary answer Laboratory outcomes, ploidy rates and clinical results after euploid blastocyst transfer are similar between the two DualStim/PGTA strategies. What is known already Luteal phase ovarian stimulation (LPOS) after follicular phase ovarian stimulation (FPOS) is an effective strategy to obtain more oocytes in a single cycle and reduce time-to-pregnancy. The main goal of controlled ovarian stimulation in PGTA patients is to obtain a sufficient number of oocytes to have at least one euploid blastocyst. The standard treatment for achieving this goal is DuoStim plus oocyte cryopreservation. However, this method may decrease oocyte competence in older patients. To address this, we propose comparing the outcomes of two consecutive fresh ICSI-PGTA cycles to our standard protocol in older patients, as an alternative approach. Study design, size, duration This multicentric retrospective study evaluated 91 poor-prognosis PGTA cycles (≤ 5 antral follicle count) using the DualStim approach (182 oocyte pick-ups) in the same menstrual cycle, from January to October 2022. Fifty-three patients (group 1; 746 oocytes retrieved) underwent two consecutive egg retrievals, ICSI, and blastocyst biopsy, after FPOS and LPOS, while 38 patients (group 2; 743 oocytes retrieved) underwent oocyte vitrification after FPOS, followed by LPOS, egg retrieval, oocyte warming, ICSI, and blastocyst biopsy. Participants/materials, setting, methods We compared various parameters including oocyte number, maturity, fertilization rates, blastocyst quality, biopsy rates, and genetic results between two groups after both egg retrievals. Additionally, clinical outcomes after euploid embryo cryotransfer were also compared. Oocytes were microinjected, cultured in Geri® time-lapse incubators, and underwent embryo biopsy on day 5/6. Statistical analysis was conducted using R software (v.4.2.0), including maternal age as a confounding factor. Results were considered significant with a p-value less than 0.05. Main results and the role of chance Patient age was found to be significantly different between groups (40.5±2.2 in group-1 vs 38.5±2.7 in group-2; p < 0.001) and was used as a confounding factor in statistical comparisons. No significant differences were found in terms of oocytes retrieved per patient (14±7.1 vs 13.6±5.8), maturity rate (79.4 % vs 77.9%), fertilization rate (75.3 % vs 74.6%), or day-5 blastocyst rate (54.8% vs 51.7%). However, a significant difference was observed in the percentage of blastocysts biopsied per group (50.2% group-1 vs 43.0% group-2; p = 0.023). After genetic analysis, euploidy rate was found to be lower in group-1 compared to group-2 although not statistically significant (19.8% and 32.6%, respectively, p = 0.007/p=0.309 after adjusting for maternal age), and this led to similar average number of euploid embryos per DuoStim cycle (1.1±1.3 group-1 vs 1.3±1.4 group-2). Aneuploidy (73.9% vs 62%) and mosaicism rates (6.3% vs 5.4%) were also comparable between the two groups. No statistical differences were observed between the follicular and luteal phases in each group in terms of laboratory and genetic results. Preliminary clinical outcomes showed similar pregnancy rates with 12 pregnancies achieved in group 1 after 23 euploid embryo transfers (52.2%), and 14/20 in group 2 (70.0%) with no significant difference between groups. Limitations, reasons for caution This study is a retrospective analysis and further validation through prospective study is recommended. Additionally, while the average age of the study groups is statistically different due to bias in the clinical prescription of treatment, this difference was statistically corrected. Wider implications of the findings Cryopreserving oocytes obtained after FPOS and warming them after the subsequent LPOS does not negatively impact the quality or final results of a DuoStim PGTA cycle. This approach should be considered as it can reduce workload for the IVF laboratory and minimize costs for patients. Trial registration number none

P-755 Timeless importance of the embryo transfer technique: a real-life retrospective study comparing direct versus afterload catheters

Abstract Study question Can the embryo transfer (ET) technique influence clinical pregnancy rate (CPR)? Summary answer Our study demonstrated that CPR is significantly affected by the ET technique. In particular, with the afterload technique both CPR and easy transfer rates increased. What is known already Despite it having widely demonstrated that the occurrence of difficult ETs reduces the success of assisted reproductive technology (ART) cycles and that afterload technique has showed a decrease in the number of difficult transfers compared to the direct one, few data are available assessing pregnancy outcomes in the two different techniques. The present study represents the progression of a published prospective randomized controlled trial (RCT) performed in the same setting on a smaller population comparing direct and afterload techniques on the difficult transfer rate, but not designed to demonstrate differences in terms of CPR. Study design, size, duration Single center retrospective cohort analysis of 8,189 fresh and frozen single blastocyst transfers performed between January 2016 and December 2021 in a tertiary University affiliated fertility center. Of the total, 2,000 ETs were performed with the direct technique and 6,189 with the afterload one. All fresh single day-5 and single day-5 and day-6 frozen blastocyst transfers performed during the study timeframe were included. Participants/materials, setting, methods Direct technique was the only one used from January 2016 to September 2017. In the clinical trial recruitment period, the choice was given by randomization. From April 2019, only the afterload technique was used: the operator never had chance to choose the technique. Preimplantation genetic testing cycles, gamete donation and day-7 blastocyst transfers were excluded. CPR was the primary outcome, while difficult transfer rate the secondary one. Univariate and multivariate logistic regression have been performed. Main results and the role of chance During the period, 8,189 single blastocyst transfers were performed. Afterload ET showed a percentage of difficult transfers which was almost one third compared to that of the direct ET, being respectively 9.06% and 26.85% (OR 0.27, 95% CI 0.24-0.31p <0.001). Overall, the CPR between the two groups was significantly different: 44.69% in afterload ET and 41.65% in direct ET (OR 1.13, 95% CI 1.02-1.25, p = 0.017). As for the interaction between the two techniques and difficult transfer on CPR, only the direct difficult ET resulted in a significantly lower CPR with an OR of 0.62 (95%CI 0.49-0.77, p < 0.001). Lastly, among the 15 operators that performed the ETs, a range of difficult transfers from 3.8% to 45.4 % in the direct group and 0.8% to 20.5% in the afterload group was found (p < 0.001). Limitations, reasons for caution The main limitations of the study are the difference in sample sizes between the two groups, the wide range of time considered, and the 15 operators included. A further possible reason is the high prevalence of difficult transfer, probably due to the broad definition we used. Wider implications of the findings The study points out how in embryo transfers the use of the afterload technique instead of the direct one significantly improves clinical pregnancy rate and easy transfer rate. Since there is a limited knowledge on the embryo transfer technique impact, our findings aim to increase the effectiveness of embryo transfers. Trial registration number NCT05364528.

Predicting the likelihood of live birth: an objective and user-friendly blastocyst grading system

Research questionCan day-5 blastocysts be ranked according to their likelihood of live birth using an objective and user-friendly grading system? DesignA retrospective multicentre study conducted between 2017 and 2019, including 1044 day-5 blastocysts. Blastocyst expansion degree, trophectoderm and inner cell mass quality were assessed morphologically and morphometrically. Several analyses were conducted: the association between the qualitative and quantitative assessment for the blastocyst expansion degree and the number of trophectoderm cells; the effect of the embryo quality on day 3 and the contribution of the three blastocyst parameters to live birth, with logistic regression; and a decision tree with the most significant variables to create the new scoring system. ResultsCut-off points were found to discriminate between expanding and expanded blastocysts (165 µm for blastocyst diameter) and between trophectoderm grades (A: ≥14 cells; B: 11–13 cells; C: ≤10 cells). When the embryos reached the blastocyst stage, their quality on day 3 did not add predictive value for implantation and live birth. In the logistic regression analysis, the only parameter capable of significantly predicting the live birth likelihood was the trophectoderm grade: A versus C (OR 1.95, 95% CI 1.26 to 3.0); B versus C (OR 1.71, 95% CI 1.22 to 2.4). The decision tree supported the finding that the trophectoderm grade had the highest predictive value for a live birth, followed by the blastocyst expansion degree in a second step. ConclusionsThis new method makes objective blastocyst assessment feasible, allowing for standardization and exportation to other laboratories worldwide.

Modulation of Apoptosis and Autophagy by Melatonin in Juglone-Exposed Bovine Oocytes.

Melatonin, an antioxidant hormone secreted by the pineal gland, has been recognized as a regulator for numerous biological events. The deleterious effects of juglone, a polyphenolic extract of walnut trees, on embryo development has been previously reported. In the current study, we aimed to display the impact of melatonin administrated during in vitro oocyte maturation (IVM) on juglone-treated oocytes. Thus, in vitro matured oocytes were collected after 24 h post incubation with juglone in the presence or absence of melatonin. Reactive oxygen species (ROS), glutathione (GSH) content, mitochondrial distribution, and the relative abundance of mRNA transcription levels were assessed in oocytes, in addition, oocytes were in vitro fertilized to check the competency levels of oocytes to generate embryos. We found that administration of melatonin during the maturation of oocytes under juglone stress significantly improved the cleavage rate, 8-16 cell-stage embryos and day-8 blastocysts when compared to the sole juglone treatment. In addition, the fluorescence intensity of ROS increased, whereas the GSH decreased in juglone-treated oocytes compared to melatonin-juglone co-treated and untreated ones. Additionally, a significant increase in the mitochondrial aberrant pattern, the pattern that was normalized following melatonin supplementation, was observed following juglone administration. The mRNA analysis using RT-qPCR revealed a significant upregulation of autophagy and oxidative-stress-specific markers in the juglone-treated group compared to the co-treatment and control. In conclusion, the study reveals, for the first time, a protective effect of melatonin against the oxidative stress initiated following juglone treatment during the in vitro maturation of oocytes.

Comparison of clinical outcomes for different morphological scores of D5 and D6 blastocysts in the frozen-thawed cycle

BackgroundBoth embryo development speed and embryo morphology score played a significant role in frozen-thawed embryo transfer cycle (FET) outcomes. Most of the literature indicates that D5 embryos performed better than D6 embryos, although a few also indicate that there is no difference in clinical outcomes between D5 and D6 embryos. Clinically, D5 embryos are preferred for equal morphological scores. But how to choose embryos when the morphological score of D6 embryos is better than D5?MethodsA retrospective study including 8199 frozen-thawed embryo transfers (FETs) was conducted to analyze patients who underwent IVF-FET from January 2018 to December 2020. Patients were divided into 8 groups according to the rate of embryonic development and morphological scores to compare pregnancy outcomes. We further compared clinical pregnancy outcomes and neonatal outcomes between BC embryos on day 5 (D5) and BA/BB embryos on day 6 (D6).ResultsOur study found no difference in clinical pregnancy rate (CPR) and live birth rate (LBR) between AA/AB blastocysts in D5 or D6 frozen blastocysts. However, for BA/BB/BC blastocysts, embryonic pregnancy outcome was significantly better in D5 than in D6. In our further analysis and comparison of BC embryos in D5 and BA/BB embryos in D6, we found no difference in clinical pregnancy outcomes and neonatal outcomes, but D6 BA/BB embryos had a higher rate of miscarriage. After adjusting for confounding factors, none of the indicators differed between groups.ConclusionOur study provides suggestions for embryo selection: AA/AB embryos are preferred, regardless of the embryo development day, and the second choice is BA or BB embryos on D5. BA/BB embryos in D6 had a higher miscarriage rate than BC embryos in D5 but were not statistically significant after adjusting for confounding factors.

Live birth rate following frozen-thawed blastocyst transfer is higher in high-grade day 6 blastocysts than in low-grade day 5 blastocysts.

Day 5 (D5) blastocysts are generally given priority to transfer than day 6 (D6) blastocysts; however, which one should be prioritized to transfer when only low-grade D5 and high-grade D6 blastocysts are available? A large retrospective cohort study was carried out to evaluate the live birth rate (LBR) following D5 and D6 blastocysts in single frozen-thawed blastocyst transfer (FBT) during January 2014 and December 2018.A multivariate logistic regression was conducted to evaluate the combined impact of expansion day (D5 and D6) and blastocyst quality (high grade/low grade) on LBR, accounting for the potential confounding factors. The biopsied blastocysts from a consecutive PGT-A case series during February 2013 to December 2021 were analyzed in a supplementary study. The LBR achieved in high-grade D6 blastocyst transfer was significantly higher than that in low-grade D5 blastocyst transfer (50.43% vs. 40.70%, aOR 1.54, 95% CI 1.05-2.26, p = 0.027). There were no significant differences in preterm birth rate, very preterm birth rate, mean live birth weight, and birth weight <1,500 g and >4,000 g between the two cohorts. As for aneuploidy analysis in PGT, there were 54.55% of euploid blastocysts (30/55) among high-grade D6 blastocysts, significantly higher than the 41.39% of euploid blastocysts (565/1,365) among low-grade D5 blastocysts (p < 0.001). Our data suggest that D6 blastocysts with high morphology grading are preferred than D5 blastocysts with low morphology grading when selecting blastocyst transfer to shorten the time of conception.

Correlations between clinical parameters, blastocyst morphological classification and embryo euploidy.

The aim of the present study was to evaluate clinical and embryo parameters to predict embryo ploidy. In this retrospective analysis, we studied 838 biopsied day-5 blastocysts from 219 patients in the period from May 2021 to July 2022. All embryos were morphologically classified before biopsy and were divided into two groups according to genetic test results. Euploid embryos (299) were compared with aneuploid embryos (539) based on maternal age, anti-Mullerian hormone, antral follicle count, and embryo morphology. Maternal age (36.2±3.0) of euploid embryos was lower than maternal age (37.1±2.5) of aneuploid embryos (p<0.0001). AMH levels were higher (3.9±1.2) in the group of euploid embryos than in the group of aneuploid embryos (3.6±1.3, p<0.0001). However, the AFC was not different in the group of euploid embryos (15.3±6.0) compared to the group of aneuploid embryos (14.5±5.9, p=0.07). The presence of aneuploidy was negatively correlated with top embryo quality (embryos 4AA and 4AB). All euploid embryos (299) were top quality versus 331 of 539 (61.49%) aneuploid embryos (p<0.0001). We found that euploid embryos were associated with lower maternal age, higher AMH levels, and higher quality embryos.

Clinical outcome analysis of frozen-thawed embryo transfer on Day 7.

To investigate the clinical outcomes of Day 7 (D7) frozen-thawed embryo transfer (FET) and to provide a reference value for clinical work. This was a retrospective cohort study. Patients undergoing FET cycles in the Reproductive Medicine Center of the Third Affiliated Hospital of Zhengzhou University between December 2015 and January 2021 were included. According to the developmental stage of the embryos at transfer, the embryos were divided into three groups: Day (D) 5, D6 and D7 blastocysts. Group D7 was compared with Groups D5 and D6. Simultaneously, the preimplantation genetic testing (PGT) and non-PGT cycles in Group D7 were analyzed and compared. The main outcomes were the clinical pregnancy, live birth and miscarriage rates. The secondary outcomes were the implantation and euploidy rates. In total, 5945, 4094 and 137 FET cycles were included in the D5, D6 and D7 groups, respectively. The clinical pregnancy rate was significantly lower in Group D7 than in Groups D5 (13.9% vs 62.9%, P <0.001) and D6 (13.9% vs 51.4%, P <0.001). Additionally, the live birth rate was significantly lower in Group D7 than in Groups D5 (7.3% vs 50.7%, P <0.001) and D6 (7.3% vs 40.5%, P <0.001). However, the miscarriage rate was significantly higher in Group D7 than in Groups D5 (47.4% vs 18.2%, P =0.001) and D6 (47.4% vs 20.6%, P =0.004). The clinical pregnancy and live birth rates for D7 blastocysts were significantly higher in the PGT group than in the non-PGT group (41.7% vs 13.9%, P=0.012; 33.3% vs 7.3%, P =0.003). D7 blastocyst transfer can yield a live birth rate that is lower than that for D5 and D6 blastocysts but has value for transfer. PGT for D7 blastocysts may reduce the number of ineffective transfers and improve the outcome of D7 blastocyst transfer, which can be performed according to a patient's situation.

Clinical application of noninvasive chromosomal screening for elective single-blastocyst transfer in frozen-thawed cycles

BackgroundThe objective of this study was to explore the clinical application of noninvasive chromosomal screening (NICS) for elective single-blastocyst transfer (eSBT) in frozen-thawed cycles.MethodsThis study retrospectively analysed the data of 212 frozen-thawed single-blastocyst transfers performed in our centre from January 2019 to July 2019. The frozen embryos were selected based on morphological grades and placed in preincubation for 6 h after warming. Then spent microdroplet culture media of frozen-thawed blastocysts were harvested and subjected to NICS. The clinical outcomes were evaluated and further stratified analysis were performed, especially different fertilization approaches.ResultsThe clinical pregnancy, ongoing pregnancy, and live birth rates in the euploidy group were significantly higher than those in the aneuploidy group (56.2% versus 29.4%) but were nonsignificantly different from those in the chaotic abnormal/NA embryos group (56.2% versus 60.4%). Compared with day6 (D6) blastocysts, D5 blastocysts had a nonsignificantly different euploidy rate (40.4% versus 48.1%, P = 0.320) but significantly increased clinical pregnancy (57.7% versus 22.2%, P < 0.001), ongoing pregnancy (48.1% versus 14.8%, P < 0.001), and live birth rates (48.1% versus 13.0%, P < 0.001). The percentage of chaotic abnormal/NA embryos group was significantly higher among D5 embryos than among D6 embryos (30.1% versus 11.1%, P = 0.006). The percentage of aneuploid embryos was higher among the embryos with lower morphological quality(21.5% among ‘good’ embryos versus 34.6% among ‘fair’ embryos versus 46.0% among ‘poor’ embryos, P = 0.013); correspondingly, the overall clinical pregnancy, ongoing pregnancy and live birth rate rates showed similar declines.ConclusionsNICS combined with morphological assessment is an effective tool to guide frozen-thawed SBT. The optimal embryo for SBT is a ‘euploid embryo with good morphology’, followed sequentially by a ‘chaotic abnormal/NA embryo with good morphology’, ‘euploid embryo with fair morphology’, and ‘chaotic abnormal/NA embryo with fair morphology’.

ICSI in non-male factor infertility patients does not alter metabolomic signature in sibling embryos as evidenced by sensitivity enhanced nuclear magnetic resonance (NMR) spectroscopy.

Intracytoplasmic sperm injection (ICSI) was developed to overcome male factor infertility, however, there recently has been an increasing trend in ICSI usage irrespective of the etiology, demonstrating an overuse of this insemination technique. There is a limited knowledge on the behaviour of ICSI derived embryos in non-male factor infertility patients. Metabolomic assessment of preimplantation embryos in conjunction with morphological evaluation can provide better understanding of embryonic behaviour. Hence, this study was undertaken to explore if there are any metabolomic differences between IVF and ICSI derived sibling day-5 blastocysts from non-male factor infertility patients. This prospective study included nineteen couples with non-male factor infertility undergoing Assisted Reproductive Technology. The sibling oocytes retrieved from each patient were randomly assigned to two groups and inseminated either by IVF or ICSI. Spent culture media (SCM) in which embryos were cultured up to day 5 were collected and investigated using sensitivity enhanced NMR based metabolite profiling utilizing high resolution (800 MHz) NMR equipped with cryogenically cooled micro-coil (1.7 mm) probe. The metabolomic signature between IVF and ICSI derived sibling blastocysts was assessed. A significant reduction in the concentrations of pyruvate, citrate, glucose and lysine were observed in both IVF and ICSI sibling embryos compared to medium control (P< 0.05–0.001). Further, histidine and valine level was found lower in ICSI embryos compared to medium control (P<0.05) during 96 hours of in vitro culture. Notably, between IVF and ICSI SCM, no significant difference in the concentration of the metabolites was found. Our results suggest that ICSI in non-male factor does not alter the SCM metabolomic signature during 96 hours of embryonic development.

Developmental potential of different embryos on day 3: a retrospective study

To investigate how different quality of day 3 (D3) embryos affect blastocyst formation and clinical outcomes. This retrospective study analysed 699 patients undergoing assisted reproductive technology (ART) between January 2017 and February 2021. A total of 2517 D3 embryos were transferred to blastocyst medium for extended culture. D3 embryos were divided into five groups. Grade A, 6–10 cells, symmetrical blastomeres and <20% fragmentation; grade B, 6–10 cells, uneven blastomeres and ≥20% fragmentation; grade C, >10 cells, symmetrical blastomeres and <20% fragmentation; grade D, >10 cells, uneven blastomeres and ≥20% fragmentation; grade E, <6 cells. Status of day 5 (D5) and day 6 (D6) blastocysts and the clinical outcomes (blastocyst transfer) of each D3 embryo were recorded. The grade C group showed a higher D5 blastocyst formation rate and a high-quality blastocyst rate than other groups (p<.05). However, the clinical pregnancy rates in the grade A group were higher than other groups (p<.05). Embryos with low speed of development (grade E group) showed considerable clinical outcomes that were still worth investigating. D3 embryos with less fragmentation and ≥6 symmetrical blastomeres revealed a higher developmental potential, while embryos with 6–10 blastomeres showed the ideal clinical outcomes. Impact Statement What is already known on this subject? Accurate embryo evaluation can effectively reflect the developmental potential of different embryos. The number of blastomeres, proportion of fragmentation, and blastomere symmetry are three important and popular morphologic parameters used for evaluating day 3 (D3) embryos. However, in existing reports, combining these three parameters for embryo evaluation often results in different results. This is because different researchers have chosen different criteria for these three parameters. What do the results of this study add? In this retrospective study, we summarised the medical records of our reproductive centre in the past three years, redefined the evaluation method of the D3 embryos, and analysed the corresponding developmental potential and clinical outcomes. We conclude that although the embryonic development potential of grade C embryos (>10 cells, symmetrical blastomeres and/or <20% fragmentation) is relatively good, the results of grade A embryos (6–10 cells, symmetrical blastomeres and/or <20% fragmentation) are better in terms of clinical outcomes. What are the implications of these findings for clinical practice and/or further research? We believe this is meaningful for embryologists to choose embryos for transfer and predict the clinical outcome of IVF cycles.