Time-lapse System Research Articles

PREDICTING WHICH BLASTOCYSTS WILL DEVELOP INTO FETUSES: TIME INTERVAL FROM MORULAE TO BLASTOCYSTS IS A NOVEL AND USEFUL INDICATOR FOR SUCCESSFUL PREGNANCY

The increased use of blastocyst transfer has emphasized the importance of selecting high-quality embryos. Analyses of embryos in a time-lapse incubator system showed that zygotes with the shortest intervals between the first and second or the second and third mitotic divisions were higher blastocyst rate. Additionally, embryos that developed relatively faster to the morulae and blastocysts had higher implantation rates. However, the useful indicators that predict which embryos will develop into fetuses without pregnancy loss have not yet been identified. Do time to morulae and interval between morulae and blastocysts predict which blastocysts will develop into fetuses? We analyzed 208 blastocysts derived from ICSI in 177 patients from September 2019 to September 2020. All embryos were monitored by a time-lapse system. The outcome of vitrified-thawed single blastocyst transfers was classified into pregnancy and non-pregnancy groups. The pregnancy group was divided into ongoing pregnancy and pregnancy loss. Data were analyzed using multiple logistics regressions. According to the logistic analysis of women age, the data were classified as less than or more than 39 years old. Time-lapse analyses provided data on time from pronuclear fading to morulae (tM), time to expanded blastocysts (tEB), interval between morulae and blastocysts (tB-tM), the number of equatorial trophectoderm cells (eTE), and the KIDScore D5 v3 (KS5). Data from pregnancy (hCG > 50 mIU/ml at 4 weeks) versus non-pregnancy were compared, as well those from ongoing pregnancy (a gestational sac at 12 weeks) versus pregnancy loss groups. In this study, 51.4% of total blastocyst transfers resulted in pregnancy. Significant differences were found between pregnancy and non-pregnancy groups: tM, 63.0 vs. 65.8 h; tEB, 92.4 vs. 95.5 h; tM-t8, 29.5 vs. 32.2 h; eTE, 6.5 vs. 5.5; and KS5, 6.7 vs. 5.8. The pregnancy rate in women less than 39 years old was significantly higher than in women over 39 years old (67.4% vs. 38.1%). Under 39-years-old, ongoing pregnancy rate was 79.7%, and pregnancy loss rate was 20.3%. A faster tM, a longer tB-tM, and a more eTE was found in the ongoing pregnancy group compared to the pregnancy loss group. The potential of tM, tB-tM and eTE as indicators for pregnancy was predicted with an AUC of 0.79. Over 39-years-old, ongoing pregnancy rate was 72.1%, and pregnancy loss rate was 27.9%. In ongoing pregnancy group, t2, t4 and tM were significantly different to the pregnancy loss group, which suggested delay of early embryonic development increases pregnancy loss risk. Under 39 years old, tM, tB-tM and eTE are effective predictors for selection of high-quality blastocysts that will be successful pregnant. The time interval tB-tM is a new indicator for identifying high-quality blastocysts. Over 39 years old, faster embryo development is a critical indicator for pregnancy.

INCREASED AGE AND BODY MASS INDEX RELATES TO INCREASED TIME-LAPSE VARIABILITY AMONG EMBRYOS FROM THE SAME CYCLE

The negative effect of the increased age and obesity on women’s fertility, natural conception and assisted reproduction is already well known. To date, it is still not fully clear how these important factors could influence different aspects of embryo development and morphokinetics. The aim of the present study was to assess the association between the inter-embryo variability of morphokinetic parameters and patient age and body mass index (BMI). A total of 305 women (967 embryos) were included for analysis in this study. All embryos were cultured in a single culture medium (Life Global, Brussels, Belgium) and a time-lapse system (EmbryoScope, Vitrolife, Sweden). Only women with more than 2 embryos per cycle were included in this study. Fifteen morphokinetic parameters were analyzed: time of pronuclei appearance (tPNa), time of pronuclei fading (tPNf), cleavage times (t2, t3, t4, t5, t6, t7, t8, t9), morulae formation time (tM), starting blastulation (tSB), full blastocyst stage (tB), expansion (tEB) and hatching timing (tHB). Statistical analysis was performed using IBM SPSS Software v.21. Inter-embryo variability for each woman/cycle was measured as the coefficient of variation (CV). Spearman correlation analysis was done to find the association between the coefficients of variation of morphokinetic variables per cycle and female age and BMI. Women age showed low but significant positive correlation with the inter-embryo variability (CV) of certain morphokinetic variables – t7 (r=0.17, p=0.006), t8 (r=0.22, p=0.006), t9 (r=0.13, p=0.048), tB (r=0.18, p=0.022). Moreover, BMI also showed low but significant positive correlation with relatively higher number of CV of time-lapse variables, including earlier events – t3 (r=0.20, p=0.038), t4 (r=0.25, p=0.012), t5 (r=0.24, p=0.02), t6 (r=0.26, p=0.012), t8 (r=0.23, p=0.03) and t9 (r=0.23, p=0.045). In conclusion, time-lapse variability of human embryos is associated with women age and BMI. Embryo morphokinetics variability differs in the initial stages of embryo development according to BMI and in the later stages according to female age.

TIME-LAPSE VARIABILITY OF HUMAN EMBRYOS IS ASSOCIATED WITH IVF OUTCOME

Many morphokinetic parameters obtained from human embryos cultured in time-lapse imaging systems have been evaluated as biomarkers for optimal selection of embryos with high implantation potential. However, it is not clear whether the variability of time-lapse parameters among embryos from the same cycle differ between cases with successful and unsuccessful implantation. The purpose of this study was to quantify and compare the variability of time-lapse parameters among embryos from the same cycle between women with successful and unsuccessful implantation. 1712 blastocysts generated from ICSI between January 2017 and November 2020, derived from 437 patients were included in this retrospective study. Among them, 170 women had successful implantation (positive beta hCG test) and 267 women had unsuccessful implantation (negative beta hCG test) after embryo transfer with embryos from the studied group. Only women with more than 2 embryos per cycle were included in this study. All embryos were cultured in single culture medium (Life Global, Brussels, Belgium) and a time-lapse system (EmbryoScope, Vitrolife, Sweden). Fifteen morphokinetic parameters were analyzed: time of pronuclei appearance (tPNa), time of pronuclei fading (tPNf), cleavage times (t2, t3, t4, t5, t6, t7, t8, t9), morulae formation time (tM), starting blastulation (tSB), full blastocyst stage (tB), expansion (tEB) and hatching timing (tHB). Statistical analysis was performed by IBM SPSS Software v.21. Inter-embryo variability of each woman/cycle was measured as the coefficient of variation (CV), expressed as percentage. The CV value of each patient was compared between women with and without successful implantation using Student t-test. P<0.05 was considered statistically significant. There were no significant differences in women age, 3 Day FSH, BMI and morphological embryo quality between the compared groups (p>0.05, Student t-test). However, there was a significant difference of the CV values between women with successful and unsuccessful implantation in 8 of the studied time-lapse variables – tPNa (4% vs. 8%, respectively, P=0.001), t3 (6% and 9%, P=0.021), t4 (5% and 8%, P=0.004), t6 (6% and 8%, P=0.012), t7 (6% and 9%, P=0.005), tM (5% and 7%, P=0.010), tSB (1% and 2%, P=0.033), and tB (0.7% and 1%, P=0.024). Lower inter-embryo variability among embryos from the same cycle is associated with higher chance of successful implantation. The inter-embryo variation of time-lapse parameters could be considered as a potential predictor for successful implantation.

STUDY ON THE RELATIONSHIP BETWEEN THE ANGLE OF MII SPINDLE AND THE DIFFERENCE IN THE SIZE OF 2PN IN HUMAN ZYGOTE USING TIME-LAPSE SYSTEM

Previous studies have shown that the angle of spindle in MII oocyte is associated with embryo development. The incidence of zygotes with significant pronucleus size asynchrony is considered to be an indication of the poor embryo development. However, the correlation between angle the of spindle and difference in the size of 2PN have not been studied. In the present study, we investigated the correlation between the angle of spindle and the difference in the size of 2PN, and the effect of its correlation on clinical outcomes. Data were retrospectively collected from polarized light microscopy and time-lapse system performed from March 2018 to February 2020 in the Fertility center of CHA Gangnam Medical Center. We analyzed 235 zygotes from 66 patients. We measured the angle of spindle prior to ICSI using a LC-PolscopeTM (CRI). Oocytes were divided into two groups according to the angle of MII spindle (<30° group =189, ≥30° group =46) and were cultured in EmbryoscopeTM (Vitrolife). The size of 2PN were analyzed by measuring the horizontal diameter at the same focal plane before PN fade. Also, an abnormal division (direct division and not available cell number due to poor quality)in 2-cell embryo was checked. The average of difference in the size of 2PN was significantly increased in ≥30° group (<30° group; 1.4 um vs. ≥30° group; 3.5 um, p< 0.05). The rate of abnormal division (8.5% vs.23.9%) was highly increased but the rate of good quality (63.5% vs. 47.8%) was decreased in the ≥30° group compared with <30° group (p <0.05). When only embryos from the <30° group were selected for transfer, the pregnancy and implantation rates were 67.7% and 57.1%. But when only embryos from the ≥30° group were transferred, those rates were 25.0% and 20.0%, respectively (p <0.05). This is the first study that has analyzed the correlation between spindle angle and difference in the size of 2PN. Further studies with larger sample size are needed. This study could provide a new insight for the embryo selection by enabling noninvasive judgement of embryos that are more likely to become pregnant at the earliest time point. Also, it could be especially useful to assess zygote quality in countries with strict policies.

P–144 Undisturbed culture in time-lapse systems improves embryo development and quality

Abstract Study question Does culture in integrated time-lapse systems (TLS) improve embryo development and blastocyst quality compared to conventional benchtop incubators (CI), within the same IVF laboratory? Summary answer Under similar conditions, culture in TLS resulted in a significant increase in blastocyst rate, top quality blastocyst rate and proportion of biopsied embryos per treatment What is known already Integrated TLS have the potential of delivering a stable and undisturbed environment throughout the whole embryo culture, avoiding taking them out for assessment. However, there is still lack of quality evidence of the performance of these incubators compared to CI at supporting embryo culture until blastocyst stage. Studies abording this issue are still scarce, heterogeneous and have a small sample size. Although some authors have reported an improvement in embryo development and quality using TLS, global results are inconsistent. To our knowledge, the present study evaluates the effect of TLS on embryo quality on the largest sample size yet. Study design, size, duration Unicentric retrospective cohort study including 14248 ICSI treatments from 2016 to October 2020, with both autologous and donated oocytes. We compared blastocyst rate (BR) and proportion of top-quality blastocysts (TQB=Morphology ASEBIR score A) per treatment between those using TLS (N = 7500) and CI (N = 6748), and the proportion of embryos biopsied (EB) in cycles with pre-implantation genetic testing (PGT-A; N = 2642). We performed a sub-analysis in treatments using single-step culture medium (N-TLS=4398, N-CI=1140) in both types of incubators. Participants/materials, setting, methods Embryo cohorts were cultured until blastocyst stage in one of 3 TLS: EmbryoScope, EmbryoScope Plus (Vitrolife,) and Geri (Genea Biomedx), or in a CI (ASTEC). Embryo quality was assessed following ASEBIR morphological criteria. Culture protocols and media changed during the included time period. For that reason, we did a sub-study in the treatments performed since the implementation of Gems® (Genea Biomedx) single-step (SS) culture medium in all incubators. Statistical analysis was done using ANOVA tests. Main results and the role of chance Treatments were differently distributed and heterogeneous in terms of number of oocytes obtained per patient, so we stratified the analysis according to ovum origin and compared mean rates per cycle instead of total number of embryos per group. BR was statistically higher (P &amp;lt; 0,001) in the TLS group, in both autologous (62,98±29,37% vs 59,49±31,09% in CI) and oocyte donation treatments (69,25±22,07% vs 66,27±23,28% in CI). Proportion of TQB was also significantly higher in the TLS in both types of cycles (P &amp;lt; 0,001): 3,60±12,29% in TLS vs 2,27±9,71% in CI in autologous cycles, 8,68±15,31% in TLS vs 7,32±14,02% CI in ovum donation cycles. Results were corroborated in the SS media sub-study (P &amp;lt; 0,05): BR was 63,87±29,23% in TLS vs 57,53±30,61% in CI with autologous oocytes, and 70,76±21,63% in TLS vs 67,39±22,68% in CI with donated oocytes; TQB rates were 3,66±12,06% in TLS vs 2,05±9,26% in CI in autologous treatments and 8,81±15,21% in TLS vs 6,84±12,91% in CI in ovum donation treatments. Regarding PGT-A treatments, we found no significant difference in the biopsy rate in the total comparison, although the rate significantly increased in the TLS group since the implementation of single-step medium (52,36±24,69% in TLS vs 48,63±22,56% in CI; P = 0,007) Limitations, reasons for caution Not only culture conditions varied over time, but also the number of TLS in the laboratory, which increased lately. Hence, even though the most recent treatments included in the all-SS sub-study are more homogeneous in terms of culture conditions, they are unbalanced regarding the distribution among incubators. Wider implications of the findings: Our results demonstrate the superiority of TLS coupled with single-step culture media against traditional embryo culture systems at supporting embryo development. The optimal environment provided by TLS enhances embryo development until blastocyst stage as well as their quality, increasing the cumulative chances of getting a life-birth for each patien. Trial registration number Not applicable

O-214 Undisturbed embryo culture under High Humidity atmosphere in a time-lapse system increases pregnancy rates

Abstract Study question Does culture in high relative humidity conditions (HC) improve pregnancy rates when using a time-lapse system (TLS) and single-step (SS) culture medium? Summary answer Using an integrated-TLS and SS medium, culture under HC increases the likelihood of embryos to achieve a pregnancy with respect to those cultured in DC. What is known already Many variables affect embryo development, and need to be precisely tuned in every IVF laboratory, especially inside the incubators. TLS provide stability during embryo culture, which is a well-known key factor for a proper embryo development. The humidity content of culture atmosphere is especially relevant in order to avoid oscillations in culture media osmolality. It has been previously reported that culture under HC has a significant effect on embryo quality and morphokinetics. However, studies assessing the effect of HC in clinical outcome are rare and inconclusive, mostly due to the variability in the incubator device used and insufficient sample size. Study design, size, duration The present is a retrospective study performed over 1624 ICSI treatments from 3 fertility clinics from December 2017 to October 2020. Zygote cohorts were randomly assigned to dry (N = 794) or humid conditions (N = 830). It includes autologous treatments with (N = 555) and without (N = 368) pre-implantation genetic testing (PGT) and egg donation treatments (N = 701). Following selection by combining morphological and morphokinetic criteria, 1611 mostly single embryo transfers (92%) were performed, 779 from DC and 832 from HC. Participants/materials, setting, methods Stimulation, oocyte pickup and fertilization were performed according to the standard procedures of the clinic. We used a GERI incubator (Genea Biomedx), with 6 separated chambers for individual patients, 3 of them configured to work in DC, and 3 in HC. Embryos were cultured in specific 16-well GERI trays with single-step Gems® culture medium (Genea Biomedx). The effect of HC in pregnancy rate was assessed by multivariate logistic regression and Pearson Chi Square Test. Main results and the role of chance Types of treatment and patient demographics were homogeneously distributed in the two study groups. Mean patient age was 39.88±4.47 years, BMI: 23.54±4.21 Kg/m2 and number of correctly fertilized oocytes: 7.86±3.87. A logistic regression was performed, including other possible affecting factors: ovum age and origin, transfer day, fresh or frozen-warmed embryo transfer, number of transferred embryos and the use of PGT. Said analysis revealed that embryos cultured in HC are more likely to achieve a pregnancy than those cultured in DC (OR = 1.30, 95% CI (1.05-1.59), p=0.014). Pregnancy rate was significantly higher in HC (66.7%) than in DC (60.9%) in the total embryo transfers (p = 0.017). Pregnancy rate was also higher in HC in fresh embryo transfers (68.6% in HC vs 63.2% in DC; p = 0.133) and frozen-thawed transfers (65.2% in HC vs 59.1% in DC; p = 0.062), although differences were not statistically significant due to the reduced sample size. Stratifying the results, the significant difference remained in transfers belonging to autologous cycles (68.4% HC vs 56.5% in DC; p = 0.030) and in treatments in which PGT was performed (67.1% HC vs 56.0% in DC; p = 0.023), but the difference in egg donation procedures was not statistically significant (66.4% in HC vs 64.7% in DC, p = 0.577). Limitations, reasons for caution This is a retrospective analysis performed over the clinics’ treatments, so it might be compromised by some bias, although multivariable analysis may overcome them. For further assessing the effect of HC in clinical results a prospective controlled study, with a larger sample size could be performed, also comparing life-birth rates. Wider implications of the findings These results, alongside our previous findings (Valera et al. 2020, Albert et al. 2020), support that HC contributes to optimize embryo development and clinical results in undisturbed culture in TLS with single-step medium. To our knowledge, this is the largest study on the matter and the first performing multivariable analysis. Trial registration number Not applicable

P–241 Construction of a Machine Learning algorithm based on early morphokinetics for human blastocyst development prediction: a retrospective analysis of 575 cleavage-stage embryos

Abstract Study question Can morphokinetic features included into Machine Learning (ML) algorithms identify cleavage-stage embryos with the best chance to reach the expanded blastocyst stage on day 5? Summary answer A ML algorithm based on early morphokinetic features can identify cleaving embryos that will reach the expanded blastocyst stage on day 5. What is known already To date, the conventional morphology assessment of cleaving human embryos has a limited predictive power on further embryo developmental potential. The morphokinetic analysis using Time-Lapse systems (TLS) was introduced in order to provide a new tool to identify dynamic biomarkers of embryo quality. More recently, ML approach has been applied for the analysis of specific embryo-related features, aiming at developing predictive algorithms to assess the embryo development potential. Study design, size, duration We retrospectively analysed 575 embryos obtained from 80 women aged 25–42 years, with normal BMI, AFC≥8, day 3 FSH&amp;lt;12 IU/l, AMH&amp;gt;2.5 ng/ml, no diagnosis of polycystic ovary syndrome or endometriosis. These patients underwent IVF at our IVF Unit between March 2018 and March 2020; their embryos were cultured using the Geri plus® TLS and a single blastocyst transfer was performed. Participants/materials, setting, methods A total number of 29 morphological and morphokinetic parameters were considered to build six different ML algorithms. The performance to assess which was the best-fitting algorithm was calculated using the ROC curve considering accuracy (% of embryos correctly classified by the algorithm), Cohen-kappa coefficient (measurement of the agreement among features), mean number of TP (embryos correctly classified as undergoing developmental arrest), mean number of TN (embryos uncorrectly classified as undergoing developmental arrest). Main results and the role of chance Overall, 210 embryos progressed to the expanded blastocyst stage on day 5 (BL group), whereas 365 displayed developmental delay or arrest at any stage (nBL group). Among the six different algorithms, the best-fitting algorithm was obtained using the Kbest features selection approach combined with a Random Forrest evaluation strategy. This algorithm was based on 7 variables: embryo morphological score on day 2, pronuclear fading time (tPNf), completion time of cleavage to two, four and eight cells (t2, t4, and t8 respectively), time intervals t4-t3 and t8-t4. The algorithm showed an AUC of 0.78, with an accuracy of 0.73, a Cohen-kappa of 0.41, a mean TP number of 302/365 embryos in the nBL group and a mean TN number of 120/210 embryos in the BL group. Mean false positive (FP) and false negative (FN) numbers were of 63 and 90.2, respectively. Limitations, reasons for caution The results obtained in this study may not be generalizable to patients with other clinical characteristics, to other time-lapse systems or different laboratory settings. The predictive power of the algorithm should be validated prospectively on a larger number of embryos. Wider implications of the findings: The current study represents a preliminary analysis for the development of hierarchical predictive models for embryo assessment based on their developmental potential, that embryologists will be able to apply as a support for decision-making. Trial registration number Not applicable

P–197 Two different strategies for embryo culture and selection: time-lapse with single-step medium and conventional incubator with sequential media. Are there differences in clinical results?

Abstract Study question Is there a significant difference in the clinical results of embryos cultured in time-lapse systems with single-step medium and conventional benchtop incubators with sequential media? Summary answer Embryos cultured in time-lapse systems and single-step media are more likely to achieve an ongoing pregnancy and have higher implantation rates than those cultured otherwise. What is known already One of the strategies for embryo culture in IVF consisted in conventional benchtop incubators combined with sequential culture media (CI-Seq). New generation time-lapse systems provide useful information on the morphokinetics of embryo development, but also a stable culture environment where embryos can develop undisturbed until blastocyst stage when paired with single-step culture media (TLS-SS). These features have the potential to improve embryo development and selection. Nonetheless, there is inconclusive evidence of whether this new culture strategy has a significant effect on clinical results of ICSI treatments. Studies on the matter are heterogeneous and reduced in both number and sample size. Study design, size, duration Unicentric retrospective cohort study. We compared the results of 11471 blastocyst transferences from 10276 ICSI treatments performed during 4 consecutive years, where embryos were cultured either on CI with sequential media (N = 5255) or a TLS with single-step medium (N = 5021). 3922 of the totals were fresh embryo transfers (ET) and 7549 frozen-thawed ET. We compared the implantation rate (IR) and ongoing pregnancy rate (OGPR) in both study groups, stratifying by ovum origin. Participants/materials, setting, methods Three models of TLS were used for embryo culture: EmbryoScope, EmbryoScope Plus (Vitrolife) and GERI (Genea Biomedx), as well as one CI (ASTEC). Sequential media: Cook, Origio, Vitrolife; Single-step media: Gems, Irvine, Life Global. Embryo scoring and selection was performed by ASEBIR criteria in the CI group, and by morphological and morphokinetic assessment for embryos cultured in TLS. Embryos were extracted from the CI only for media change. Statistical analysis: ANOVA tests and Logistic regressions. Main results and the role of chance A general Logistic Regression was performed, including egg origin, PGT-A and culture strategy to explain their impact in OGPR. Egg origin (OR = 1,094 (95%CI: 1,015–1,179); P = 0,019) and culture strategy (OR = 1,141 (95%CI: 1,060–1,229); P &amp;lt; 0,001) were statistically significant, which confirms the need for stratification due to the heterogeneity of the groups. The total IR in the TLS-SS group was 54,68±48,84%, significantly higher than that of CI-Seq (49,18±47,91%; P &amp;lt; 0,001). In ovum-donation treatments, a complete Logistic Regression for OGPR, with all typical confounding variables (age, BMI, nº oocytes, fresh/frozen transfer, number and day of ET) resulted in an OR = 1,187 (95%CI: 1,074–1,313; P = 0,001) favoring culture in TL-SS. IR in these treatments were 61,98±47,68% in TL-SS vs 55,08±46,58% in CI-Seq (P &amp;lt; 0,001) in fresh transfers and 51,48±48,91% in TL-SS vs 44,39±47,67% in CI-Seq (P &amp;lt; 0,001) in frozen-thawed ET. In autologous treatments with PGT a similar regression yielded an OR = 1,055 (95%CI: 0,889–1,252; P = 0,542) for culture strategy. The IR of genetically tested ET was not significantly different: 53,08±49,49% for TL-SS, 50,90±49,07% for CI-Seq, P = 0,246. In autologous procedures without PGT, culture strategy was not significant for OGPR (OR = 0,998 (95%CI: 0,835–1,191), P = 0,979) nor IR of fresh (49,75±48,91% TL-SS vs 44,23±47,36% CI-Seq; P = 0,081) nor frozen-thawed transferences (50,77±48,33% TL-SS vs 50,67±47,33% CI-Seq; P = 0,970). Limitations, reasons for caution After fertilization check, embryos were evaluated exclusively on D5/6. On D3, embryos cultured in CI were taken out only for a quick media change, but not for evaluation, and all handling was done in isolette cabins with controlled environmental conditions. Being a retrospective study, there is high variability in population. Wider implications of the findings: A more homogenous prospective study, including comparison in life-birth rates, is necessary to extract final conclusions. However, our results suggest that the introduction of TLS and SS media in IVF laboratories might be a valid strategy to increase clinical results, especially in fresh embryo, thanks to an improved embryo selection. Trial registration number Not applicable

P–133 Monopronuclear (1PN) embryos can derive in healthy pregnancies

Abstract Study question Should 1PN embryos be considered suitable for transfer when normal development is observed at day 3 or day 5? Summary answer In IVF/ICSI cycles, 1PN zygotes are encountered in 2.7% of inseminated oocytes. Transfer of 1PN-embryos should be considered in the absence of suitable 2PN embryos. What is known already During in vitro fertilisation (IVF) and intracytoplasmic sperm injection (ICSI) zygotes containing only a single pronucleus (monopronuclear, 1PN) are encountered in 1–7.7% of cases, while the display of two pronuclei is expected in a normally fertilised oocyte. A 1PN zygote can be of gynogenetic or androgenetic origin, but it can also be biparental. Gynogenetic and androgenetic 1PN embryos can be haploid or diploid, so a diploid 1PN embryo is not guaranteed to be normally fertilised. Generally, 1PN are discarded, as they have an increased risk for aneuploidy. However, sporadically they can develop into healthyl babies. Study design, size, duration 1PN-zygotes (n = 1287, 2.7% from all inseminated oocytes) from 1–1–2016 up to 15–12–2020 were retrospectively evaluated. The development and fate (discarded/transferred/cryopreserved) of all embryos were recorded. Embryos were evaluated at day 2, 3 or 5 of development. The policy of our unit is that, in absence of 2PN embryos, normal developed 1PN-embryos can be transferred on day 3. Supernumerary 1PN embryos can be cryopreserved at blastocyst stage. Ongoing pregnancies from fresh embryo transfers (ET) were analysed. Participants/materials, setting, methods In 946 IVF/ICSI cycles, at least one 1PN zygote was observed (total 1287 embryos). ICSI with ejaculated, PESA or TESE sperm counted for a total of 795 embryos, IVF cycles for 494 embryos. Embryo evaluation was performed using a home-made numerical algorithm: A (top embryo; 150–200 points), B (regular embryo; 100–149 points) or C (poor embryo; 0–99 points). Monopronuclear embryos always scored lower than equal developed 2PN embryos. Blastocyst evaluation was according to Gardner score. Main results and the role of chance From the 795 ICSI embryos, 49 (6.1%) were used for fresh ET (26 scored quality A or B), and a total of 60 embryos developed to blastocyst and were cryopreserved. From these 49 ICSI transfers, 4 (8.1%) ongoing pregnancies were obtained, all 4 from DET (1PN+2PN embryo), from which one twin pregnancy was confirmed. From the 494 IVF embryos, 41 (8.3%) were used for fresh ET (24 scored A or B), and 62 blastocysts were cryopreserved. A total of 9/41 (22%) ongoing pregnancies were obtained: 5 from SET (1PN) and 4 from DET (1PN+ 2PN embryo). Therefore, in only five IVF cycles a confirmed pregnancy was observed from a 1PN embryo (all A-quality embryos). Considering six ongoing pregnancies with complete certainty of monopronuclear origin from fresh tranfers could be confirmed from our retrospective data, we can conclude that although the live birth rate of these embryos is very low (around 0,5- 1.0%), they should not be discarded when development is normal and no dipronuclear embryos are present. Limitations, reasons for caution Cryo-thawing data is missing as these embryos were not differentially marked at freezing. Therefore, the cumulative pregnancies from monopronuclear embryos could be higher. Embryos were not evaluated in a time lapse system, so asynchronicity of PN formation could explain missing the right moment for evaluation, while normal fertilized. Wider implications of the findings: Notably, IVF monopronuclear embryos display a higher developmental potential than those derived from ICSI. We suggest that, in absence of dipronuclear embryos, culture to blastocyst stage before considering fresh ET or cryopreservation will help differentiate viable 1PN embryos, reducing the higher chance of genetic anomalies and miscarriages. Trial registration number N.A.

P–566 Advanced paternal age can influence aneuploidy rate in egg donation cycles with poor sperm quality

Abstract Study question Could advanced paternal age influences the embryos aneuploidy rate in eggs donation cycles with poor sperm quality? Summary answer In case of severe male factors increased paternal age can affect embryos aneuploidy rate in egg donation cycles. What is known already While the impact of advanced maternal age on reproductive is well understood, the effect of paternal age on reproductive function is controversial. Many studies have shown that Advanced Paternal Age (APA) could impact on male fertility potential affecting testicular function and sperm quality. Moreover, APA also has been associated with increased epigenetics changes and DNA mutations. Increased paternal age could be associated with different types of disorders such as autism, schizopherenia and bipolar disorders. Egg donation cycles, controlling female variables, represent the ideal model for the study of the impact of paternal age on reproductive outcomes. Study design, size, duration We retrospectively analyzed 43 egg donation cycles (October 2014-January 2020) with ≥ 50% survival rate of vitrified/warmed oocyte. Only cycles with poor sperm quality were considered. Cycles were divided in two GROUPS: group–1 included male paternal age ≤ 45 while group–2 included male paternal age &amp;gt;45. Data, shown as avarage±SD, were analyzed with Chi square or Student-t test. Participants/materials, setting, methods Group–1 included 20 cycles and 219 oocytes, male age was 40,89 ±6.12; Group–2 included 17 cycles and 173 oocytes, male age was 51±6.06. Respectively, in Group 1 and in Group 2, donor age were 22.4±2.65 and 24.8±3.88 (NS). All oocytes were injected with abnormal sperm samples according to WHO 2010. Embryos were cultured in time-lapse system until blastocyst stage. Trophectoderm biopsy and PGT-A analysis were performed according to standardized laboratory protocols. Main results and the role of chance Oocytes survival rates in Group1 and 2 were 86% (188/219) and 90.7% (157/173) (NS), respectively. Fertilization rates in Group1 and –2 were 71.42 (135/189) and 73.45% (119/162) (NS), respectively. The total number of obtained embryos (transferred + frozen) were 81 and 801 in Group–1 and –2, respectively. The rates of obtained embryos per reiceved occytes were 37% (81/219) and 46.24% (80/173) in Group–1 and –2 (p &amp;lt; 0.7), respectively. The PGT-A analysis showed 38.7% (31/80) and 31.17% (24/77) of euploid (NS) and 25% (20/80)and 42.85% (33/77) of aneuploid embryos (P &amp;lt; 0.05) in Group–1 and –2, respectively. Mosaic embryos were 33.5% (26/80) and 27.27%(21/77), in Group–1 and –2, respectively. (NS). These results indicate that in presence of severe male factor, advanced paternal age could increase embryos aneuploidy rate raising incidence of chromosomal abnormalities. Limitations, reasons for caution Each donor was stimulated with different protocols according to her history and hormones levels. Nothing is known about which type of sperm parameters (semen amount, morphology or motility) have a major impact when focusing on the embryos genetic outcome. Wider implications of the findings: To better known the effect of APA, it could be necessary identify embryos chromosomal abnormalities and the correlation with specific sperm parameters. Further studies should be done to confirm the APA effect in patients with severe male factors and define a cut-off male age where PGT-A should be recommended. Trial registration number Not applicable

P–221 Prospective randomized sibling study on gamete preparation, insemination and subsequent culture of human oocytes in a time-lapse system using media systems with and without antioxidants

Abstract Study question Does the addition of antioxidants for gamete preparation, insemination and embryo culture lead to differences in embryo development and clinical outcome Summary answer Using an antioxidant-containing media system for sperm preparation, insemination and embryo culture imparts significantly higher good-quality blastocyst rates and improved clinical outcome in elderly patients. What is known already A previous study showed that adding combined antioxidants for sequential embryo culture in conventional incubators (interrupted culture) improves embryo viability and clinical outcome, especially for elderly patients. Here we investigated the combined effect of three antioxidants Acetyl-L-Carnitine (10 µM), N-Acetyl-L-Cysteine (10 µM), and α-Lipoic Acid (5 µM) during sperm preparation, insemination, and time-lapse culture in a single step medium on human embryo development and clinical outcome. Study design, size, duration Prospective randomized single center study including 143 couples for IVF/ICSI between August 2018 and December 2019. Inclusion required at least eight cumulus-oocyte-complexes (COCs) after retrieval. Cycles involving PGT, split IVF/ICSI, and surgically retrieved sperm were excluded. Immediately after retrieval oocytes were randomly distributed to a study or control media system with or without antioxidants (Vitrolife). Similarly, ejaculates were split and prepared with and without antioxidants. Participants/materials, setting, methods Sibling oocytes were inseminated in the respective group with accordingly prepared sperm. Single step embryo culture was conducted in medium with (Gx-TL) and without (G-TL) antioxidants in the EmbryoScope+. Embryo quality and clinical outcome were assessed in relation to maternal age (&amp;lt;35/&amp;gt;35 years). Good-quality embryos on day 3 were defined as 8- to 10-cells with even cells and low fragmentation; good-quality blastocysts as &amp;gt; 3BB. Clinical outcome was assessed after single vitrified blastocyst transfer (SVBT). Main results and the role of chance From 143 participants (female age, 34.7±3.2 years), a total of 2424 COCs were collected; 1180 COCs/916 metaphase-II (MII) oocytes were allocated to Gx-TL media and 1244 COCs/981 MII oocytes to G-TL media. Age-related analysis in Gx-TL compared with G-TL in relation to allocated MII oocytes revealed a trend for higher fertilization rates in Gx-TL for both age groups (&amp;lt;35: 72.1% vs. 66.9%; &amp;gt;35: 70.7% vs. 64.9%, P &amp;lt; 0.1). Good-quality day 3 embryo development/MII oocytes was higher, albeit not significant, in the elderly patients in Gx-TL (&amp;lt;35: 35.9% vs. 34.4%; &amp;gt;35: 31.1% vs. 27.9%). Overall day 5/6 blastocyst rate was similar for both media (&amp;lt;35: 48.2% vs. 49.9%; &amp;gt;35: 42.3% vs. 39.5%). Day 5/6 GQB rate was comparable for younger patients (&amp;lt;35: 23.8% for Gx-TL vs. 26.0% for G-TL) but significantly higher in Gx-TL in elderly patients (&amp;gt;35: 20.7% vs. 14.4%; P &amp;lt; 0.05). A total of 200 SVBT were performed; 99 in the Gx-TL- and 101 in the G-TL-arm. We noted almost similar implantation and ongoing pregnancy rates between Gx-TL vs G-TL in the younger (&amp;lt;35) age group (50.0% vs. 55.4%; 50.0% vs. 55.6%) but higher albeit not significant rates for Gx-TL in older (&amp;gt;35) patients (44.1% vs. 33.3%; 44.1% vs. 33.3%). Limitations, reasons for caution In almost 95% of the cycles, oocytes were inseminated by ICSI; thus results may not equally apply for cycles with IVF. The use of a closed time-lapse system may have prevented from some environmental oxidative stress. Therefore results may come out different with a similar study using standard incubation. Wider implications of the findings: Supplementation of antioxidants to media for gamete isolation and preparation, as well as subsequent single step time-lapse culture may improve GQE/B rates and clinical outcomes in certain age groups, plausibly through the reduction of oxidative stress. Further studies in selected sub-groups (severe OAT syndrome / testicular cases) may be indicated. Trial registration number UMIN000034482

P–247 Application of deep learning for automated measurement of key morphological features of human zygotes for IVF

Abstract Study question Can deep learning (DL) algorithms trained on time-lapse videos be used to detect and track the size and gender of pronuclei in developing human zygotes? Summary answer Our DL algorithm not only outperforms state-of-the-art models in detecting the pronuclei but can also accurately identify and track its gender and size over time. What is known already Recent researches have explored the use of DL to extract key morphological features of human embryos. Existing studies, however, focus either on blastocysts’ morphological measurements (Au et al. 2020) or on embryos’ general developmental stages classification (Gingold et al. 2018, Liu et al. 2019, Lau et al. 2019). So far, only one paper attempted to evaluate zygotes’ morphological components but stopped short of identifying the existence and location of their pronuclei (Leahy et al. 2020). We address this research gap by training a DL model that can detect, classify the gender, and quantify the size of zygotes’ pronuclei over time. Study design, size, duration A retrospective analysis using 91 fertilized oocytes from infertile patients undergoing IVF or ICSI treatment at Hanabusa Women’s Clinic between January 2011 and August 2019 was conducted. Each embryo was time-lapse monitored using Vitrolife which records an image every 15 minutes at 7 focal planes. For our study, we used videos of the first 1–2 days of the embryo from its 3 central focal planes, corresponding to 70–150 images per focal plane. Participants/materials, setting, methods All 273 timelapse videos were split into 30,387 grayscale still images at a 15-minute interval. Each image was checked and annotated by experienced embryologists where every pixel of the image was classified into 3 categories: male pronuclei, female pronuclei, and others. Images were converted into grayscale, resized into 500x500 pixels, and then fed into a neural network with the Mask R-CNN architecture and a ResNet101 backbone to produce a pronuclei instance segmentation model. Main results and the role of chance The 91 embryos were split into training (∼70% or 63 embryos) and validation (∼30% or 28 embryos). Our pronuclei model takes as input a single image and outputs a bounding box, mask, category, confidence score, and size measured in terms of pixel for each detected candidate. For prediction, we run the model on the 3 middle focal planes and merge candidates by using the one with the highest confidence score. We used the mean-average precision (mAP) score to evaluate our model’s ability to detect pronuclei and used the mean absolute percentage error (MAPE) between the actual size (as annotated by the embryologist) and the predicted one to check the model’s performance in tracking the pronuclei’s size. The mAP for detecting pronuclei, regardless of its gender, achieved by our model was 0.698, higher than the 0.680 value reported in the Leahy et al. paper (2020). Breakdown by gender, our model’s mAP for male and female pronuclei are 0.734 and 0.661 respectively. The overall MAPE for tracking pronuclei’s size is 21.8%. Breakdown by gender, our model’s MAPE for male and female pronuclei are 19.4% and 24.3% respectively. Limitations, reasons for caution Samples were collected from one clinic with videos recorded from one time-lapse system which can limit our results’ reproducibility. The accuracy of our DL model is also limited by the small number of embryos that we used. Wider implications of the findings: Even with a limited training dataset, our results indicate that we can accurately detect and track the gender and the size of zygotes’ pronuclei using time-lapse videos. In future models, we will increase our training dataset as well as include other time-lapse systems to improve our models’ accuracy and reproducibility. Trial registration number Not applicable

P–804 Morphokinetic development by time-lapse imaging of conceptuses generated from artificial oocytes

Abstract Study question What are the ideal culture conditions to enhance full preimplantation development of embryos generated by FVB somatic cell haploidization (SCH) in the mouse model? Summary answer The presence of a histone deacetylase inhibitor yielded the best morphokinetic development of expanded blastocysts generated by FVB SCH, comparable to control blastocysts. What is known already Various culture conditions and medium supplements have been proposed to promote preimplantation development of embryos generated by SCH, including supplementation with trichostatin A (TSA), fasudil, scriptaid, and RAD–51 stimulatory compound–1 (RS–1). TSA and scriptaid, both histone-deacetylase inhibitors, have been found to improve embryo development following nuclear transfer by enhancing histone acetylation and cellular reprogramming. Additionally, fasudil is a Rho-associated kinase inhibitor that has been shown to reduce apoptosis and promote cell proliferation. Finally, RS–1 stimulates RAD51 activity, which promotes the repair of DNA damage and increases the efficacy of somatic cell reprogramming. Study design, size, duration B6D2F1 mouse metaphase II (MII) oocytes underwent enucleation and nuclear transfer, or were ICSI inseminated serving as controls. Reconstituted oocytes showing development of a meiotic-like spindle demonstrated successful SCH, and were ICSI inseminated. SCH conceptuses were cultured in one of three groups: KSOM, KSOM supplemented with TSA (TSA), or KSOM supplemented with fasudil, scriptaid, and RS–1 (Cocktail). ICSI controls (ICSIC) were cultured in KSOM medium. Fertilization and full preimplantation development were compared among all groups. Participants/materials, setting, methods Ooplasts were generated from MII oocytes by removing spindle complexes under OosightÔ visualization and cytochalasin B exposure. A single FVB mouse cumulus cell was transferred into the perivitelline space and fused with the ooplast, facilitated by Sendai virus. Reconstructed oocytes with novel pseudo-meiotic spindles underwent piezo-ICSI and were cultured in different media conditions in a time-lapse imaging system up to 96h. TSA and Cocktail embryos had media changed to regular KSOM 10 hours after insemination. Main results and the role of chance A total of 274 B6D2F1 MII oocytes were enucleated, resulting in a 95.9% survival rate. All ooplasts survived nuclear transfer and 62.1% successfully haploidized after 2 hours. ICSIC and reconstituted SCH oocytes survived piezo-ICSI at rates of 81.5% and 57.0%, respectively (P &amp;lt; 0.01). SCH embryos were then allocated into KSOM, TSA supplied, and Cocktail media. Fertilization rates for ICSIC, KSOM, and TSA embryos were 92.4%, 90.7%, and 94.4%, respectively, while the rate for embryos cultured in Cocktail was only 71.9% (P &amp;lt; 0.03). While embryos cultured in Cocktail had a comparable 2-cell timing to ICSIC, embryos in TSA reached developmental milestones with a closer timing to the ICSIC, having minor delays at the 3-, 4-, and 6-cell stages (P &amp;lt; 0.05). KSOM- and Cocktail-cultured embryos were delayed at most of the stages (P &amp;lt; 0.01), except for the two-pronuclei appearance. Although the TSA group displayed the best embryo developmental pattern, the final rate of blastocyst development was somewhat homogeneous with rates of 15.4%, 23.5%, and 13.0% for the KSOM, TSA, and Cocktail groups, respectively (P &amp;lt; 0.001), and remarkably lower than the ICSIC (81.6%). Limitations, reasons for caution Although live pups have been obtained using BDF cumulus cells, embryos generated by FVB cumulus cells show a remarkably lower blastocyst development, but maintain morphokinetic characteristics similar to ICSIC in the presence of TSA. Wider implications of the findings: While using different strains to enhance genetic variance, the morphokinetic analysis of preimplantation embryos in ideal culture conditions is paramount to the progress of neogametogenesis. The implementation of this technique may soon help create genotyped oocytes for women with compromised ovarian reserve. Trial registration number N/A

O-003 The puzzling unknowns of abnormal fertilization and first cleavage

Abstract text Fertilization is a critical event in development in that it provides the connection between the gametes and the earliest stages of embryogenesis. Yet, despite the central importance of this process in contributing to embryo developmental fate, clinical embryologists have historically assessed fertilization merely by the number of pronuclei and, if two are present, perhaps, by the presence of two polar bodies. Even though over 20 years ago, time lapse imaging was applied for defining early events of fertilization (Payne et al., 1997), it is only with contemporary time-lapse imaging systems in the last few years that detailed evaluation of spatial and temporal events of fertilization have been described (Iwata &amp; Yasuyuki, 2016; Cottichio et al., 2018). These careful analyses allow us to describe typical and atypical events of fertilization and how they are each associated with timing of the first cleavage division and subsequent embryo development. In this lecture, we will first describe the fundamental underpinnings of fertilization and highlight the normal events associated with this process. We will then discuss gross morphological abnormalities as visualized by light microscopy and highlight the unknowns associated with these events. Finally, we will focus on time-lapse imaging studies, which have revealed the remarkable spatial and temporal coordination of meiotic resumption, pronuclear dynamics, chromatin organization and cytoplasmic/cortical modifications that occur during fertilization and the implications of aberrations for the first cleavage division. At the conclusion of this presentation, attendees should be able to: Review the normal events associated with fertilization and the first cleavage division. 1 Describe gross morphological aberrations of these two fundamental processes. 2 Discuss temporal and spatial abnormalities in the coordinated sequence of events that underly these processes. 3 State the potential application of these abnormalities as predictors of abnormal embryo development. 4 Summarize the puzzling unknowns that underly these abnormalities.

P–201 The beneficial effects of ZP-free culture on cytoplasmic fragmentation in human embryos. : An innovative trial using 3PN zygotes

Abstract Study question Is it possible to culture ZP-free embryos to eliminate perivitelline threads, which are known to be involved in generating cytoplasmic fragments at the first cleavage? Summary answer ZP-free culturing, an innovative system that decreases the amount of cytoplasmic fragments without disrupting the blastomeres, using incubators with time-lapse imaging. What is known already A study in 2017 observed perivitelline threads in more than 50% of cleavage-stage human embryos using time-lapse imaging, and the rate of cytoplasmic fragmentation (at the first cleavage) was significantly decreased in embryos without perivitelline threads (P &amp;lt; 0.001). While it has been proposed that perivitelline threads play an important role in crosslinking the cumulus cells and oocyte during maturation, the mechanism underlying such a role remains unclear. It is also unknown whether the threads still function in mature MII oocytes. Study design, size, duration A prospective study was conducted using 2,852 normal (2PN/2PB) embryos from c-IVF/ICSI and 113 abnormal (3PN) embryos obtained from c-IVF between 2017 and 2019. The zona pellucida (ZP) of 71 abnormal embryos was removed at the pronuclear stage (“ZP-free”), and the rest (n = 42) were cultured as “ZP-intact”. Normal and abnormal embryos were cultured for five days in bench-top incubators (MINC, COOK) and an incubator equipped with a time-lapse imaging system. Participants/materials, setting, methods Embryos used in this study were donated by 412 couples who underwent c-IVF cycles in our clinic between 2017 and 2019. For ZP removal, 3PN embryos were placed in 0.125M sucrose-containing HEPES media drops to reduce the ooplasm size. Then, ooplasms were completely separated from ZPs by a laser and pipetting. Embryo development and morphology of the three groups (normal, ZP-intact and ZP-free abnormal) were compared based on the degree of cytoplasmic fragmentation. Main results and the role of chance The first cleavage occurred in 97.8% (n = 2,790/2,852) of 2PN/2PB, 83.3% (n = 35/42) of ZP-intact 3PN and 97.2% (n = 69/71) of ZP-free 3PN. Normal (2PN/2PB), ZP-intact and ZP-free 3PN embryos were classified into three groups based on the modified Veeck’s criteria thus: &amp;lt;20% fragmented compared to the total volume of cytoplasm at the first cleavage (Grade 1 and 2, Good); 20–39% fragmented (Grade 3, Fair) and ≧40% fragmented (Grade 4, Poor). Of 69 cleaved ZP-free 3PN embryos, 68.1% (n = 47) showed less than 20% fragments which was significantly higher than 2PN/2PB (43.7%, n = 1,218/2,790) and ZP-intact 3PN (45.7%, n = 16/35; P &amp;lt; 0.05). Furthermore, 24.6% (n = 17/69) of ZP-free 3PN embryos showed 20–39% fragments which was significantly lower than 2PN/2PB (45.9%, n = 1,281/2,790; P &amp;lt; 0.05). In addition, 50.7% of ZP-free 3PN embryos (n = 36) developed to the morula stage after the third cleavage, and 29.6% (n = 21) formed blastocoel and became blastocysts. Thus, removing the ZP before the first cleavage did not adversely affect embryo development and decreased the cytoplasmic fragmentation. Limitations, reasons for caution Due to ethical and clinical limitations, we only examined abnormally fertilized embryos in this study. Moreover, since the relationship between the perivitelline threads and cytoplasmic fragments is unclear, we plan to conduct molecular biological analysis of the perivitelline threads in further studies. Wider implications of the findings: This study revealed that ZP is not always necessary after the pronuclear stage because ZP-free embryos studied herein developed normally and maintained cell adhesion well. This innovative culture method might provide the breakthrough needed for patients to improve embryo quality who obtain embryos with severe fragmentation caused by perivitelline threads. Trial registration number Not applicable

P-805 Artificial oocytes: from somatic cells to fertile pups

Abstract Study question We analyzed the efficacy of generating artificial oocytes using somatic cells (SCs) from two mouse strains (B6D2F1 and FVB) and followed their full pre-/post-implantation development. Summary answer While artificial oocytes generated from the new strain (FVB) had higher fertilization rates, those from the standard strain (B6D2F1) provided expanded blastocysts and fertile pups. What is known already B6D2F1 is a popular hybrid mouse strain for cloning and transgenic creation due to its geno-/pheno-typic uniformity and high oocyte yield and quality. Indeed, B6D2F1 oocytes have a distinct metaphase II (MII) spindle complex, making them an ideal candidate to generate ooplasts used in SC nuclear transfer (SCNT). However, because they lack genetic variance, they are less suitable for reciprocal SCNT studies. In contrast, FVB mice have single nucleotide polymorphisms and indels on each chromosome that can aid in tracing the pedigree of progeny. Study design, size, duration A total of 10 experiments were performed over the course of 3 months, using 30 stimulated mice. SCs were retrieved from cumulus oophorus harvested from FVB and B6D2F1 mice. SCs from both strains were injected into enucleated MII B6D2F1 oocytes. Unmanipulated B6D2F1 oocytes were piezo-ICSI inseminated, serving as controls. The occurrence of haploidization, fertilization, and full preimplantation development was compared. Some blastocysts were transferred into pseudo-pregnant CD-1 mice to obtain offspring. Participants/materials, setting, methods Oocyte enucleation was performed under Oosight™ visualization and cytochalasin B exposure. An FVB or B6D2F1 SC was transferred into the perivitelline space of the ooplast with Sendai virus to promote fusion. Haploidization was monitored by pseudo-meiotic spindle formation followed by extrusion of a pseudo-polar body after insemination. Conceptuses were cultured in a time-lapse imaging system, with piezo-ICSI controls. Expanded blastocysts were transferred into uterine horns of pseudo-pregnant mice. Offspring were mated to test their fertility. Main results and the role of chance FVB (n = 278) and B6D2F1 (n = 905) SCs at G0 phase, with a diameter &amp;lt;10 mm, were chosen for SCNT and transferred into enucleated B6D2F1 ooplasts. Enucleation of 1,212 oocytes yielded a survival rate of 97.6%. Both FVB and B6D2F1 SCNT resulted in similar survival rates of 100% and 98.5%, respectively. Successful haploidization, determined by the presence of a pseudo-meiotic spindle 2 hours after SCNT, was also comparable, with 59.9% of FVB and 63.7% of B6D2F1. Survival after piezo-ICSI was also comparable between FVB- and B6D2F1-reconstituted oocytes, with rates of 64.3% and 60.3%, respectively, albeit lower than the control (75.2%, P&amp;lt;0.00001). FVB embryos fertilized at a rate of 88.7%, comparable to the control zygotes at 85.8%, while B6D2F1 conceptuses demonstrated a lower fertilization rate (70.8%, P &amp;lt; 0.00001). Blastulation of FVB- and B6D2F1-derived embryos was 15.1% and 24.0%, respectively, while the control was 80.7% (P&amp;lt;0.00001). Whole-genome karyotyping of 9 B6D2F1-derived blastocysts confirmed 5 of the samples to be euploid. FVB blastocysts (N = 8) and B6D2F1 blastocysts (N = 81) were transferred into pseudo-pregnant mice, resulting in 3 fertile offspring only from the B6D2F1 conceptuses. Limitations, reasons for caution This is still a limited number of observations, and pups were delivered only from the B6D2F1 strain. The utilization of a strain with higher genetic variance may help facilitate offspring fingerprinting. Wider implications of the findings This study demonstrates the ability to generate artificial genotyped conceptuses, yielding live offspring. The identification of a feasible donor cell, together with optimization of cell cycle stage and standardization of post-implantation development, will help promote this technique for human reproduction in couples with age-related infertility or poor ovarian reserve. Trial registration number N/A

O-208 High sperm DNA fragmentation index negatively impacts embryo morphokinetics, but not embryo morphology and development rates: the importance of time-lapse imaging system

Abstract Study question Can time-lapse imaging (TLI) identify morphokinetic events impacted by high sperm DNA fragmentation index (DFI), irrespective of conventional morphological embryo assessment and development rate? Summary answer Embryo morphokinetic parameters are negatively impacted by high DFI, whereas conventional morphological embryo assessment and blastocyst development rate are not related to DNA integrity. What is known already Paternal genome activation occurs late in the embryo, and therefore, the negative impact of sperm factors on embryo development is more often observed in the outcomes of pregnancy, such as embryonic implantation and pregnancy loss, than in the potential for embryonic development itself, such as successful development to the blastocyst stage. With the development of TLI technology, and the possibility of assessing complete embryonic development, we hypothesized that sperm factors related to DNA fragmentation may interfere with the speed and pattern of cell divisions, leading to slower embryos; something that would not be detected by conventional morphological embryo assessment. Study design, size, duration The study included 978 zygotes cultured until day five in a TLS incubator between March/2019 and August/2020, derived from 118 patients undergoing ICSI in a private university-affiliated IVF center. Kinetic markers from the point of insemination were recorded. Generalized linear models adjusted for potential confounders, followed by Bonferroni post hoc were used to compare timing of specific events in patients with low (&amp;lt;30%) of high (≥30%) DFI. The post hoc achieved power was &amp;gt; 90%. Participants/materials, setting, methods Recorded kinetic markers were: timing to pronuclei appearance and fading (tPNa and tPNf), timing to two (t2), three (t3), four (t4), five (t5), six (t6), seven (t7), and eight cells (t8), and timing to morulae (tM), start of blastulation (tSB) and blastulation (tB). Durations of second and third cell cycles (cc2 and cc3) and timing to complete synchronous divisions s1, s2, and s3 were calculated. The KIDScore ranking was also recorded. Main results and the role of chance Blastocyst development (53.1% ± 1.3 vs. 55.1% ± 1.5, p = 0.380) and high-quality rates (87.9% ± 2.9 vs. 86.2% ± 3.6, p = 0.749) were similar between embryos derived from sperm samples with &amp;lt;30% DFI (n = 592) and ≥30% DFI (n = 386), respectively. Embryos derived from sperm samples with ≥30% DFI showed significantly slower divisions compared to those from &amp;lt;30% DFI: tPNa (6.1h ± 0.2 vs. 6.8h ± 0.2, p = 0.030), tPNf (23.0h ± 0.3 vs. 24.2h ± 0.3, p = 0.009), t2 (25.4h ± 0.3 vs. 26.9h ± 0.3, p = 0.002), t3 (34.8h ± 0.3 vs. 37.3h ± 0.4, p &amp;gt; 0.001), t4 (37.5h ± 0.4 vs. 39.3h ± 0.4, p = 0.003), t5 (46.2h ± 0.5 vs. 49.5h ± 0.6, p &amp;lt; 0.001), t6 (49.7h ± 0.5 vs. 52.8h ± 0.6, p = 0.001), t7 (52.4h ± 0.6 vs. 55.6h ± 0.7, p = 0.001), t8 (56.2h ± 0.7 vs. 58.9h ± 0.8, p = 0.017), tSB (97.5h ± 1.5 vs. 105.9h ± 1.7, p = 0.002), tB (108.6h ± 0.8 vs. 112.4h ± 1.2, p = 0.016). The KIDScore ranked significantly different between embryos derived from samples with &amp;lt;30% or ≥ 30% DFI (4.5 ± 0.1 vs. 3.9 ± 0.2, p = 0.033, respectively). A significant difference was observed in implantation rate (&amp;lt;30% DFI: 51.5% ± 2.2 vs. ≥30% DFI: 30.5% ± 1.3, p &amp;lt; 0.001). Limitations, reasons for caution Retrospective nature of this study and the small sample size may be a reason for caution, despite adequate power has been achieved. Wider implications of the findings Increasing DFI correlates with delayed cell cleavage and blastulation, leading to reduced implantation rates, without compromising blastulation rate and quality. This finding highlights the importance of TLI for the identification and de-selection of slow-growing embryos for transfer, in cycles with high DFI. Trial registration number Not applicable

P–805 Artificial oocytes: from somatic cells to fertile pups

Abstract Study question We analyzed the efficacy of generating artificial oocytes using somatic cells (SCs) from two mouse strains (B6D2F1 and FVB) and followed their full pre-/post-implantation development. Summary answer While artificial oocytes generated from the new strain (FVB) had higher fertilization rates, those from the standard strain (B6D2F1) provided expanded blastocysts and fertile pups. What is known already B6D2F1 is a popular hybrid mouse strain for cloning and transgenic creation due to its geno-/pheno-typic uniformity and high oocyte yield and quality. Indeed, B6D2F1 oocytes have a distinct metaphase II (MII) spindle complex, making them an ideal candidate to generate ooplasts used in SC nuclear transfer (SCNT). However, because they lack genetic variance, they are less suitable for reciprocal SCNT studies. In contrast, FVB mice have single nucleotide polymorphisms and indels on each chromosome that can aid in tracing the pedigree of progeny. Study design, size, duration A total of 10 experiments were performed over the course of 3 months, using 30 stimulated mice. SCs were retrieved from cumulus oophorus harvested from FVB and B6D2F1 mice. SCs from both strains were injected into enucleated MII B6D2F1 oocytes. Unmanipulated B6D2F1 oocytes were piezo-ICSI inseminated, serving as controls. The occurrence of haploidization, fertilization, and full preimplantation development was compared. Some blastocysts were transferred into pseudo-pregnant CD–1 mice to obtain offspring. Participants/materials, setting, methods Oocyte enucleation was performed under Oosight™ visualization and cytochalasin B exposure. An FVB or B6D2F1 SC was transferred into the perivitelline space of the ooplast with Sendai virus to promote fusion. Haploidization was monitored by pseudo-meiotic spindle formation followed by extrusion of a pseudo-polar body after insemination. Conceptuses were cultured in a time-lapse imaging system, with piezo-ICSI controls. Expanded blastocysts were transferred into uterine horns of pseudo-pregnant mice. Offspring were mated to test their fertility. Main results and the role of chance FVB (n = 278) and B6D2F1 (n = 905) SCs at G0 phase, with a diameter &amp;lt;10 mm, were chosen for SCNT and transferred into enucleated B6D2F1 ooplasts. Enucleation of 1,212 oocytes yielded a survival rate of 97.6%. Both FVB and B6D2F1 SCNT resulted in similar survival rates of 100% and 98.5%, respectively. Successful haploidization, determined by the presence of a pseudo-meiotic spindle 2 hours after SCNT, was also comparable, with 59.9% of FVB and 63.7% of B6D2F1. Survival after piezo-ICSI was also comparable between FVB- and B6D2F1-reconstituted oocytes, with rates of 64.3% and 60.3%, respectively, albeit lower than the control (75.2%, P &amp;lt; 0.00001). FVB embryos fertilized at a rate of 88.7%, comparable to the control zygotes at 85.8%, while B6D2F1 conceptuses demonstrated a lower fertilization rate (70.8%, P &amp;lt; 0.00001). Blastulation of FVB- and B6D2F1-derived embryos was 15.1% and 24.0%, respectively, while the control was 80.7% (P &amp;lt; 0.00001). Whole-genome karyotyping of 9 B6D2F1-derived blastocysts confirmed 5 of the samples to be euploid. FVB blastocysts (N = 8) and B6D2F1 blastocysts (N = 81) were transferred into pseudo-pregnant mice, resulting in 3 fertile offspring only from the B6D2F1 conceptuses. Limitations, reasons for caution This is still a limited number of observations, and pups were delivered only from the B6D2F1 strain. The utilization of a strain with higher genetic variance may help facilitate offspring fingerprinting. Wider implications of the findings: This study demonstrates the ability to generate artificial genotyped conceptuses, yielding live offspring. The identification of a feasible donor cell, together with optimization of cell cycle stage and standardization of post-implantation development, will help promote this technique for human reproduction in couples with age-related infertility or poor ovarian reserve. Trial registration number N/A

P–150 Does trophectoderm biopsy performed on different blastocyst stages affect the clinical outcome?

Abstract Study question The objective of this study is to evaluate the effect of trophectoderm (TE) biopsy on different blastocyst stages and its clinical outcome. Summary answer Our results showed that TE biopsy significantly reduced the clinical outcome of fully hatched blastocyst. What is known already: TE biopsy is a method widely practiced to harvest cells to determine the chromosomal constitution of a blastocyst, ensuring higher implantation and healthy pregnancies. The effect on clinical outcome after transferring blastocysts biopsied at different blastocysts stages has not been extensively studied. Study design, size, duration This retrospective study was conducted from January 2017 until July 2019 at Alpha IVF &amp; Women’s Specialists. Following laser assisted hatching on day 3, TE biopsy was performed on unhatched, hatching and fully hatched day–5 blastocysts. A total of 1,020 single euploid blastocysts transfer (SBT) were performed. The average maternal age was 31.7. Implantation rates (IR) were evaluated for all stages of hatching (Unhatched: BG3 &amp; 4; hatching: BG5; fully hatched: BG6). Participants/materials, setting, methods Laser assisted hatching (Hamilton Thorne Bioscience, USA) was performed on day–3 and subsequently cultured to blastocyst-stage. Different hatching stages were observed using embryoscope time-lapse system (Vitrolife, Sweden) and were recorded. Day–5 blastocysts with at least BG3BB grade (Gardner’s System) were selected for TE biopsy and the biopsied cells were sent for preimplantation genetic testing for aneuploidy (PGT-A) using Next-Generation Sequencing (Life Technologies, USA). All blastocysts were vitrified and warmed using the Cryotec Method (Cryotech, Japan). Main results and the role of chance: All 1,020 blastocysts survived post-warmed (post-warm survival rate= 100%) and were transferred in frozen transfer cycles. TE biopsy performed on unhatched blastocysts showed a comparable IR to hatching blastocysts (60.0% [15/25] and 65.2% [627/961]). While fully hatched blastocysts (44.12% [15/34]) show a significantly lower IR when compared to hatching blastocysts (65.2% [627/961]), no significant difference was seen when comparing unhatched blastocysts to fully hatched blastocysts (60.0% [15/25] and 44.12% [15/34]; p = 0.2949). Limitations, reasons for caution The sample size was comparatively smaller in the unhatched and fully hatched group than the hatching group. Further studies with a larger sample size is recommended to ascertain the clinical outcome. Since this is a retrospective study and biopsy was done by different embryologists, the biopsy technique was not controlled. Wider implications of the findings: To achieve higher clinical pregnancy, it is recommended to perform TE biopsy before the blastocysts is fully hatched. Trial registration number Not applicable

O-086 End-to-end deep learning for recognition of ploidy status using time-lapse videos

Abstract Study question Our Retrospective study is to investigate an end-to-end deep learning model in identifying ploidy status through raw time-lapse video. Summary answer Our deep learning model demonstrates a proof of concept and potential in recognizing the ploidy status. What is known already Since the time-lapse system has been introduced into the IVF lab, the relationship between morphogenetic and ploidy status has been often discussed. However, the result has not yet reached a united conclusion due to some limitations such as human labeling. Besides the statistical approach, deep learning models have been utilized for ploidy prediction. As such approaches are single image-based, the performance remains unpromising as previous statistical-based research. Therefore, in order to move further toward clinical application, better research design and approach are needed. Study design, size, duration A retrospective analysis of the time-lapse videos and chromosomal status from 690 biopsied blastocysts cultured in a time-lapse incubator (EmbryoScope+, Vitrolife) between January 2017 and August 2018 in the Lee Women’s Hospital were assessed. The ploidy status of the blastocyst was derived from the PGT-A using high-resolution next-generation sequencing (hr-NGS). Embryo videos were obtained after normal fertilization through the intracytoplasmic sperm injection or conventional insemination. Participants/materials, setting, methods By randomly dividing the data into 80% and 20%, we developed our deep learning model based on Two-Stream Inflated 3D ConvNets(I3D) network. This model was trained by the 80% time-lapse videos and the PGT-A result. The remaining 20% has been tested by feeding the time-lapse video as input and the PGT-A prediction as output. Ploidy status was classified as Group 1 (aneuploidy) and Group 2 (euploidy and mosaicism). Main results and the role of chance Time-lapse videos were divided into 3-time partitions: day 1, day 1 to 3, and day 1 to 5. Deep learning models have been fed by RGB and optical flow. Combining 3 different time partitions with RGB, optical flow, and fused result from RGB and optical flow, we received nine sets of test results. According to the results, the longest time partition with the fusion method has the highest AUC result as 0.74, which appeared higher than the other eight experimental settings with a maximum increase of 0.17. Limitations, reasons for caution The present study is retrospective and future prospective research would help us to identify more key factors and improve this model. In addition, expanding sample size combined with cross-centered validation will also be considered in our future approach. Wider implications of the findings Group 1 and Group 2 approach provided deselection of aneuploidy embryos, while future deep learning approaches toward high mosaicism, low mosaicism, and euploidy will be needed, in order to provide a better clinical application. Trial registration number CS18082