Start Of Blastulation Research Articles

P-120 Morphokinetic behavior in embryos leading to biochemical pregnancy

Abstract Study question Does embryos leading to biochemical pregnancy behave morphokinetically different than those leading to positive or negative pregnancy result? Summary answer Embryos that resulted in a biochemical pregnancy did not display evidence of abnormal morphokinetics on time-lapse imaging. What is known already One of the possible outcomes of the pregnancy test is a biochemical pregnancy; where the pregnancy test is positive but does not progress into a clinical pregnancy. Despite its unknown etiology, several associated factors have been suggested such as embryo aneuploidy, abnormal uterine lining, sperm DNA damage and so on. Embryo monitoring with timelapse imaging (TLI) may be a valuable approach to evaluate whether embryonic morphokinetics can predict the occurrence of a biochemical pregnancy. The aim of this study was to investigate whether embryos leading to biochemical pregnancy behave morphokinetically different than those leading to positive or negative pregnancy result. Study design, size, duration This cohort study was performed in a private university–affiliated IVF center between March/2019 and April/22. 753 women undergoing ICSI were split into three groups according to the pregnancy outcome: Biochemical Group (n = 30 cycles and 54 transferred embryos); Positive Group (n = 255 cycles and 444 embryos); and Negative Group (n = 468 cycles and 750 embryos). Generalized mixed models followed by Bonferroni post-hoc test were used to compare morphokinetics among the groups (post hoc achieved power > 90%). Participants/materials, setting, methods Injected oocytes were cultured in the EmbryoScope+ incubator, which recorded the following kinetic markers: timing to pronuclei appearance and fading (tPNa and tPNf), two (t2), three (t3), four (t4), five (t5), six (t6), seven (t7), and eight cells (t8), 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 recorded. Main results and the role of chance Embryos resulting in biochemical pregnancy behaved similarly to those embryos resulting in a clinical pregnancy in terms of tPNa, tPNf, t2, t3, t4, t5, t6, t7, t8, tM, tsB, cc2, cc3, s1, s3 and KIDScore ranking, but tended to present slower tB (p = 0.089) and s2 (p = 0.084). Embryos resulting in a negative pregnancy showed significantly slower embryo development in terms of tPNa, tPNf, t2, t3, t4, cc2, and KIDScore ranking compared to both Biochemical and Positive groups and differed from the Positive group only in terms of t6, t7, t8, s1, s2, and s3. Limitations, reasons for caution The use of historical cohort groups is a drawback. Despite the eligibility criteria for inclusion in the analysis, potential differences in the baseline characteristics cannot be ruled out. Wider implications of the findings Biochemical pregnancy is likely multifactorial, including both embryo and endometrial factors. Further research is needed to identify factors that can predict and prevent biochemical pregnancy. Trial registration number N/A

P-122 The impact of severe male factor on morphokinetic embryo development in low-prognosis patients according to the POSEIDON criteria: an analysis of 10366 injected oocytes

Abstract Study question Does severe male factor infertility (SMF) impact embryo morphokinetic behavior in low-prognosis women as stratified by the Patient-Oriented Strategies Encompassing IndividualizeD Oocyte Number (POSEIDON) criteria? Summary answer Embryos from SMF patients showed lower KIDScore ranking than those from non-SMF (nSMF) patients, which may justify the reduced clinical outcomes contrasted to Control group. What is known already It has been reported that 37.1% of poor responder patients (POR) have infertile male partners. While the maternal influence has been well-investigated, the role of seminal quality on reproductive outcomes in POR remains questionable. Embryo monitoring with timelapse imaging (TLI) may be a valuable approach to evaluate whether sperm quality relates to association between POSEIDON classification and the competences of developing embryos. The aim of this study was to investigate whether SMF impacts embryo quality and morphokinetic behavior in the four groups of low-prognosis women as stratified by the POSEIDON criteria. Study design, size, duration This historical cohort-study was performed in a private university–affiliated IVF center, between Mar/2019 and Apr/2022. Kinetic data were analyzed in 10366 embryos cultured in a TLI incubator until day five, derived from 2272 patients undergoing ICSI. Patients were split into eight groups according to the POSEIDON criteria and the presence or absence of SMF (<5 × 106 sperms/mL). Embryo morphokinetics were compared among the groups (post hoc achieved power > 90%). Participants/materials, setting, methods Injected oocytes were cultured in the EmbryoScope+ incubator, which recorded the following kinetic markers: timing to pronuclei appearance and fading (tPNa and tPNf), two (t2), three (t3), four (t4), five (t5), six (t6), seven (t7), and eight cells (t8), 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 recorded. Main results and the role of chance POSEIDON group 1 embryos showed significantly slower tPNa, tPNf, t2, t3, t4, t6, and t7 compared to those from Control group. POSEIDON group 2 embryos showed significantly slower tPNa, t4, t6, t7, t8, and tM compared to those from Control group. Embryos in POSEIDON 2 SMF subgroup took longer than those in POSEIDON 2 nSMF subgroup, and those in both Control subgroups, to achieve tPNf, t2, t3, t5, tSB. POSEIDON group 3 embryos showed significantly slower t8, and cc2 compared to those from Control group. POSEIDON group 4 embryos showed significantly slower tPNf, t2, t3, t4, t5, t6, t7, t8, s2, and s3 compared to those from Control group. For POSEIDON groups 1, 2 and 4, and Control group, the KIDScore ranking was significantly different between SMF and nSFM subgroups. KIDScore ranking was significantly different across all the subgroups in POSEIDON 3. Irrespective of sperm quality, implantation and miscarriage rates were significantly improved in Control subgroups compared to POSEIDON 2 subgroups, there were trends toward improved clinical outcomes in Control subgroups compared to POSEIDON 3 subgroups, and pregnancy, implantation and miscarriage rates were significantly improved in Control subgroups compared to POSEIDON 4 subgroups. Limitations, reasons for caution The use of historical cohort groups is a drawback. Despite the eligibility criteria for inclusion in the analysis, potential differences in the baseline characteristics cannot be ruled out. Wider implications of the findings The results presented contribute to the understating of the relationship between sperm quality and embryo morphokinetic behavior in POSEIDON patients. Embryo quality, as assessed by TLI-imaging, and clinical outcomes were significantly lower in POSEIDON SMF patients, suggesting that both oocyte (quantity and quality) and sperm are determinant of embryo development. Trial registration number N/A

P-258 Not too big, not too small: euploid blastocysts that lead to live birth have an optimal surface area, diameter and optimal pace of development

Abstract Study question Does embryo diameter and surface area at tsB and tB as determined by AI predict live birth (LB) potential amongst euploid blastocysts? Summary answer Euploid blastocysts with optimal diameter (tsB:120-129; tB:136-140μm) and surface area (tsB:11771-13577; tB:15000-15643μm2) are more likely to lead to LB than larger or smaller blastocysts. What is known already Current embryology consensus favours larger blastocysts based on Gardner grading, associating expansion with higher quality. Human assessment of embryos is subjective, and it is unknown whether this dogma holds when assessing pre-expansion blastocyst stages. The impact of blastocyst surface area and diameter at tSB and tB; and pace of development on LB remains unclear. This may help clarify whether large blastocysts are associated with accelerated embryo growth and abnormal metabolic processes. The study’s objective was to use AI to quantitatively determine the optimal embryo surface area and diameter of euploid blastocysts that lead to live birth. Study design, size, duration Retrospective study including 97 time-lapse videos of euploid embryos with known live birth outcomes from January to December 2021 Participants/materials, setting, methods Embryo diameter and surface area were measured at start of blastulation (tSB) and at time of blastulation (tB) by an AI model (CHLOE-EQ, Fairtility Ltd). Live birth (LB) outcomes were manually annotated by embryologists. An Optimal embryo surface area and diameter were determined by the highest live birth rate and the suboptimal range was determine by a decreased Live birth rate. A comparison between the optimal and suboptimal live birth rate was measured using chi-square. Main results and the role of chance Moderately sized embryos were found to have higher LB outcomes. At tSB, optimal surface area was 11771-13577 um2, while optimal embryo diameter was 120-129μm. LB rate for optimal embryo surface area was higher than for suboptimal [Optimal 80% (20/25) vs Suboptimal 55% (40/72), p < 0.001]. In addition, optimal embryo diameter had higher LB rates than embryos with suboptimal diameter [80% (16/20) vs 52% (31/60), p < 0.05). At tB, optimal embryo area was 15000-15643μm2 and optimal embryo diameter 136-140μm. LB rate for optimal embryo surface area was higher than for suboptimal 82% (14/17) vs 49% (36/73), p < 0.05]. Optimal embryo diameter had higher LB rates than embryos with suboptimal diameter (77% 20/26 vs 47% 30/64, p < 0.05). Embryos with an optimal surface area and diameter were slower than bigger suboptimal embryos: t2 (26±2 vs 25±2), t8 (59±8 vs 53±6), t9 + (71±6 vs 67±6), tM (82±7 vs 77±8), tSB (99±6 vs 93±6), tB (107±7 vs 100±6), tEB (137±2 vs 122±12); t8-t5 (12±10 vs 6±5), t8-t2 (33±8 vs 28±5), tEB-tSB (34±2 vs 23±9), p < 0.05. Optimal vs small suboptimal embryos were faster in t9 + (71±6 vs 76±4), tEB (121±11 vs 137±2, tEB-tSB (20±10 vs 34±1). But were slower in t8-t5 (12±10 vs 5±6), t8-t2 (33±8 vs 28±5), p < 0.05. Limitations, reasons for caution This is the first time a quantitative embryo surface area and diameter has been studied in its correlation with live birth. Generalisation requires the replication across diverse centers with varying demographic profiles to validate findings and establish broader applicability within the context of assisted reproductive technologies. Wider implications of the findings This data-driven approach can support the embryo selection process, correlating quantitative parameters with live birth outcomes, thereby refining the precision of the decision-making in IVF. This level of quantitative data of embryo surface area and diameter is only accessible through AI tools. Trial registration number IRB-007CITY-FL21.08.2023

O-118 Morphokinetic embryo behavior in low-prognosis patients according to the POSEIDON criteria: an analysis of 3326 injected oocytes

Abstract Study question Do embryo quality and morphokinetic behavior differ in the four groups of low-prognosis women as stratified by the POSEIDON criteria? Summary answer Embryo quality and morphokinetic behavior differ across the POSEIDON groups, being more favorable in POSEIDON group 1, as well as were implantation and miscarriage rates. What is known already Poor ovarian responders (POR) represent 9-24% of patients undergoing ovarian stimulation, and the clinical management of these patients remains a challenge in everyday practice. In an attempt to standardize the diagnosis of POR, the medical community developed the Bologna Criteria and then shifted to the Patient-Oriented Strategies Encompassing IndividualizeD Oocyte Number (POSEIDON). Embryo monitoring with timelapse imaging (TLI) may allow the identification of morphokinetic events potentially affected by ovarian response. The aim of this study was to compare embryo quality, and speed and pattern of cell divisions in the four groups of POR as stratified by the POSEIDON criteria. Study design, size, duration This cohort study included low-prognosis patients undergoing their first intracytoplasmic sperm injection (ICSI) from Mar/2019 to Apr/2022 at a private-university affiliated IVF center, who met the POSEIDON criteria. A total of 3326 injected oocytes from 846 women were analyzed. Kinetic markers from the point of insemination were recorded. Generalized mixed models followed by Bonferroni post hoc were used to compare morphokinetics among the POSEIDON groups. The post hoc achieved power was > 80%. Participants/materials, setting, methods Injected oocytes were cultured in the EmbryoScope+ incubator, which recorded the following kinetic markers: timing to pronuclei appearance and fading (tPNa and tPNf), two (t2), three (t3), four (t4), five (t5), six (t6), seven (t7), and eight cells (t8), 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 recorded. Main results and the role of chance The results from the generalized mixed models followed by Bonferroni post hoc for the comparison of means among groups showed that embryos derived from patients in the POSEIDON groups 2, 3 and 4 showed significantly slower divisions compared to those from POSEIDON 1 group (Table 1). The KIDScore rank was significantly lower for embryos derived from POSEIDON groups 2, 3 and 4 (2: 4.4 ± 0.7 vs. 3: 4.2 ± 0.2 vs. 4: 3.0 ± 0.4) compared to those derived from POSEIDON 1 group (4.8 ± 0.1, p < 0.001). Group POSEIDON 1 also showed improved implantation (26.9% vs. 2: 22.4% vs. 3: 20.0% vs. 4: 14.0, p < 0.001) and miscarriage rates (5.6% vs. 2: 31.2% vs. 4: 50.0%, p = 0.013). Limitations, reasons for caution The use of historical cohort groups is a drawback. Despite the eligibility criteria for inclusion in the analysis, potential differences in the baseline characteristics cannot be ruled out. Wider implications of the findings Embryo development was more favorable in POSEIDON group 1, suggesting a direct relationship between oocyte quantity and quality, and that oocyte quality is determinant of embryo development. These show the reasonability of classifying POR by the POSEIDON criteria, providing information for the appropriate counselling of POR regarding their possible prognosis. Trial registration number N/A

O-298 Poor semen parameters alter embryo morphokinetic patterns during preimplantation development but do not compromise clinical outcomes

Abstract Study question Does semen quality affect preimplantation development and clinical outcomes in ICSI cycles using donor oocytes? Summary answer Poor semen parameters led to altered morphokinetic parameters during preimplantation development but did not compromise blastocyst quality nor clinical outcomes in oocyte donation cycles. What is known already The quality of semen used for in vitro fertilization (IVF) may compromise the kinetics and morphology of developing embryos, ultimately impairing implantation and reducing live birth rates. Such effects are particularly pronounced when testicular or epididymal spermatozoa are used for IVF treatment. However, current studies assessing the impact of poor semen parameters on embryo development and clinical outcomes are often confounded by the use of oocytes from infertile patients. Performing such evaluations in oocyte donation cycles will provide more definitive insights into the role of semen during preimplantation development. Study design, size, duration This retrospective study included 616 embryos from 99 oocyte donation ICSI cycles, performed between February 2018 and July 2019. We compared embryo morphokinetics and clinical outcomes (cumulative pregnancy and live birth rates) across three groups with variable semen parameters. These included donor semen (control group, DD, n = 294), partner sperm obtained by testicular sperm extraction (TESE group, n = 72) and partner semen with altered parameters (including poor concentration, motility and morphology, ALT group, n = 250). Participants/materials, setting, methods We assessed several morphokinetic parameters, including second polar body extrusion (tPB2), pronuclei appearance (PN1a, PN2a), 2-3-4-5-8-cell stages (t2 to t8), start of blastulation (tSB) and blastocyst formation (tB). Kaplan-Meier survival curves were built for each parameter per study group. Survival curves were analysed by Cox regression. Qualitative parameters (even size PN and blastomeres, percent fragmentation, abnormal cleavages, blastocyst grade) were assessed using a Chi-Squared test, while Fisher’s exact test was used to evaluate clinical outcomes. Main results and the role of chance Interestingly, TESE embryos reached several developmental stages faster than the DD group: tPB2 (3.03 vs. 3.08, p < 0.001), PN1a (5.55 vs. 6.19, p < 0.001), PN2a (6.78 vs. 7.36, p < 0.001), tPNf (21.30 vs. 22.20, p = 0.001), t3 (34.02 vs. 35.79, p = 0.016), t4 (35.90 vs. 36.92, p = 0.015), t5 (46.70 vs. 48.07, p = 0.036), t8 (52.48 vs. 55.28, p = 0.049), tSB (85.53 vs. 94.62, p < 0.001) and tB (104.09 vs. 106.60, p = 0.002). ALT embryos were faster in early stages: PN1a (6.63 vs. 6.19, p = 0.005), PN2a (7.84 vs. 7.36, p < 0.001), t5 (48.12 vs. 48.07, p = 0.028), t8 (55.57 vs. 55.28, p = 0.032), but slower in the late stages: tSB (93.71 vs. 94.62, p = 0.001), tB (105.60 vs. 106.60, p = 0.002). Compared to DD, ALT and TESE embryos showed a lower rate of even pronuclei (ALT p = 4.6x10-06; TESE p = 8x10-05) and even blastomeres at the 2-cell (ALT p < 0.001; TESE p = 0.006) and 4-cell stage (ALT p = 0.004; TESE p = 0.005). Moreover, embryos derived from testicular sperm showed significantly higher fragmentation rates at the 8-cell stage (p = 0.005), while no significant differences in the frequency of irregular divisions nor in the number of top-quality blastocysts were detected in ALT and TESE. Importantly, cumulative live birth rates per cycle were similar across all study groups (DD 31/53, ALT 31/51, TESE 8/11; p > 0.05). Limitations, reasons for caution The main limitation resides in the retrospective nature of this study and the limited number of embryos in the TESE group. We also note variability in the testicular biopsy (obstructive and non-obstructive azoospermia) and non-normozoospermic diagnoses (oligozoospermia, teratozoospermia or asthenozoospermia), which may confound the analysis. Wider implications of the findings Although poor semen parameters significantly altered embryo morphokinetics, they did not compromise embryo quality, pregnancy or cumulative live birth rates, resulting in similar clinical outcomes as with double donation. Overall, our findings support the use of autologous sperm with donor oocytes, even in severe cases of male factor infertility. Trial registration number not applicable

P-129 Morphokinetic embryo behavior in oocyte presenting with dimorphisms: an analysis of 1158 injected oocytes

Abstract Study question Are morphokinetic embryo behavior and embryo quality as per Known Implantation Diagnosis Score (KIDScore) assessment related to oocyte dimorphism in intracytoplasmic sperm injection (ICSI) cycles? Summary answer Morphokinetic embryo behavior and embryo quality as per KIDScore assessment are positively related with oocyte dimorphisms, which was demonstrated by delayed cell cleavage and blastulation. What is known already As a routine procedure in intracytoplasmic sperm injection (ICSI) cycles, denudation of retrieved oocytes allows the determination of maturation status and the assessment of morphological features of the cytoplasm, perivitelline space (PVS) and zona pellucida. Oocytes presenting extra and intracytoplasmic dimorphisms have been correlated to impaired embryo developmental potential and implantation. Time-lapse imaging (TLI) systems allow for the mapping of morphological changes or events with the exact time-point of occurrence. The aim of this study was to investigate the relationship between oocyte dimorphisms an embryo morphokinetic events. Study design, size, duration This cohort study, performed in a private university-affiliated IVF center from Mar/2019-Dec/2021, included 156 ICSI cycles and 1158 injected oocytes cultured in a TLI incubator (EmbryoScope+). The presences of intracytoplasmic dimorphisms (granulation clusters, smooth endoplasmic reticulum (SER), dark cytoplasm and vacuoles), and extracytoplasmic dimorphisms (large perivitelline space (PVS), PVS granulation, polar body (PB) fragmentation and abnormal zona pellucida) were recorded, and their potential association with embryo morphokinetic events were investigated considering clustering of data. Participants/materials, setting, methods Evaluated kinetic markers were timing to pronuclei appearance (tPNa) and fading (tPNf), to two, three, four, five, six, seven, and eight cells (t2 - t8), to morulation (tM), start of blastulation (tSB) and blastulation (tB). Durations of the second (t3-t2) and third (t5-t3) cell cycles (cc2 and cc3) and timing to complete synchronous divisions s1 (t2-tPNf), s2 (t4-t3), and s3 (t8-t5) were calculated. The KIDScore ranking was recorded. The post hoc power was > 95.0%. Main results and the role of chance Morphologically normal oocytes (n = 747) reached tPNa (6.0 ± 0.1h vs. 6.4 ± 0.1h), t3 (36.5 ± 0.3h vs. 37.5 ± 0.2h), t6 (50.9 ± 0.5h vs. 52.1 ± 0.3h), tM (88.3 ± 0.8h vs. 91.6 ± 1.2h), tSB (97.4 ± 0.8h vs. 108.8 ± 3.6h), tB (104.7 ± 0.8h vs. 109.3 ± 0.5h) and cc2 (10.0 ± 0.3h vs 10.7 ± 0.2h) significantly faster than oocytes showing intracytoplasmic dimorphisms (n = 411). Significantly faster t5 (48.4 ± 0.3h vs. 50.5 ± 0.8h), t6 (51.5 ± 0.3h vs. 53.0 ± 0.7h), t7 (54.0 ± 0.3h vs. 57.1 ± 0.8h), t8 (57.9 ± 0.4h vs. 60.5 ± 0.9h), tB (107.9 ± 0.5h vs. 113.2 ± 1.1h) and cc3 (11.6 ± 0.2h vs. 13.5 ± 0.5h) were observed in normal oocytes compared to oocyte showing extracytoplasmic dimorphisms (n = 162). The incidence of extracytoplasmic dimorphisms per cohort was associated with decreased implantation (B: −23.9, CI: −37.8 – −10.1) and odds of pregnancy (OR: 0.360, CI: 0.163 – 0.796). KIDScore ranked significantly different between normal oocytes and those presenting ZP (3.8 ± 0.8 vs. 2.7 ± 0.8) and shape dimorphisms (4.1 ± 0.7 vs. 2.4 ± 1.0), and non-resistant membranes (4.4 ± 0.1 vs. 2.8 ± 0.1). Limitations, reasons for caution Despite the use of a nested statistical analysis that accounted for the fact that embryos from the same patient share comparable developmental behavior, this study has limitations, such as the retrospective design and small sample size, though an adequate power has been achieved. Wider implications of the findings This study adds to knowledge of oocyte quality role in embryo development. Dimorphic oocytes may have inefficient biological machinery. Significant differences, that could not have been noticed in a conventional incubator, were observed in embryo morphokinetic development when normal and abnormal oocytes were compared. Trial registration number N/A

P-130 Progesterone-primed cycles result in slower embryos without compromising the implantation potential, with the advantage of oral administration and potential cost reduction: A time-lapse imaging study

Abstract Study question Could exogenous progestin replace the use of an antagonist of GnRH (GNRH-antagonist) without any effects on embryo morphokinetics and implantation rate in freeze-all cycles? Summary answer Exogenous progestin leads to slower embryo development and increased cyclés cancellation rate, with increased implantation rate when compared to cycles using an GnRH-antagonist. What is known already Improvements in cryopreservation techniques associated with the expansion of elective-single embryo transfer have steadily increased the use of deferred embryo transfers. This gives the opportunity to break away from the standard sequence of stimulation–retrieval–transfer, and to consider new strategies for pharmacological control of follicle growth. Usual ovarian stimulation regimens use an analog of the GnRH to prevent the LH surge and premature ovulation. Since progesterone is able to block the LH surge question remains on whether exogenous progesterone may replace the use of an GnRH analogue without compromising embryo development, in cycles followed by embryo cryopreservation. Study design, size, duration This cohort study, performed in a private university-affiliated IVF center, from Mar/2019 - Mar/2021, included 288 freeze-all ICSI cycles and its 2,768 respective embryos. Patients were age matched, according to the age, into groups depending on the protocol used to prevent the LH surge: Progestin-Primed-Group (n = 144 cycles and 1,360 embryos) and GnRH-Antagonist-Group (n = 144 cycles and 1,408 embryos). Embryos were cultured in a time-lapse imaging incubation system (TLI) and embryo morphokinetics were compared among the groups Participants/materials, setting, methods Evaluated kinetic markers were timing to pronuclei appearance (tPNa) and fading (tPNf), to two, three, four, five, six, seven, and eight cells (t2 - t8), to morulation (tM), start of blastulation (tSB) and blastulation (tB). Durations of the second (t3-t2) and third (t5-t3) cell cycles (cc2 and cc3) and timing to complete synchronous divisions s1 (t2-tPNf), s2 (t4-t3), and s3 (t8-t5) were also calculated. The KID-Score ranking, laboratorial and clinical outcomes were also evaluated. Main results and the role of chance Mean maternal and paternal ages were 37.0 ± 3.8 and 39.0 ± 6.4 years old. Slower tPNa (6.2 ± 0.2 vs. 7.0 ± 0.2, p = 0.008), t2 (27.2 ± 0.3 vs. 26.2 ± 0.3, p = 0.045), t7 (56.4 ± vs. 54.7 ± 0.5, p = 0.046), tM (89.3 ± 0.8 vs. 87.1 ± 0.6, p = 0.045), tSB (101.5 ± 0.8 vs. 110.8 ± 0.1, p = 0.012), tB (111.0 ± 0.8 vs. 108.5 ± 0.7, p = 0.034), were observed among embryos derived from Progestin-Primed cycles. Similar tPNf, t3, t4, t5, t6, t8, timing to cc2, cc3, s1, s2, and s3 were observed among the groups. No significant differences were noted in the Kid-score D5, number of aspirated follicles, retrieved oocytes, oocyte yield, mature oocytes, and rates of mature oocytes, fertilization, and blastocyst formation. The cyclés cancelation (15.7% vs. 2.0%, p = 0.004) and implantation rates (63.1% ± 6.1 vs. 44.4% ± 6.3, p = 0.004) were increased in the Progestin-Primed-Group, however no significant differences were noted in the cumulative pregnancy (64.4% vs. 49.0%, p = 0.104) and miscarriage rate (2.6% vs. 8.6%, p = 0.554) for Progestin-Primed and GnRH-Antagonist groups, respectively. The expense for the ovulation prevention using the GnRH-Antagonist was U$318.18, while by using progestins U$ 11.05 was sufficient to inhibit the premature LH surge. Limitations, reasons for caution This study has limitations, such as the retrospective design and small sample size. Wider implications of the findings Despite the increased implantation rate and potential cost reduction, the high cancellation rate and slower embryo development in the progestin-protocol cannot be ignored. Delayed embryo transfer due to the freeze-only approach may also be inconvenient. Before considering any protocols for LH surge prevention, pros and cons must be carefully evaluated. Trial registration number N/A

Circulating maternal prorenin and oocyte and preimplantation embryo development: the Rotterdam Periconception Cohort.

Could circulating maternal prorenin serve as a proxy for oocyte and preimplantation embryo development, assessed by time-lapse parameters and clinical treatment outcomes? High circulating maternal prorenin concentrations after ovarian stimulation associate with a larger oocyte area, faster cleavage divisions from the five-cell stage onwards and increased chance of successful implantation. After ovarian stimulation, circulating prorenin (renin's precursor), is largely ovary-derived. Prorenin may contribute to ovarian angiotensin synthesis, which is relevant in reproduction given its role in follicular development and oocyte maturation. Prospective observational cohort study including couples requiring fertility treatment from May 2017 as a subcohort of the ongoing Rotterdam Periconception Cohort conducted in a tertiary referral hospital. Between May 2017 and July 2020, 309 couples with an indication for IVF treatment or ICSI were included. Resulting embryos (n = 1024) were submitted to time-lapse embryo culture. Time of fertilization (t0), pronuclear appearance (tPNa), and fading (tPNf) as well as the exact timing of reaching the two- to eight-cell stage (t2-t8), the start of blastulation (tSB), reaching the full (tB), and expanded blastocyst (tEB) were retrospectively recorded. Oocyte area was measured at t0, tPNa, and tPNf. Prorenin was determined at the day of embryo transfer. After adjustment for patient- and treatment-related factors, linear mixed modeling showed that higher prorenin concentrations associate with a larger oocyte area at tPNa (β 64.45 µm2, 95% CI 3.26; 125.64, P = 0.04), and faster progression from five-cell stage onwards (e.g. β8-cell -1.37 h, 95% CI -2.48; -0.26, P = 0.02). Prorenin associated positively with pre-transfer outcomes (e.g. βfertilized oocytes 2.09, 95% CI 1.43; 2.75, P < 0.001) and implantation (odds ratio+β-hCG-test: 1.79, 95% CI 1.06; 3.08, P = 0.03), but not with live birth. This prospective observational study provides associations and therefore residual confounding cannot be excluded and causality has to be shown in intervention studies. Theca cell-derived factors, such as prorenin, may help to clarify the underlying endocrine mechanism of oocyte maturation and embryo development, with a special focus on the (patho)physiological reproductive role of prorenin and the identification of factors influencing its secretion and activity, which is of great added value for improving embryo selection and predicting implantation and pregnancy outcomes. This will bring us to investigate which determinants of oocyte quality and embryo development should take center stage in developing preconception care strategies. This research was funded by the Department of Obstetrics and Gynecology of the Erasmus MC, University Medical Center, Rotterdam, the Netherlands, and the Erasmus MC Medical Research Advisor Committee's 'Health Care Efficiency Research' program (OZBS72.16080). The authors have no competing interests to disclose. N/A.

PREVIOUS INFECTION WITH SARS-COV-2 IMPACTS EMBRYO MORPHOKINETICS IN INTRACYTOPLASMIC SPERM INJECTION CYCLES

Coronavirus disease 2019 (COVID-19) is caused by a novel coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which may infect any cell type expressing ACE2 and TMPRSS2 receptors. ACE2 is expressed in several human ovarian compartments, and relatively highly expressed in oocytes. Therefore, the ovary and oocyte might be potential targets of SARS-CoV-2. Information concerning the susceptibility of the female reproductive systems to SARS-CoV-2 infection, and possible effects on embryo development remain inconsistent. Time-lapse imaging (TLI) systems allow for the mapping of morphological changes or events with the exact time-point of occurrence. The aim of this study was to investigate the impact of SARS-CoV-2 infection on embryo morphokinetic development. This historical cohort study enrolled 884 injected oocytes from 88 female patients undergoing ICSI from Mar/2019 to Jun/2021. Female patients were age matched in a 1:3 ratio, to either a COVID group, including patients with a positive SARS-CoV-2 immunoglobulin test (n=22 patients, 152 injected oocytes), or a control group, including patients with a negative SARS-COV-2 immunoglobulin test (n=66, 732 injected oocytes). A sample of at least 111 embryos had 95% power to detect a 20% effect with alpha of 5%. Embryos were cultured in the EmbryoScope incubator, which recorded the following kinetic markers: timing to pronuclei appearance and fading (tPNa and tPNf), two (t2), three (t3), four (t4), five (t5), six (t6), seven (t7), and eight cells (t8), 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 recorded. The impact of SARS-COV-2 on embryo morphokinetic events and ICSI outcomes were investigated considering clustering of data. Embryos derived from patients in the COVID group presented longer tPNa (7.4 ± 0.26 vs. 6.71 ± 0.18, p< 0.001), tPNf (24.503 ± 0.47 vs. 23.386 ± 0.336, p=0.003), t2 (26.980 ± 0.489 vs. 26.001 ± 0.344, p=0.032), t3 (37.992 ± 0.659 vs. 36.146 ± 0.461, p=0.005), t4 (41.027 ± 0.677 vs. 37.745 ± 0.471, p< 0.001), t5 (51.856 ± 1.067 vs. 48.636 ± 0.722, p< 0.001) and tB (109.122 ± 0.804 vs. 106.244 ± 1.429, p=0.028). Durations of cc3 (14.187 ± 0.65 vs. 12.806 ± 0.25, p=0.049), s1 (2.397 ± 0.895 vs. 2.732 ± 0.306, p< 0.001), s2 (3.073 ± 0.36 vs. 1.871 ± 0.146, p=0.002) were also significantly increased in the COVID group compared with the control group, while KIDScore D5 ranked significantly lower in the COVID group (5.3 ± 0.3 vs. 6.3 ± 0.1, P=0.005). Previous SARS-CoV-2 maternal infection had significant impacts on embryo morphokinetic events and KIDScore rank.

P-528 Time-lapse monitoring: an adjunct tool to select embryos for preimplantation genetic testing

Abstract Study question Does embryo aneuploidy have any impact on embryo morphokinetic events in a time-lapse imaging (TLI) culture system? Summary answer Aneuploidy had a significant impact on early and late embryo morphokinetic events. Mosaic and aneuploid embryos behaved similarly in terms of morphokinetics. What is known already The preimplantation genetic testing (PGT) emerged as an approach to analyse embryos’ DNA for determining genetic abnormalities, distinguishing chromosomally normal embryos, considered to have high developmental potential, from their aneuploid siblings. Although shown to be efficient and clinically relevant, the invasive nature of the technique limits its success. Given the challenges associated with invasive embryo biopsy, ongoing studies are now seeking for non-invasive assessments of the embryo DNA. Timelapse imaging (TLI) may allow the identification of morphokinetics events affected by aneuploidy, which could be a powerful tool for improving embryo selection for transfer, without the detrimental effects of embryo biopsy. Study design, size, duration This case-control study was performed in a private university–affiliated IVF center, between 2019 March and 2020 December. Kinetic data were analyzed in 957 embryos, derived from 316 patients undergoing ICSI cycle with PGT-A. Kinetic markers from the point of insemination were recorded. Generalized linear models followed by Bonferroni post hoc were used to compare timing of specific events in euploid (n = 352), aneuploid (n = 583) and mosaic embryos (n = 22). The post hoc achieved power was &amp;gt; 80%. Participants/materials, setting, methods Embryos were cultured in the EmbryoScope incubator, which recorded the following kinetic markers: timing to pronuclei appearance and fading (tPNa and tPNf), two (t2), three (t3), four (t4), five (t5), six (t6), seven (t7), and eight cells (t8), 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 recorded. Main results and the role of chance Aneuploid embryos showed significantly longer tPNf, t2, t3, t4, t6, t7, t8, tM, tSB, tB, s2 and s3 compared to euploid embryos. The KIDScore day 5 also rank was significantly lower in aneuploid embryos compared to euploid ones. As for mosaic embryos no significant differences were observed in the comparisons of means of morphokinetic events and KIDScore rank, but regression analyses showed an increased chance of mosaicism among embryos with longer tPNf, t2, t3, t6, t7, tM, tB, as well as a inverse association with KIDScore rank, when compared with euploid embryos. Embryos were hierarchically distributed into quartiles according to the KIDScore rank: Q1 ≤3.9, Q2 4-5.6, Q3 5.7-7.5, and Q4 ≥7.6. Euploidy incidence was significantly different among the KIDScore quartiles, in which the percentage of euploidy increased directly proportional to the kidscore rank. For patients &amp;gt;35 years old, the logistic regression analysis demonstrated that comparing with embryos in the Q4, embryos in the Q1, Q2, and Q3 have a decreased chance of being euploid. For patients ≤35 years old, comparing with embryos in the Q4, embryos with KIDScore D5 in the Q1 and Q2, but not in Q3, have a decreased chance of being euploid. Limitations, reasons for caution Our study is limited by its retrospective nature and small casuistic, despite adequate power has been achieved. The reason for the lack of consistency with some previous findings can be explained by variations in culture systems, studied population, embryo stage for biopsy and wide range of tests used for PGT. Wider implications of the findings Our evidence suggests that TLI monitoring may be an adjunct approach to select embryos for PGT, however, cautious investigation is still needed. The mechanism by which embryo aneuploidy may alter the cleavage timing of embryos is undefined, but disruptions of mitosis, cell-cell interactions and blastocyst differentiation are possible candidates. Trial registration number not applicable

P-287 An assessment of agreement between automated embryo annotation, through artificial intelligence, and manual embryo annotation

Abstract Study question How strong is the agreement between embryo morphokinetic annotations performed by experienced embryologists compared to an automated embryo annotation system based on artificial intelligence (AI)? Summary answer Agreement between manual and automated annotation as determined by the interclass correlation coefficient (ICC) revealed strong or very strong agreement for all analysed morphokinetic variables. What is known already Transitioning from time-lapse imaging to embryo selection for transfer, freezing or discard involves annotation; the action of converting images to numerical data. Numerical data can be used as input to selection models quantifying embryo viability. Currently, embryos are manually annotated by the embryologist which can be subjective and time-consuming. As such, clinics prioritise a manageable number of variables to annotate, leading to a range of clinic practices. There is the additional challenge of operator variation, despite the development of standardised definitions and quality assurance schemes. AI may help resolve these challenges. Study design, size, duration Retrospective comparative analysis, including 2442 embryos from IVF and ICSI cycles, from four private fertility clinics belonging to the same group in the UK. All the embryos cultured in a time-lapse incubator (EmbryoScope,Vitrolife) between January 2016 and 2019 were included in the study. Manual annotations (MA) versus automated annotations (AA) were compared using a two-way, mixed interclass correlation coefficient (ICC), which produced five categories of agreement, very weak(0-0.20), weak(0.21-0.40), moderate(0.41-0.60), strong(0.61-0.80), very strong(0.81-1.00). Participants/materials, setting, methods Videos were manually annotated by experienced embryologists from pronuclei fading (tPNf) to time of expanded blastocyst (tEB) with all cell stages annotated in between (time to two-cell (t2), three-cell (t3), four-cell (t4), five-cell (t5), six-cell (t6), seven-cell (t7), eight-cell (t8), nine-cell (t9), morula (tM), start of blastulation (tSB) and full blastocyst (tB)). Blind to human annotations, and without any training, the same videos were annotated by CHLOE (Fairtility) to produce automated annotation data. Main results and the role of chance Of the expected annotations, AA did not provide a result for 15.4% of the MA(3235/21008). Very strong agreement(0.81-1.00) between MA and AA was found for tPNf, t2, t3, t5, t6, tM, tSB, tB, tEB. Strong agreement(0.61-0.80) was found for t4, t7, t8 and t9+. Outliers in the AA data, defined as one standard deviation from the MA, were interrogated further for five key morphokinetic parameters; t2, t5, t8, tSB and tB. A total of 269 outliers were identified. For t2 outliers(n = 14,6%), the average time difference was 5.97h(range;5.50-24.44h). All embryos with a t2 outlier were classed as either poor(PQ) or average quality(AQ). The t5 outliers(n = 45,19%) had an average time difference of 2.84h(range;9.33-36.69h). 96%(n = 43) of these embryos were classed as PQ(n = 25,56%) or AQ(n = 18,40%). Outliers for t8(138,58%) were, on average, 17.53h different between MA and AA(range;12.68-40.35h). 94%(n = 130)of these embryos were classed as PQ(n = 77,56%) or AQ(n = 53,38%). The tSB outliers(n = 28,12%) had an average time difference of 3.58h(range;0.71-14.39h). 89%(n = 25) of these embryos were classed as PQ(n = 16,57%) or AQ(n = 9,32%). Finally, outliers associated with tB(n = 44,18%) had an average time difference of 6.39h(range;0.02-33.67h). 95%(n = 42) of these embryos were classed as PQ(n = 38,86%) or AQ(n = 4,9%). Almost 15%(n = 40) of the embryos had outliers in more than one of the five morphokinetic parameters. Limitations, reasons for caution The findings for this study reflect the capabilities of a specific AI-based annotation algorithm against the practice in multiple clinics in the same group and country. The automated annotation algorithm was not trained on this dataset prior to validation, which is encouraging for generalisability. Wider implications of the findings AI is ideally suited to resolve annotation challenges. This study demonstrates that where embryo quality is poor, annotation could be skewed both when performed manually and automatically. Once robustness is demonstrated, AI tools such as CHLOE, may allow clinics to process clinical data efficiently, objectively and consistently. Trial registration number None

P-176 Morphokinetic Expressions Predict Day Five Embryo Quality and Implantation - A Prospective Observational Study

Abstract Study question The aim of this prospective study was to investigate the value of human embryo morphokinetics as predictors of day 5 embryo quality and implantation. Summary answer Day 5 embryo quality was predicted best by Cleavage Synchronicity 2-8-cell stage (CS2-8). Implantation was predicted best by the time to the expanded blastocyst (tEB-tPNf). What is known already Time-lapse imaging technology enables embryologists to observe and assess not only embryo morphology but also kinetics and cleavage synchronicity. It was found that time intervals like time from 5- to 8-cell stage (s3 = t8 - t5) can predict blastocyst formation. Cetinkaya et al. defined four relative morphokinetic expressions describing cleavage synchronicity: Cleavage Synchronicity 2-8-, 4-8-, 2-4-cell stage (CS2-8, CS4-8, CS2-4) and DNA Replication time ratio (DR). In a retrospective study they found that CS2-8 followed by s3, and CS4-8 were better predictors of blastocyst quality than absolute cleavage time points (https://doi.org/10.1007/s10815-014-0341-x). Study design, size, duration This prospective observational study was conducted between December 2020 and October 2021 at a single university hospital. Patients between 18 and 45 years who underwent stimulated IVF or ICSI treatment with embryo culture in a time-lapse incubator were included. Exclusion criteria were endometriosis, PCOS, history of chemotherapy, PGT cycles and end of culture before day 5. Every patient was included only once (n = 100). Implantation was assessed for all patients with embryo transfer. Participants/materials, setting, methods Embryos (n = 298) reaching at least 8-cell stage were annotated for morphokinetics and divided into four groups based on day 5 morphology: top (TQ; n = 60), good (GQ; n = 69), poor quality (PQ; n = 105) or not reaching blastocyst stage (AE; n = 64). Absolute times were normalized to fading of pronuclei (tPNf). ROC-analysis was performed to test how morphokinetics predict quality and implantation (only known-implantation-data; n = 110). Main results and the role of chance To predict day 5 embryo quality, four morphokinetic expressions had an Area Under the Curve (AUC) of more than 0.7, including CS2-8 (AUC = 0.732), CS4-8 (AUC = 0.717), s3 (AUC = 0.724) and the start of blastulation (tSB-tPNf) (AUC = 0.725). All morphokinetic expressions mentioned were significantly different between TQ plus GQ versus PQ plus AE embryos (p &amp;lt; 0.001). Additionally, the data of each morphokinetic parameter were divided into four quarters using the 25th, 50th and 75th percentile. CS2-8 had a rate of 67.6% TQ plus GQ blastocysts in the fourth quarter (CS2-8 &amp;gt; 0.86). CS4-8, s3 and tSB-tPNf had the highest rate of TQ plus GQ blastocysts (64.3 %, 64.3 %, 72.9 %) in the first quarter (CS4-8≤0.20, s3≤3.01 h, tSB-tPNf ≤ 67.06 h). Implantation was predicted best by the time to reach the morula stage (tM-tPNf; AUC = 0.636, p = 0.049), start blastulation (tSB-tPNf; AUC = 0.660, p = 0.022) and expanded blastocyst (tEB-tPNf; AUC = 0.688, p = 0.009). The relative expressions CS2-8 and CS4-8 and the interval s3 were not significantly different between implanted and non-implanted embryos. Limitations, reasons for caution Morphokinetic annotation is open to inter- and intraobserver variability, although partially controlled by prospectivity. The ROC-analysis does not adjust for statistical dependence between embryos of one patient. The used morphological groups do not necessarily measure developmental potential. Embryos with KID showed less morphokinetic variation because of pre-selection for transfer. Wider implications of the findings The findings could contribute to an early decision model on transfer hierarchy that would help avoiding costs and a potential negative influence of prolonged blastocyst culture. Observing the kinetics of blastocyst formation (tM, tSB, tEB) might allow an improved prediction of implantation potential. Trial registration number DRKS00022983

O-208 Use of epididymal spermatozoa in in vitro fertilization cycles impacts the morphokinetics of embryos cultured in a time-lapse imaging incubator system

Abstract Study question Does sperm origin impact embryo morphokinetics (the moments at which an embryo reaches a developmental milestone) and clinical outcomes of intracytoplasmic sperm injection (ICSI) cycles? Summary answer The use of epididymal sperm correlates with late cell cleavages, and higher multinucleation and abnormal cleavage rates compared to those from men without infertility factor. What is known already ICSI with sperm surgically retrieved from the epididymis (PESA) dramatically altered the pregnancy prognosis in couples with severe male factor infertility, including obstructive azoospermia. The integration of time-lapse imaging (TLI) into the in vitro fertilization (IVF) laboratory allowed for an in-depth analysis of embryonic development. However, the impact of the use of epididymal sperm on embryonic morphokinetics has been poorly studied. We hypothesized that factors related to sperm origin may interfere with the speed and pattern of cell divisions. Study design, size, duration Women undergoing ICSI with epididymal sperm between Jan/2019 and Dec/2020 in a private university-affiliated IVF center were included (PESA group, n = 32 cycles/276 embryos). A matching was performed to form two groups of women undergoing ICSI using ejaculated sperm from partners with idiopathic male factor infertility (IMF group, n = 32 cycles/284 embryos) or without male factor infertility (Control group, n = 32 cycles/246 embryos). Generalized linear models followed by Bonferroni post hoc were used to compare morphokinetics between groups. Participants/materials, setting, methods Embryos were cultured in the EmbryoScope incubator, which recorded the following kinetic markers: timing to pronuclei appearance and fading (tPNa and tPNf), two (t2), three (t3), four (t4), five (t5), six (t6), seven (t7), and eight cells (t8), 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 recorded. Main results and the role of chance Embryos derived from epididymal sperm showed significantly slower divisions compared to those from ejaculated sperm (Table 1). The KIDScore rank was significantly lower for embryos deriving from epididymal sperm (3,1 ± 0.2) compared to those deriving from ejaculated sperm from IMF and control groups (5.4 ± 0.1 and 5.6 ± 0.2, p &amp;lt; 0.001, respectively). The incidence of multinucleation was significantly higher in embryos deriving from epididymal sperm (23.2%) compared to those deriving from ejaculated sperm from IMF and control groups (2.8% and 3.7%, p &amp;lt; 0.001, respectively). The incidence of abnormal cleavage patterns (direct or reverse) was significantly higher in embryos deriving from epididymal sperm (11.1%) compared to those deriving from ejaculated sperm from control group (4.3%, p = 0.001). Similar clinical outcomes were observed between the groups. Limitations, reasons for caution Retrospective nature of this study and the small sample size may be a reason for caution. Wider implications of the findings The use of epididymal sperm for ICSI correlates with delayed cell cleavage, and increased incidences of multinucleation and abnormal cleavage patterns. This finding highlights the importance of TLI for the identification and de-selection of slow-growing embryos for transfer, in ICSI cycles with surgical sperm retrieval. Trial registration number not applicable

P-167 Male infertility and polycystic ovary syndrome (PCOS) affect absolute and relative embryo morphokinetics observed by time-lapse imaging

Abstract Study question This study aimed to test if morphokinetics are altered in embryos from patients with male infertility, polycystic ovary syndrome (PCOS) or recurrent pregnancy loss (RPL). Summary answer Morphokinetics differed significantly between embryos from patients with male infertility or PCOS compared to the control groups, possibly suggesting a negative impact on development potential. What is known already Time-lapse imaging technology allows continuous observation of embryonic morphology and developmental kinetics during the preimplantation period. The ESHRE Working group on Time-lapse technology recently summarised common absolute morphokinetic time points and intervals [DOI: 10.1093/hropen/hoaa008]. Additionally, four relative morphokinetic expressions were introduced by Cetinkaya et al.: Cleavage Synchronicity 2-8-, 4-8-, 2-4-cell stage (CS2-8, CS4-8, CS2-4) and DNA Replication time ratio (DR) [DOI: 10.1007/s10815-014-0341-x]. A study by Freis et al. observed worse CS2-8 and CS4-8 ratios in embryos from patients with endometriosis [DOI: 10.1177/1933719117741373]. Other studies have found that male infertility and PCOS may affect morphokinetic time points. Study design, size, duration In this retrospective observational study n = 1433 two-pronuclei oocytes from n = 433 patients between 18 and 45 years undergoing IVF/ICSI-treatment between 09/2016 and 12/2019 were examined. The use of a time-lapse incubator was obligatory. Exclusion criteria were the presence of endometriosis, history of chemotherapy, preimplantation genetic testing (PGT) cycles and genetic abnormalities. Every patient was included only once. Control groups were uterine, tubal factor and idiopathic infertility. Participants/materials, setting, methods Male infertility (n = 928 oocytes; 288 patients) versus control (n = 400; 115), PCOS (n = 48; 14) versus control (n = 400; 115) and RPL (n = 79; 21) versus control (n = 321; 94) were compared. Patient and treatment characteristics were compared using Mann-Whitney-U and Fisher’s exact test. Times normalised to fading of pronuclei (tPNf), intervals and ratios were analysed using a Generalised Linear Mixed Model (GLMM). Main results and the role of chance The male infertility group was significantly different (p &amp;lt; 0.05) from the control group regarding male age (p = 0.009), fertilisation rate (p = 0.008), number of embryos per patient (p = 0.033), treatment method (p &amp;lt; 0.001) and use of Calcium-Ionophore (p = 0.049) or SpermMobile (p = 0.003). DR was significantly higher in the male infertility group compared to the control group (p = 0.031). The start of blastulation (tSB-tPNf; p = 0.008) and start of expansion of the blastocyst (tEB-tPNf; p = 0.002) were significantly delayed. The PCOS group was significantly different concerning female age (p = 0.023) and AMH blood level (p &amp;lt; 0.001). CS2-8 was significantly lower (p = 0.003) and CS2-4 significantly higher (p = 0.001) in the PCOS group compared to the control group. The time intervals from three- to four-cell (s2; p = 0.013), from five- to eight-cell (s3; p = 0.032) and from four- to eight-cell stage (ECC3; p = 0.043) were significantly longer in the PCOS group. There was no significant difference in patient or treatment related characteristics between the RPL group and the control group. Also, none of the morphokinetic parameters analysed in this study was significantly different between the groups. Limitations, reasons for caution Small group sizes decreased further at later developmental time points. In general, time-lapse data show a high intra- and inter-observer variability. A GLMM was performed to take statistical dependencies between embryos of one patient into account. Its Gaussian assumption was partially violated by the true distribution of the data. Wider implications of the findings These data support the hypothesis that patients’ co-morbidities may affect embryo morphokinetics. The impact on clinical outcome should be examined further. Accordingly, our working group conducted another study about the predictive value of morphokinetics on blastocyst quality and implantation (DRKS-ID: DRKS00022983). The long-term goal is to improve embryo assessment. Trial registration number not applicable

P-191 Differences in morphokinetic patterns and clinical outcomes between fresh and frozen oocytes; a retrospective analysis

Abstract Study question Is there a difference in morphokinetics and clinical outcomes between embryos from fresh and vitrified oocytes? Summary answer Embryos from vitrified versus fresh oocytes showed a delay at the cellular stage, but no impact on time to blastulation or clinical outcomes was evident. What is known already Oocyte vitrification has greatly impacted assisted reproduction, with the number of treatments cycles using frozen oocytes more than doubling in the UK since 2013. Studies of thawed vitrified oocytes have shown similar success rates and outcomes compared to fresh, allowing the technique to be considered safe and effective. However, vitrification and thawing subjects the oocyte to stress and osmotic changes that may be evident in alterations in the timing of their morphological events. Analysis of morphokinetic markers using time-lapse incubators was performed to investigate this. Study design, size, duration Matched cohort study. A total of 823 embryos were analysed, 414 embryos from fresh oocytes and 409 from vitrified. The embryos were from the 288 ICSI treatment cycles performed at LWC in 2019. Fresh oocytes were from women less than 35 years old undergoing fertility treatment and vitrified oocytes were from egg donors under 35. Participants/materials, setting, methods Embryos graded AA, BB, BA, AB, were selected and annotated retrospectively on the Embryoscope for the following events: pronuclei appearance (tPNa) and disappearance (tPNf), time until two (t2), four (t4) and eight cells (t8), compaction initiation (tSC), the start of blastulation (tSB) and time to expanded blastocyst (tEB). PN duration, second and third embryo cell cycle (ECC), compaction and blastulation duration were also calculated as well as differences in clinical outcomes. Main results and the role of chance Embryos derived from vitrified oocytes (EVO) were observed to have a statistically significant delay in 4/8 morphokinetic events studied: t4 (p = 0.03), t8 (p &amp;lt; 0.01), tSC (p &amp;lt; 0.01) and tSB (p = 0.01). A mean delay of 1h50min was observed when compared to embryos from fresh oocytes (EFO). ECC duration showed a statistically significant difference with a delay of 48 minutes in the vitrified group. However, compaction occurred on average just 84min faster in this group, meaning no differences were observed in the time needed to achieve a full expanded blastocyst. Regression analysis revealed a correlation between the age of the oocyte and morphokinetic timings. Oocytes from older women demonstrated slower development, with age having a statistically significant impact in the following categories: tPNa, tPNf, t2 and t4. No differences found between fresh and vitrified groups in fertilization rate (80% EFO vs 79% EVO) (p = 0.841), embryo utilization rate (60% EFO and 61% EVO) (p = 0.432), implantation rate (54% EFO vs 52% EVO) (p = 0.837) and clinical pregnancy rates (49% EFO vs 42% EVO) (p = 0.502). Limitations, reasons for caution Limitations of the present study include the retrospective analysis, small sample size and the lack of adjustment for potential contributory/confounding factors such as semen quality, body mass index (BMI), antimüllerian hormone (AMH) levels, type of ovarian stimulation or type of infertility which are known possible influencers of embryo morphokinetics. Wider implications of the findings The delay observed at the cellular stage by EVO had no impact on the time the embryos needed to achieve full expansion. While vitrification affects embryo morphokinetics, it does not seem to impact the ability of the oocyte to be fertilized, activated, or to produce a viable blastocyst and pregnancy. Trial registration number Not applicable

P-182 The impact of ethnic differences on embryo morphokinetics and clinical outcomes: the importance of racial admixture

Abstract Study question Can ethnic differences affect embryo morphokinetics and clinical outcomes in a time-lapse imaging (TLI) embryo culture system? Summary answer Embryos from Mestizos develop faster and have a higher implantation rate than those from Caucasian and Asian patients. What is known already Over the past decades, the use of assisted reproductive technology (ART) has made pregnancy possible for many infertile couples. However, certain infertile couples exhaust all forms of ART, without achieving success. Differences in treatment success have been described as varying by race and ethnicity. In fact, it has been reported that ethnicity is a major determinant of ART outcome as indicated by significantly lower live birth rates in some ethnic minority groups, however the precise reason for this fact remains unknown. TLI may allow the identification of morphokinetics events potentially affected by ethnicity, which may impact embryo development and implantation. Study design, size, duration This historical cohort study enrolled 3,304 embryos from 466 patients undergoing ICSI cycles from Feb/2019 to Ago/2021. Female patients were split into groups according to the patient’s ethnicity: (i) Caucasian (356 patients and 2556 embryos), Mestizo (82 patients and 592 embryos), and Asian (156 patients and 28 embryos). Embryos were cultured in a TLI incubation system and morphokinetics factors and clinical outcomes were compared between the groups using generalized linear models followed by Bonferroni post-hoc. 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. Additionally, the known implantation data score (KIDScore) day-5 was recorded. Main results and the role of chance Embryos derived from Mestizo patients completed several key-point stages faster, followed by Caucasians, while Asiatic embryos were the slower ones. Mestizo presented shorter t6 (54.8 ± 0.92, 52.6 ± 0.20, 51.0 ± 0.42, for Asians, Caucasians, and Mestizos respectively, p &amp;lt; 0.001), t7 (58.4 ± 1.05, 55.4 ± 0.22, 54.1 ± 0.45, for Asians, Caucasians, and Mestizos respectively, p &amp;lt; 0.001), t8 (59.3 ± 1.2, 58.7 ± 0.24, 57.3 ± 0.49, for Asians, Caucasians, and Mestizos respectively, p = 0.019), tM (99.3 ± 2.1, 90.8 ± 0.37, 87.5 ± 0.79, for Asians, Caucasians, and Mestizos respectively, p &amp;lt; 0.001), tsB (106.4 ± 1.9, 101.4.0 ± 0.54, 98.0 ± 1.4, for Asians, Caucasians, and Mestizos respectively, p &amp;lt; 0.001), and tB (109.1 ± 0.31,116.2 ± 2.0, 106.0 ± 0.64, for Asians, Caucasians, and Mestizos respectively, p &amp;lt; 0.001). In addition, the KIDScore-D5 ranked significantly higher for Mestizos, followed by Caucasians, while Asian patients presented the lower KIDScore-D5 (2.9 ± 0.27, 5.3 ± 0.1, 6.3 ± 0.4, p &amp;lt; 0.001). Pregnancy (25.0 ± 4.9, 33.0 ± 13.2, 58.0 ± 11.5, p = 0.038) and implantation rates (30.0 ± 1.2, 16.3 ± 0.4, 50.0 ±1.5, p &amp;lt; 0.001) were also highest among Mestizos, followed by Caucasians, whereas the Asian patients had the lowest rates. Limitations, reasons for caution The retrospective nature of the study and the small sample size may have biased the results. Wider implications of the findings Despite the increasing number of studies investigating the effect of ethnicity on ART outcomes, most of the literature has focused on White, Black and Asians. However, racial admixture has markedly increased, making attempts at strict racial categorisation confusing and out-dated. Apparently, miscegenation benefits embryonic development, which may impact clinical outcomes. Trial registration number Not applicable

P–233 The spatial arrangement of blastomeres and time of cavitation forming as predictors of blastocyst quality

Abstract Study question Does the spatial arrangement of blastomeres and the start of blastulation affect blastocyst quality? Summary answer Better blastocyst quality is associated with the spatial arrangement of the embryo and the shorter time frame of blastulation (cavitation). What is known already The ability to select the human embryo with the highest implantation potential remains one of the greatest challenges in the management of In Vitro Fertilization patients. Several publications have proposed that additional morphological evaluations of blastomere arrangement and the dynamics of late-stage embryonic divisions might be a useful non-invasive way for embryo selection. In the last decade, the introduction of time-lapse technology enables continuous monitoring of embryo development, which leads to better outcomes than a selection based on the traditional morphology assessment. Study design, size, duration The spatial arrangement was defined as tetrahedrally if the cleavage planes were perpendicularly orientated, while embryos with rather parallelly orientated cleavage axes were considered as non-tetrahedral embryos. The injection time of ICSI was designated as “time zero” (t0), and EmbryoViewer software was used to calculate the time duration between injection and start of blastulation (cavitation). Obtained results were later correlated with the embryo’s capability to form a blastocyst as well as with blastocyst quality. Participants/materials, setting, methods A total of 195 oocytes from 40 patients undergoing the antagonist cycle for ICSI treatment were evaluated. All blastocysts were cultured in Embryoscope™ according to the manufacturer’s specifications (Vitrolife, Sweden). The Gardner and Schoolcraft scoring system was used to describe blastocyst quality. Statistical analyses were performed using IBM SPSS version 24. Data were reported as median and range. Differences between groups were tested using the Mann-Whitney U test. Statistical significance was defined as p &amp;lt; 0.05. Main results and the role of chance Obtained data showed that 83.6% (61/73) of embryos with tetrahedral arrangement formed blastocysts compared to 42.4% (50/116) of embryos with the non-tetrahedral arrangement (p &amp;lt; 0,001). Moreover, tetrahedral embryos more frequently formed good quality blastocyst compare to the non-tetrahedral [59% (36/61) vs 18 (9/50)% respectively; p &amp;lt; 0,001]. In addition, we found that good quality blastocyst had a significantly shorter time frame between injection and blastulation start, compared with blastocysts which did not reach good quality [95.00h (84–118) vs 102h (77–121) respectively; p = 0,006]. Limitations, reasons for caution The limitation of the present study was that due to the double-embryo transfer correlation between those morphokinetic parameters and pregnancy rate can not be calculated. Further research should link these morphokinetic parameters with pregnancy rate and live birth rate as well. Wider implications of the findings: The potential of our findings is considerable, especially for countries with strict Embryo Law Regulation. Obtained results might be highly useful for selecting embryos with high implantation potential. In addition, the present work illustrates the possibility of additional information that can potentially be incorporated into an embryo classification model. Trial registration number Not applicable

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–278 The embryo score provided by the KIDScoreD5 is correlated with implantation rate and clinical outcomes in fresh transfer

Abstract Study question Can the algorithm used by EmbryoScopePlus software predict implantation and clinical pregnancy in women of different age groups on fresh transfer? Summary answer The embryo score generated by KIDScoreD5 is highly related to the rates of implantation and clinical pregnancy in fresh transfers in women of different age. What is known already Artificial Intelligence algorithms use statistics to find patterns in large amounts of data and describe a non-biased approach to multiparameter analysis. Several algorithms have been described, but none has been adopted for universal use. KIDScoreD5 is the algorithm included in the EmbryoScopePlus system and classifies embryos according to the cleavage times and morphology of the blastocyst. Version 3, more current, includes the annotations of the number of pronuclei, the time of division for 2, 3, 4 and 5 cells, time to start of blastulation, and morphology of the Internal Cell Mass and trophectoderm. Study design, size, duration Retrospective study evaluated 86 embryos from January to December 2019 at the Reproferty clinic, grown at EmbryoScopePlus and transferred fresh on the fifth day of embryo development. The morphological and morphokinetic parameters were automatically evaluated by the software and in case of any mistake, they were manually corrected. The embryos were evaluated by KIDScoreD5 v3 in different scores from 0.0 to 9.9 and divided into 4 groups (0.0–2.5; 2.6–5.0; 5.1–7.5; 7.6 –9.9). Participants/materials, setting, methods The inclusion criterion was transfer of a single embryo with 1 gestational sac and positive FHB and transfer of two embryos with 2 gestational sac and positive FHB. Patients with progesterone on the trigger day ≥ 1.5ng/mL and/or with endometrium ≤7mm were excluded. The implantation and clinical pregnancy rates were calculated according to age group, G1: ≤35 years; G2: between 36 and 39 years old; G3: ≥40 years, within the embryo classification. Main results and the role of chance For patients in group 1 (n = 31 embryos), 33.4% of the embryos were classified between 2.6–5.0; 69.20% of embryos with scores between 5.1–7.5 and 57.10% of embryos with scores between 7.6–9.9, with 100% of embryos that implanted, regardless of classification, resulting in clinical pregnancy . For group 2 (n = 35 embryos), they only showed an implantation rate for embryos where the scores were 5.1–7.5 (33.4%) and 7.6 - 9.9 (71.4%) , with 100% being the clinical pregnancy rate in these groups. For patients in group 3 (n = 24 embryos), we also observed implantation only in groups of embryos with a score of 5.1–7.5 (37.5%) and 7.6–9.9 (18.5%) , but the clinical pregnancy rate was lower when compared to the other age groups of the patients, with 33.5% for embryos having a score between 5.1–7.5 and 50% for the group 7.6–9.9. Regarding the average score given by the classification of KIDScore Day 5 v. 3 for embryos that implanted, for patients aged 35 years or less, the average was 6.92; for patients between 36 and 39 years old, the average was 8.06 and for patients aged 40 years or older, the average was 7.32. Limitations, reasons for caution This project is limited because it is a retrospective study and evaluated embryos from a single breeding center. Multicenter and prospective studies are necessary to validate the universal use of the KIDScoreD5 v3 algorithm in time-lapse incubators. Wider implications of the findings: The study showed the ability of KIDScoreD5 v3 to assist the embryologist in deciding which embryo to transfer fresh, according to the patient’s age, in addition to the software being effective in automatic annotation of morphological and morphokinetic parameters. Validating an algorithm universally will improve embryonic selection. Trial registration number Not applicable

P–137 Male embryos take longer to develop to the blastocysts stage

Abstract Study question Are there any differences in developmental timings between male and female preimplantation embryos? Summary answer There is a tendency for statistical difference in the time to reach blastocyst stage for male embryos compared to female embryos What is known already Differences in gene expression and metabolic uptake between male and female preimplantation embryos have been found in animal models and humans. These differences could affect the developmental timings of embryos resulting in differences in either sex. Morphokinetic parameters can precisely assess developmental timings. Only a few studies have analyzed morphokinetic parameters between male and female preimplantation embryos and no consensus has been reached on whether there is any sex-specific difference. The objective of this study is to compare morphokinetic parameters between male and female preimplantation embryos to determine any sex-specific developmental differences. Study design, size, duration This is a retrospective study including 102 preimplantation embryos from February 2018 to February 2020. The morphokinetic parameters obtained from time-lapse records of each embryo were: time to pronuclear fading (tPNf), times to 2–8 cells (t2, t3, t4, t5, t6, t7, t8), time to start of blastulation (tSB) and time to full blastocyst stage (tB). A two-tailed Student’s t-test was used to compare morphokinetic parameters between embryo sexes. A p &amp;lt; 0.05 was considered statistically significant. Participants/materials, setting, methods The study included retrospective time-lapse data from preimplantation embryos giving rise to 51 baby boys and 51 baby girls, as seen at birth. This is a single-center study with standardized culture conditions. Embryos in both study groups issued from cycles with donated oocytes. Only elective blastocyst stage single-embryo transfers (SET) on day 5 were assessed. Main results and the role of chance A tendency to statistical difference (p = [0.1–0.05]) was observed for blastocyst-related morphokinetic parameters: tSB (mean time was 89.6±6.3 hours in male embryos vs. 86.9±8.1 hours in female embryos, p = 0.06) and tB (100.2±5.9 hours versus 97.9±6.5 hours, p = 0.07). Male embryos showed an increased average time of 2.7 hours to tSB and 2.3 hours to tB, while no differences were found in the mean times of all the other morphokinetic paraments measured (p &amp;gt; 0.50): tPNf (∼21.8±3.0 hours) t2 (∼24.4±3.2 hours); t3 (∼35.6±3.9 hours); t4 (∼36.6±4.6 hours); t5 (∼46.9±6.0 hours); t6 (∼53.5±7.0 hours); t7 (∼54.1±7.3 hours) and t8 (∼54.1±7.3 hours). This finding suggests a sex-specific difference in reaching blastocyst stages. Limitations, reasons for caution The main limitation of the study is its retrospective nature and the small sample size. We analyzed the data of embryos leading to a live birth (high-quality embryos), therefore, caution should be made when generalizing results to non-implanting embryos (of potentially lower quality). Wider implications of the findings: Sex-specific differences in developmental timings of preimplantation embryos at blastocyst stage, as evidenced by time-lapse data, should be considered to avoid selection biases during embryo transfers in ART clinic. Trial registration number Not applicable