Time-lapse Incubator Research Articles

P-301 Assessment of ongoing clinical outcomes prediction of an AI system on retrospective SET data

Abstract Study question Would patients with fresh and frozen embryo transfer had achieved pregnancy before if the embryo was chosen by AI? Summary answer CHLOE (AI) can predict pregnancy, ongoing clinical pregnancy, and miscarriage following a single embryo transfer (SET). What is known already The use of time-lapse incubators has provided embryologists with more information to evaluate embryo development, resulting in varying clinical practices among clinics in prioritizing this information. However, manual annotation of time-lapse videos is time-consuming and prone to interoperator inconsistencies. To overcome these challenges, AI tools like CHLOE (Fairtility) can be utilized. CHLOE uses AI-based predictors to predict implantation and provides clarity on the biological factors driving these predictions. However, before incorporating AI tools into clinical practice, it is important to validate their effectiveness. Study design, size, duration Single study center that took place between July of 2021 and December 2022 at a private clinic in Spain. This was a retrospective cohort analysis that reviewed 118 time-lapse videos from single fresh embryo transfers and 92 time-lapse videos from single frozen embryo transfers with known ongoing clinical pregnancy outcome. CHLOE EQ score and CHLOE Rank efficacy of prediction of clinical outcomes and miscarriage was quantified using the metric AUC. Participants/materials, setting, methods Time-lapse videos were evaluated using CHLOE (Fairtility), an AI tool, to determine CHLOE EQ score and rank related to clinical outcomes (biochemical pregnancy, clinical pregnancy, and miscarriage) following fresh and frozen SET. CHLOE rank and embryology were compared with chi-square and AUC was calculated with logistic regression to measure prediction accuracy. T-test was used to check differences in CHLOE EQ score in different outcomes. Main results and the role of chance Embryologist vs CHLOE Ranking weren’t significant (p > 0.05). In fresh SET the mean EQ score was 7.76, and in frozen SET was 7.07. Following fresh SET, CHLOE EQ score was not-significantly predictive of biochemical pregnancy (AUC = 0.53, n = 104, p = 0.462), clinical pregnancy (AUC = 0.51, n = 79, p = 0.949), and miscarriage rate (AUC = 0.50, n = 68, p = 0.949); CHLOE Ranking was more predictive than embryologist rank for biochemical pregnancy (embryologist vs CHLOE rank: AUC = 0.51, p > 0.05 vs AUC = 0.61, p > 0.05), clinical pregnancy (embryologist vs CHLOE rank: AUC = 0.51, p > 0.05 vs AUC = 0.70, p > 0.05) and miscarriage rate (embryologist vs CHLOE rank: AUC = 0.51, p > 0.05 vs AUC = 0.75, p > 0.025). Following frozen SET, only top 1 embryos ranked by embryologists were transferred, and CHLOE EQ score was predictive of biochemical pregnancy (AUC = 0.60, n = 85, p = 0.213), clinical pregnancy (AUC = 0.64, n = 60, p = 0.919), and miscarriage rate (AUC = 0.87, n = 52, p = 0.437); CHLOE Ranking was predictive of biochemical pregnancy (AUC = 0.59, p > 0.05), clinical pregnancy (AUC = 0.64, p > 0.05) and miscarriage rate (AUC = 0.90, p > 0.05). Limitations, reasons for caution This study is a single-center retrospective analysis where embryos were chosen for transfer by human embryologists and is part of a broader effort to validate the responsible integration of AI into clinical practice. Wider implications of the findings The use of AI-based tools has the possibility to enhance the consistency, efficiency, and effectiveness of embryo selection. The information from quantitative and qualitative morphokinetics provided by AI tools like CHLOE brings greater clarity to predictions, enabling more personalized care for each individual embryo. Trial registration number Not Applicable

P-183 Blastocyst speed versus blastocyst look: Which is the better predictor of clinical pregnancy and live birth following time-lapse culture and single blastocyst transfer?

Abstract Study question To compare predictive value for pregnancy and live birth of morphological versus morphokinetic assessment of blastocysts after time-lapse culture and single blastocyst transfer. Summary answer Blastocyst morphokinetic assessment was not superior in predicting pregnancy and live birth compared to blastocyst morphology, but provides additional information, particularly for morphologically poorer-quality blastocysts. What is known already Blastocyst culture with time-lapse technology enables evaluation of both morphokinetic developmental speed and classical morphology including Gardner scoring of the blastocyst. One approach to assess the developmental speed of blastocysts is to use the morphokinetic classification proposed by Campbell, Fishel, et al., based on time to start of blastulation (tSB) and the duration of forming the blastocyst (dB). However, beyond the advantages of undisturbed embryo culture, the value of the kinetic data from time-lapse is debated. Study design, size, duration EDCOS (Embryo Developmental profiles from Controlled Ovarian Stimulation cycles using follitropin alfa) is a multicentre, prospective cohort study of patients undergoing IVF/ICSI whose embryos were cultured in time-lapse incubator devices in 10 ART centres throughout Denmark and Sweden. Patients undergoing IVF/ICSI (including donor oocyte cycles) who had ovarian stimulation with follitropin alfa (Bemfola®) and had zygotes and embryos available for monitoring in the EmbryoScope™ time-lapse incubator device (Vitrolife) were invited to join this study. Participants/materials, setting, methods Morphological and morphokinetic prognostic classifications for single blastocyst transfers (SET) were determined for three study subpopulations: fresh autologous SET (311 women), frozen autologous SET (246 women; 345 transfers) and frozen donor SET (38 women; 59 transfers). All blastocysts were graded morphologically in five grades from Amorph (best) to Emorph (worst) based on their Gardner score and morphokinetically in three grades from Akin (best) to Ckin (worst) based on tSB and dB. Main results and the role of chance Overall ongoing pregnancy and live birth rates (OPR/LBR) for morphokinetic classifications Akin, Bkin and Ckin were 36.1%, 30.0% and 16.3% (P = 0.021) and 30.4%, 21.8% and 9.1% (P = 0.000), respectively. OPR and LBR for morphological classifications Amorph, Bmorph, Cmorph, Dmorph and Emorph were 41.9%, 37.8%, 30.8%, 19.0% and 15.6% (P = 0.000) and 36.6%, 29.1%, 15.6%, 16.5% and 8.3% (P = 0.002), respectively. For Dmorph blastocysts, OPR and LBR for morphokinetic classifications Akin, Bkin and Ckin were 50%, 21.4% and 7.1% (P = 0.009) and 33.3%, 10.8% and 0% (P = 0.003), respectively. Receiver operating characteristic curve analyses achieved areas under the curve for fresh autologous SET of 55.7%, 58.9% and 67.2% for the morphokinetic, morphological and combined classifications, respectively; and 53.4%, 64.0% and 65.6%, respectively, for frozen autologous SET. Limitations, reasons for caution This was an observational study with no control group, conducted under usual clinical practice. Data were collected prospectively, although clinics could deselect and rank blastocysts according to their own procedures. However, the outcome of subsequent frozen blastocyst transfer cycles was also analysed. Wider implications of the findings Selection of a blastocyst for transfer based on morphokinetic assessment at the blastocyst stage was not superior to classical blastocyst morphological assessment after time-lapse culture but is a viable option. Combining morphokinetic and morphological evaluation provides additional supporting information to guide in blastocyst selection, especially for morphologically poorer-quality blastocysts. Trial registration number Not applicable

P-184 Morphokinetic analysis of cases with vitrified-warmed oocytes in oocyte donation program

Abstract Study question Does vitrification of oocytes in oocyte donation program alter the morphokinetic pattern of human embryos? Summary answer Both the extrusion of the second polar body (tPB2) and the appearance of the pronuclei (tPNa) occur earlier in vitrified oocytes without affecting further development. What is known already Vitrification of oocytes has been a breakthrough in oocyte cryopreservation and significantly altered practices in IVF labs. Oocyte vitrification/warming has become an integral part of daily routine and thorough assessment of its efficiency is crucial. Though, studies of vitrified-warmed oocytes have shown similar fertilization, cleavage, blastulation, pregnancy and live birth rates to fresh oocytes, the morphokinetic pattern of embryos originating from vitrified oocytes has not been thoroughly studied. According to some studies vitrification may be related to delayed blastocyst formation while others failed to see any alteration in the morphokinetic pattern between embryos derived from fresh or frozen oocytes. Study design, size, duration This retrospective observational study was performed at Embryolab Fertility Clinic, in Thessaloniki, Greece between April 2020 and September 2021 and included 590 vitrified oocytes from 68 oocyte donation cycles. Control group consisted of 31 good prognosis cases (oocyte donation cycles or homologous oocyte cycles of women under 35 years old). Participants/materials, setting, methods 68 vitrified oocyte donation cycles and 31 fresh control cycles were analyzed. All oocytes after ICSI were cultured in Embryoscope time-lapse incubator up to blastocyst stage. Key time parameters and dynamic events were analyzed using generalized estimating equations (GEE) regression analysis for the non-independent nature of the data. Moreover, the following comparisons were performed between groups: fertilization, cleavage, top cleavage, blastocyst, top blastocyst and pregnancy rates. Main results and the role of chance There was no significant difference in fertilization rates between control and frozen oocytes (77.79% ± 15.73 vs 71.26% ± 21.50 respectively, p = 0.15). Cleavage rate and top cleavage rate (more than 6 cells, equal size, with no or minor fragmentation), were higher in frozen oocytes with this difference being significant (p = 0.02 for cleavage rate and p = 0.015 for top cleavage rate). Blastocyst rate and top blastocyst (2AA, 3AA, 3AB, 4AA, 4AB according to Gardner criteria) rate, though, showed similar rates with no statistically significant difference (Blastocyst rate: 74,15% ± 17.19-control vs 76.42% ± 30.75-frozen oocytes, p = 0.6 & Top blastocyst rate: 45.54% ± 24.04-control, 45.30% ± 32.04-frozen oocytes, p = 0.82). Pregnancy rates were not significantly different (control:85.19%, frozen oocytes:75.38%, p = 0.29). The comparison for key time parameters and dynamic events that were analyzed (tPB2, tPNa, tPNf, t2, t3,t4, t5, t6, t7, t8, t9+, tSC, tM, tSB, tB, tEB, S2, S3, S5, CC1, CC2, CC3,Compaction and Blastulation) showed significant difference in 2 time parameters: tPB2 (4.62 hours-control, 3.67 hours-frozen, Coef: -1.09, p = 0.015) and tPNa (9.07 hours-control, 8.25 hours-frozen oocytes, Coef: -1.06, p = 0.043). Limitations, reasons for caution The fact that the oocytes under comparison were not sibling as well as its retrospective nature, constitute the main limitations of the present study. Moreover, the number of cases included in this study was limited and the available information about ongoing pregnancy and live births is still pending. Wider implications of the findings tPB2 and tPNa seem to occur earlier in vitrified oocytes without affecting further development, implying a need for reviewing timings between OPU/vitrification/warming/ICSI. Moreover, vitrified oocytes displayed higher cleavage and top cleavage rates with unaffected blastocyst or pregnancy rates. Overall, vitrification of oocytes seems to be safe and reliable. Trial registration number N/A

P-185 Performing Assisted Hatching on Day 4 next to the area of early cavitation reduces the chances inner cell mass (ICM) herniation and simplifies trophectoderm biopsy

Abstract Study question Can the location of the opening in the zona pellucida during Assisted Hatching reduce the herniation of cells in the ICM and simplify trophectoderm biopsy? Summary answer Performing Assisted Hatching on D4, next to an area of the zona pellucida (ZP) close to first embryo cavitation, reduces the hatching of ICM cells. What is known already Trophectoderm biopsy is performed on blastocysts to perform preimplantation genetic testing of mutations and/or aneuploidies. To simplify the excision of trophectoderm cells, Assisted Hatching (AH) is typically performed on D3 at the cleavage/morula stage by creating a hole in the ZP through which some cells will herniate. This opening is traditionally performed on a random area of the ZP and, consequently, predicting whether the trophectoderm or the ICM cells will hatch first is not possible. Should the latter be the case, the biopsy procedure would need to be adjourned until trophectoderm cells become accessible. Study design, size, duration Regular AH was performed on mouse embryos on D3 by creating a 10µm opening on a random area of their ZP. A second group of embryos was cultured until D4 and the opening of the ZP was performed in a directed way (AHD) close to where the first cavitation of the cells was observed. Both groups were compared with a third control group to which no opening was made. Participants/materials, setting, methods Thirty mouse embryos were allocated into each test group (n = 90) and cultured in an Embryoscope time-lapse incubator to better determine the first cavitation of the cells. Laser-assisted AH/AHD was performed on the Embryoslide dish. Blastocyst formation, quality and hatching rates were compared among all groups, and the belongingness of the first herniated cells to the trophectoderm/ICM was annotated individually by D5/D6. Statistical analyses were performed by Fisher’s exact test, and significance set at 5% (α = 0.05). Main results and the role of chance The blastocyst formation and hatching rates were close to 100% in all three groups with no differences among any of them, indicating that neither AH nor AHD had a detrimental effect on embryo development between D3-D6 or blastulation. In more than a third of the blastocysts of the non-treated control group, the first cells to herniate out of the ZP belonged to the ICM (38.2%), similar to what was observed in the random AH group (44.4%). By performing AHD on day 4 in the ZP next to the area of first embryo cavitation, the incidence of the ICM hatching first was significantly reduced to only 11.1% of the cases (p ≤ 0.01). Interestingly, the ratio of blastocysts that were able to completely hatch out of the ZP by day 6 was reduced both in the AH and AHD (2.8% and 5.6%, respectively) compared to the control group with no opening on the ZP (42.9%, p ≤ 0.0002). Limitations, reasons for caution The present study has been performed on the mouse model and AH/AHD timings may need to be adjusted if used in the human. Time-lapse technology has eased the recognition of early cavitation stages to perform AHD, which could result more complex in a regular culture setting. Wider implications of the findings Trophectoderm biopsy depends on the unique rate of development of each blastocyst, which complicates its integration in the laboratory workflow. The results of this study could improve the quality and simplicity of the biopsy technique by directly targeting the appropriate subgroup of cells and making them easier to be excised. Trial registration number not applicable

O-122 The first study to assess the clinical efficacy of CHLOE-EQ on the assessment of embryo viability of embryos cultured in a GERI time-lapse incubator

Abstract Study question Can an automatic AI scoring system predict ploidy, live birth and utilization? Are there differences in AI scoring between donor and own gametes? Summary answer CHLOE-EQ Score is directly associated with oocyte quality, ploidy, utilization, live birth, embryo quality, direct uneven cleavage (DUC), blastulation, utilization and selection for transfer. What is known already The integration of AI algorithms, such as CHLOE-EQ, into different Time-lapse systems requires clinical and biological validation. Geri Time-lapse videos have given the embryologists more insight into embryo development. Analyzing this information manually requires time and introduces risk of error. To tackle this issue, AI solutions like CHLOE-EQ (Fairtility) can be used to automatically assess video datapoints. CHLOE-EQ provides an embryo quality score that has been shown to predict embryo viability and ploidy, providing clarity on the underlying biological factors. Before introducing AI tools in clinical practice, it is crucial to confirm their efficacy and validate with clinical data. Study design, size, duration A retrospective cohort analysis was conducted at a private clinic in Spain from April 2021 to November 2022, involving the review of 3196 Geri time-lapse videos with a subset of known ploidy and live birth outcomes. The correlation of CHLOE-EQ score with ASEBIR clinic grading was evaluated. As well as with DUCs, oocyte quality, sperm source, blastulation, utilization, selection for transfer, ploidy and live birth. Participants/materials, setting, methods Geri time-lapse videos were automatically analyzed by CHLOE-EQ (Fairtility). CHLOE-EQ score was assessed in relation to laboratory (ploidy, clinic ASEBIR embryo scoring, utilization, selection for transfer) and clinical outcomes (live birth), as well as between own vs donor gametes (own eggs >40y vs donor eggs and testicular sperm vs donor sperm) using descriptive statistics and t-test. The accuracy of prediction was measured using binary logistic regression (AUC). Main results and the role of chance CHLOE-EQ score was positively correlated with ASEBIR embryo quality (A:8.7±1.9, n = 349 >B:6.8±2.9, n = 470 > C: 5.1±3.0, n = 124 > D:1.3±2.1, n=751; p < 0.05). Non-DUCs had higher CHLOE-EQ Score than DUCs [5.3±3.8, n=1798 vs 1.9±0.38, n=643, p<0.001]. CHLOE-EQ Score was unaffected by the quality of the sperm sample, with similar CHLOE-EQ scores between donor sperm and testicular derived sperm (4.1±3.9, n=335 vs 3.4±4.2, n=56, respectively, NS). Embryos that blastulated (yes vs no: 5.4±3.7, n=1996 vs 0.6±2.1, n=309, p < 0.001), were utilized (7.4±0.28, n=911, vs 1.0±2.1, n = 1309, p < 0.001), selected for transfer (8.7±2.4, n=153 vs 3.3±3.7, n = 2067, p < 0.001), were euploid (7.5±2.5, n=72 vs 6.3±3, n=152, p = 0.001) and resulted in live births (4.4±4.1, n = 332 vs 3.8±4, n = 499, p = 0.02) had a higher CHLOE-EQ score than embryos that did not. CHLOE-EQ Score is higher in embryos derived from oocytes from donors than own eggs, suggesting that oocyte quality affects CHLOE-EQ score (4.0±4, n = 1189 vs 2.7±3.4, n = 356). CHLOE-EQ Score is predictive of utilization (AUC=0.95, n=2220, baseline=41%, p<0.001), euploidy (AUC=0.63, n=224, baseline=32.1%, p=0.003), blastulation (AUC=0.94, n = 2305, baseline=86.6%, p < 0.001) and selection for transfer (AUC=0.89, n = 2220, baseline=41%, p<0.001). Limitations, reasons for caution This is a retrospective single-center study in which embryos for transfer were selected by human embryologists, and forms part of program to validate the responsible integration of AI into clinical practice in each individual clinic. Wider implications of the findings This is the first study presenting the efficacy of prediction of CHLOE-EQ with GERI data. AI tools have the potential to improve consistency, efficiency, and accuracy of embryo assessment and selection. CHLOE-EQ predicts through quantitative and qualitative morphological and morphokinetics information, resulting in more personalized care for each individual embryo. Trial registration number not applicable

P-216 Evaluation of embryo quality and morphokinetics following short exposure of oocytes to spermatozoa

Abstract Study question Does embryo quality and morphokinetics following short exposure of oocytes to spermatozoa differ from conventional long exposure? Summary answer No statistical differences were found in embryo quality and morphokinetics following short exposure. Short exposure technique enables better monitoring of the embryos and easier handling. What is known already Overnight exposure of oocytes to spermatozoa may lead to toxic effects associated with reactive oxygen species. In short exposure, the duration of oocyte incubation with the sperm is reduced to 2-4 hours. Few studies showed that embryo morphology as well as pregnancy rates improved by short exposure, however, this remains controversial. Embryo morphokinetics in a time-lapse incubator yielding more accurate information on embryo quality, has not been reported for embryos derived by the short exposure technique. Study design, size, duration A retrospective study (2013-2019) 536 IVF cycles were included: 110 short exposure and 426 conventional IVF (long exposure). All embryos were cultured in the EmbryoScope. Short and long exposure cycles were compared for the average embryo scores of general and adapted laboratory in-house models. Groups were analyzed according to 2, 3 and 5 days of culture and maternal age. Moreover, differences of KID (Known implanted data) embryos morphokinetics were compared for cell division timings. Participants/materials, setting, methods IVF patients with mechanical factor, normal sperm counts. In conventional IVF, the oocytes were inseminated 6-7 hours post retrieval and mechanically denuded after 18-20 hours of incubation. In short exposure, the oocytes were inseminated and denuded 2-3 hours post retrieval and immediately placed in the EmbryoScope, for accurate annotation. In both methods oocytes were washed, incubated in EmbryoScope and cultured till the day of transfer. Women were divided by age groups: <35, 35-40 and >40. Main results and the role of chance No statistical difference was found in embryo cleavage rates and number of transferred embryos per cycle between long and short exposure in all three maternal age groups. The clinical pregnancy rate was slightly higher in the short exposure compared to the long exposure group but did not reach statistical significance (37.5% vs 32.5, p = NS). In women younger than 35y, elective single embryo transfer resulted in a statistically significant higher clinical pregnancy rate in the short exposure group compared to the long (p = 0.037). No significant differences were found in the average embryo morphokinetic score according to the general model and the adapted in-house model between the two groups. Moreover, no difference was found in embryo morphokinetics between short and long exposure in 272 KID negative and 116 KID positive embryos. Limitations, reasons for caution Since the study was retrospective and the short exposure group was smaller than the conventional IVF group, additional studies are needed to support our results. Wider implications of the findings Short exposure is more convenient and easier to perform. It is as good as long exposure in terms of clinical pregnancy rates and may be beneficial in patients younger than 35 years old. Trial registration number 0015-20-CMC

P-227 ICSI outcomes after using in-situ microfluidics of fluidic walls versus DGC: a prospective non-inferiority comparative pilot study in sibling oocytes

Abstract Study question Does the novel strategy in-situ microfluidics (isM) yield comparable ICSI outcomes to the control sperm selection methodology density gradient centrifugation (DGC)? Summary answer ICSI outcomes show that handmade in-situ microfluidics of fluidic walls are as effective as DGC to achieve fertilization and usable day 5 blastocyst formation rate. What is known already Microfluidics technologies are proving their capability to select suitable sperm for ICSI. Increasingly, publications show novel and ingenious microfluidics strategies aimed to integrate sperm biomimicry during in vitro sperm selection while simplifying the IVF-workflow. We recently designed a microfluidics system which allows selecting sperm for ICSI in the same ICSI-dish, only using microfluidics and disregarding centrifugation, washing and plasticware. A previous proof-of-concept study showed that this methodology was efficient to select suitable sperm for ICSI as it could separate at least 20 progressive spermatozoa in less than 15 minutes in a clean microdroplet free of any remaining seminal plasma. Study design, size, duration The present pilot study included a total of 280 fresh MII-oocytes allocated in a 1:1 ratio to the study (isM-ICSI) and control (DGC-ICSI) groups. The statistical power was established at 80% at a CI 95%. For comparison we relied in the KPIs: ICSI normal fertilization rate (INFR) and Day 5 usable blastocyst rate (D5UBR). The non-inferiority margin (Δ) was established using 2-sigma warning limit and our historical control mean values (ΔINFR = 7% and ΔD5UBR = 10%). Participants/materials, setting, methods An informed consent was signed by all participants. The comparative study included 29 consecutive ICSI cases performed in fresh oocytes from women under 40 years old and with an oocyte yield ≥6 MII. All semen samples had ≥1 × 106/ml progressive spermatozoa and were split into three aliquots: 1-seminogram (100 µl), 2-isM (10 µl), 3-DGC (surplus). Embryos were cultured in bench-top Time-lapse incubators, using GTL-media under 6,4% CO2 and 5% O2 conditions up to blastocyst stage. Main results and the role of chance Sperm characteristics showed the following total mean values for total concentration (42,83 ± 31,1 × 106/ml), total motility (51,28 ± 14,16 %) and progressive sperm concentration (17 ± 12 × 106/ml). The mean number of MII per patient was 9,66. From the total number of oocytes (n = 280), 139 were allocated to isM (study group) and 141 to DGC (control group) groups. INFR was 72,0 % vs 76,6 %, respectively. Non-significant differences were observed (p = 0,54). We observed that the mean total number of usable blastocysts (day 5/6) per patient was 1,83 (53/29) in isM vs 1,66 (48/29) in DGC. Moreover, D5UBR was 53,0% vs 44,4 % (p = 0,26) with a difference between the study and the control of within the non-inferiority margin. We observed a lower rate of arrested embryos (up to day 6) in the study group (19,0% vs 30,0%), although results were not statistically significant (p = 0,08). Limitations, reasons for caution This pilot study includes a limited number of oocytes. The present isM protocol depends on a free-hand preparation. Although the use of templates and the training reduces inter and intra-operator variability, a ready-to-use surface would benefit the operability. Further investigation is required to evaluate clinical pregnancy and live birth outcomes. Wider implications of the findings Sperm DNA fragmentation is cited among the causes of embryo arrest. As the use of isM resulted in a lower proportion of arrested embryos, exploring its capacity to select non-fragmented sperm warrants further interest. The isM protocol streamlines sperm selection for ICSI, reduces costs and risks along the procedure. Trial registration number na

P-296 Supernumerary blastocysts resulting from abnormal cleavage patterns: freeze or not to freeze? A practical question!

Abstract Study question After eSET, the supernumerary blastocysts that showed cleavage anomalies can be vitrified as second choice embryos. Do they display enough implantation chance to be frozen? Summary answer No pregnancy was obtained after frozen transfer of blastocysts with direct uneven cleavage and chaotic cleavage. It is not recommended to freeze those spare embryos. What is known already The use of time-lapse incubation offers stable culture conditions and provides comprehensive information on dynamic embryo development. Studies found that abnormal cleavage affects the genome of human embryos and can reduce its developmental potential with embryonic arrest. Aneuploidy contributes also to failed implantation. It was also reported that cleavage anomalies such as direct uneven cleavage and chaotic cleavage affect negatively implantation and live birth. Even if the good survival rate of blastocysts after vitrification has dramatically improved clinical outcome after frozen embryo transfer, implantation and live birth are correlated to embryonic ploidy and healthy cellular machinery. Study design, size, duration Retrospective qualitative assessment of time-lapse images for 186 vitrified blastocysts resulting from extended culture of 871 embryos showing morphokinetic abnormalities such as direct uneven cleavage first mitosis (DC1), direct cleavage second mitosis (DC2), fast cleavage (FC), complete chaotic cleavage (CC) and multiple anomalies (MA), during 2021-2022 in a single private hospital, and analysis of the clinical outcome after warming and SET. Participants/materials, setting, methods From 871 embryos in Embryoscope + (Vitrolife), 445 total blastocysts and 251 top quality were obtained according to Gardner’s grading. 186 of those were vitrified for further transfer. In 5 subgroups of anomalies (DC1, DC2, FC, CC, MA) pregnancy (PR) and ongoing pregnancy rate (OPR) per warming cycle and per transfer were calculated. The results were compared by SPSS software to overall 2021 center’s results. Main results and the role of chance The mean female age was 35.4 ± 0.5 years. From the 186 top quality blastocysts that were vitrified, 96 were warmed in 94 frozen cycles with a survival rate of 98.3%, resulting in 93 SET transfers. A total of 32 pregnancies were obtained from which 21 were ongoing pregnancies. There was no statistical difference in PR per warming cycle or per transfer, and in OPR per warming cycle or per transfer between the overall study group compared to the center’s annual results 2021: 34.0% vs 40.2%, 34.4% vs 40.1%, 22.3% vs 30.1%, and 22.6% vs 30.7%. Reasonable PR and OPR per transfer were obtained with blastocysts from DC2, FC and MA patterns, with caution to small sample size: 32.0% and 20.0%, 44.1% and 32.3%, 40.0% and 25.0%. The results were statistically comparable to the center’s annual result. All the transfers from DC1 and CC pattern failed to achieve a pregnancy. Limitations, reasons for caution One limitation of the study was its retrospective nature and the other was the small sample size in each subgroup of anomaly. The results should be confirmed prospectively and should compel the live birth rate after delivery of the ongoing pregnancies obtained during the second semester of 2022. Wider implications of the findings Abnormal cleavage patterns such as DC and CC are known to affect negatively implantation and live birth. Deselection of embryos showing these specific patterns could significantly increase successful implantation for the accordingly selected embryos. It is therefore non optimal to freeze the spare blastocysts resulting from these peculiar abnormal patterns. Trial registration number NA

External validation of a model for selecting day 3 embryos for transfer based upon deep learning and time-lapse imaging

Could objective embryo assessment using iDAScore Version 2.0 perform as well as conventional morphological assessment? A retrospective cohort study of fresh day 3 embryo transfer cycles was conducted at a large reproductive medicine centre. In total, 7786 embryos from 4328 cycles with known implantation data were cultured in a time-lapse incubator and included in the study. Fetal heartbeat (FHB) rate was analysed retrospectively using iDAScore Version 2.0 and conventional morphological assessment associated with the transferred embryos. The pregnancy-prediction performance of the two assessment methods was compared using area under the curve (AUC) values for predicting FHB. AUC values were significantly higher for iDAScore compared with morphological assessment for all cycles (0.62 versus 0.60; P = 0.005), single-embryo transfer cycles (0.63 versus 0.60; P = 0.043) and double-embryo transfer cycles (0.61 versus 0.59; P = 0.012). For the age subgroups, AUC values were significantly higher for iDAScore compared with morphological assessment in the <35 years subgroup (0.62 versus 0.60; P = 0.009); however, no significant difference was found in the ≥35 years subgroup. In terms of the number of blastomeres, AUC values were significantly higher for iDAScore compared with morphological assessment for both the <8c subgroup (0.67 versus 0.56; P < 0.001) and the ≥8c subgroup (0.58 versus 0.55; P = 0.012). iDAScore Version 2.0 performed as well as, or better than, conventional morphological assessment in fresh day 3 embryo transfer cycles. iDAScore Version 2.0 may therefore constitute a promising tool for selecting embryos with the highest likelihood of implantation.

Delineating the heterogeneity of embryo preimplantation development using automated and accurate morphokinetic annotation

PurposeOur objective was to design an automated deep learning model that extracts the morphokinetic events of embryos that were recorded by time-lapse incubators. Using automated annotation, we set out to characterize the temporal heterogeneity of preimplantation development across a large number of embryos.MethodsTo perform a retrospective study, we used a dataset of video files of 67,707 embryos from four IVF clinics. A convolutional neural network (CNN) model was trained to assess the developmental states that appear in single frames from 20,253 manually-annotated embryos. Probability-weighted superposition of multiple predicted states was permitted, thus accounting for visual uncertainties. Superimposed embryo states were collapsed onto discrete series of morphokinetic events via monotonic regression of whole-embryo profiles. Unsupervised K-means clustering was applied to define subpopulations of embryos of distinctive morphokinetic profiles.ResultsWe perform automated assessment of single-frame embryo states with 97% accuracy and demonstrate whole-embryo morphokinetic annotation with R-square 0.994. High quality embryos that had been valid candidates for transfer were clustered into nine subpopulations, as characterized by distinctive developmental dynamics. Retrospective comparative analysis of transfer versus implantation rates reveals differences between embryo clusters as marked by poor synchronization of the third mitotic cell-cleavage cycle.ConclusionsBy demonstrating fully automated, accurate, and standardized morphokinetic annotation of time-lapse embryo recordings from IVF clinics, we provide practical means to overcome current limitations that hinder the implementation of morphokinetic decision-support tools within clinical IVF settings due to inter-observer and intra-observer manual annotation variations and workload constrains. Furthermore, our work provides a platform to address embryo heterogeneity using dimensionality-reduced morphokinetic descriptions of preimplantation development.

Sibling oocytes cultured in a time-lapse versus benchtop incubator: how time-lapse incubators improve blastocyst development and euploid rate.

The aim was to study whether a limited exposure of embryos outside the incubator has an effect on embryo development, blastocyst quality and euploid outcomes. This retrospective study was performed at ART Fertility Clinics, Abu Dhabi, United Arab Emirates (UAE) between March 2018 and April 2020 and included 796 mature sibling oocytes that were split randomly between two incubators after intracytoplasmic sperm injection (ICSI): an EmbryoScope™ (ES) incubator and a benchtop incubator, G185 K-SYSTEMS (KS). The fertilization, cleavage, embryo/blastocyst qualities, useable blastocyst and euploid rates were assessed to evaluate the incubator performance. In total, 503 (63.2%) mature oocytes were cultured in the EmbryoScope and 293 (36.8%) in the K-SYSTEMS. No differences were observed in fertilization rate (79.3% vs 78.8%, P = 0.932), cleavage rate (98.5% vs 99.1%, P = 0.676) and embryo quality on Day 3 (P = 0.543) between both incubators, respectively. Embryos cultured in the EmbryoScope, had a significantly higher chance of being biopsied (64.8% vs 49.6%, P < 0.001). Moreover, a significantly higher blastocyst biopsy rate was observed on Day 5 in the EmbryoScope (67.8% vs 57.0%, P = 0.037), with a highly significant increased euploid rate (63.5% vs 37.4%, P = 0.001) and improved blastocyst quality (P = 0.008). We found that exposure of embryos outside the incubator may negatively affect the in vitro blastocyst development and euploid rate on Day 5.

Morphokinetic analysis of early human embryonic development and its relationship to endometriosis resection: a retrospective time-lapse study using the KIDScore™ D3 and D5 implantation data algorithm

Research questionDoes complete resection of endometriosis improve embryo quality as assessed by morphokinetic parameters using time-lapse microscopy?DesignFor this retrospective study we analysed 237 fertilised, cultured and transferred embryos from 128 fresh IVF and/ or ICSI transfer cycles. Endometriosis was confirmed or excluded by laparoscopy. Patients were stimulated with recombinant FSH using GnRH agonist and antagonist protocols. After fertilisation, a time-lapse incubation system was used for observation. Embryo quality was assessed using the KIDScore™ D3 and D5 implantation data algorithm.ResultsThe analysis showed a median KIDScore™ D5 of 2.6 (on a scale of 1 to 9.9) for embryos from patients with endometriosis without complete resection. The control group without endometriosis achieved a score of 6.8 (p = 0.003). The median score for embryos from endometriosis patients with complete resection was 7.2, which was a significant increase compared to embryos from patients without complete resection (p = 0.002). We observed an effect size of r = 0.4 for complete resection versus no resection of endometriosis using the KIDScore™ D5. There were no differences in KIDScore™ D3 between the three patient groups. Pregnancy and miscarriage rates showed the same clinical trends. In three of our four case series of patients who underwent IVF/ ICSI cycles before and after complete resection, we found a marked improvement in embryo quality after complete resection.ConclusionsComplete resection of endometriosis could significantly improve the otherwise poor embryo quality of patients undergoing IVF-procedures. The data, therefore, strongly support recommending surgery to patients with endometriosis prior to assisted reproduction.

Novel Time-Lapse Parameters Correlate with Embryo Ploidy and Suggest an Improvement in Non-Invasive Embryo Selection.

Selecting the best embryo for transfer is key to success in assisted reproduction. The use of algorithms or artificial intelligence can already predict blastulation or implantation with good results. However, ploidy predictions still rely on invasive techniques. Embryologists are still essential, and improving their evaluation tools can enhance clinical outcomes. This study analyzed 374 blastocysts from preimplantation genetic testing cycles. Embryos were cultured in time-lapse incubators and tested for aneuploidies; images were then studied for morphokinetic parameters. We present a new parameter, "st2, start of t2", detected at the beginning of the first cell cleavage, as strongly implicated in ploidy status. We describe specific cytoplasmic movement patterns associated with ploidy status. Aneuploid embryos also present slower developmental rates (t3, t5, tSB, tB, cc3, and t5-t2). Our analysis demonstrates a positive correlation among them for euploid embryos, while aneuploids present non-sequential behaviors. A logistic regression study confirmed the implications of the described parameters, showing a ROC value of 0.69 for ploidy prediction (95% confidence interval (CI), 0.62 to 0.76). Our results show that optimizing the relevant indicators to select the most suitable blastocyst, such as by including st2, could reduce the time until the pregnancy of a euploid baby while avoiding invasive and expensive methods.

Clinical outcomes of uninterrupted embryo culture with or without time-lapse-based embryo selection versus interrupted standard culture (SelecTIMO): a three-armed, multicentre, double-blind, randomised controlled trial

Time-lapse monitoring is increasingly used in fertility laboratories to culture and select embryos for transfer. This method is offered to couples with the promise of improving pregnancy chances, even though there is currently insufficient evidence for superior clinical results. We aimed to evaluate whether a potential improvement by time-lapse monitoring is caused by the time-lapse-based embryo selection method itself or the uninterrupted culture environment that is part of the system. In this three-armed, multicentre, double-blind, randomised controlled trial, couples undergoing in-vitro fertilisation or intracytoplasmic sperm injection were recruited from 15 fertility clinics in the Netherlands and randomly assigned using a web-based, computerised randomisation service to one of three groups. Couples and physicians were masked to treatment group, but embryologists and laboratory technicians could not be. The time-lapse early embryo viability assessment (EEVA; TLE) group received embryo selection based on the EEVA time-lapse selection method and uninterrupted culture. The time-lapse routine (TLR) group received routine embryo selection and uninterrupted culture. The control group received routine embryo selection and interrupted culture. The co-primary endpoints were the cumulative ongoing pregnancy rate within 12 months in all women and the ongoing pregnancy rate after fresh single embryo transfer in a good prognosis population. Analysis was by intention to treat. This trial is registered on the ICTRP Search Portal, NTR5423, and is closed to new participants. 1731 couples were randomly assigned between June 15, 2017, and March 31, 2020 (577 to the TLE group, 579 to the TLR group, and 575 to the control group). The 12-month cumulative ongoing pregnancy rate did not differ significantly between the three groups: 50·8% (293 of 577) in the TLE group, 50·9% (295 of 579) in the TLR group, and 49·4% (284 of 575) in the control group (p=0·85). The ongoing pregnancy rates after fresh single embryo transfer in a good prognosis population were 38·2% (125 of 327) in the TLE group, 36·8% (119 of 323) in the TLR group, and 37·8% (123 of 325) in the control group (p=0·90). Ten serious adverse events were reported (five TLE, four TLR, and one in the control group), which were not related to study procedures. Neither time-lapse-based embryo selection using the EEVA test nor uninterrupted culture conditions in a time-lapse incubator improved clinical outcomes compared with routine methods. Widespread application of time-lapse monitoring for fertility treatments with the promise of improved results should be questioned. Health Care Efficiency Research programme from Netherlands Organisation for Health Research and Development and Merck.

Evaluation of fragmented embryos implantation potential using time-lapse technology.

To examine the implantation potential of fragmented embryos that underwent morphokinetic evaluation in a time-lapse incubator. A retrospective study analyzing 4210 Day 5 embryos which were incubated in a time-lapse incubator, between 2013 and 2019. Embryos with more than 5% fragmentation (379 embryos) were included in the study. Embryos selected using the general model and re-examined by our in-house model. Embryo fragmentation percentage was documented from the first cell-division (start fragmentation) to its maximal percentage (final fragmentation), and the ratio between them (fragmentation worsening). Data were analyzed with relation to embryo development, embryos transfer or freezing, clinical pregnancy, and live birth rates. Embryo fragmentation and morphokinetics were found to be independent variables for clinical pregnancy achievements. A higher fragmentation worsening was noted among discarded embryos compared to transferred or frozen embryos (p < 0.0001). Advanced maternal age had a significant negative effect on fragmentation (p < 0.001). Missed abortion rates were similar in fragmented embryos that implanted compared with the overall population. Live birth rates were comparable among embryos which were selected for transfer or freezing by their morphokinetics and had different severity of fragmentation. Our study shows that fragmented embryos have a potential to implant and therefore should be selected for transfer. Laboratories which do not use time-lapse incubators for embryo selection, should consider transferring fragmented embryos, since they have an acceptable chance for live birth. Calculation of fragmentation worsening may enhance our ability to predict embryo development. Further research with analysis of more fragmented embryo maybe beneficial. This study was approved by the local ethics committee No. 0010-19 CMC on April 18th, 2019.

Is there a relationship between morphokinetic parameters and neonatal sex in fresh embryo transfers?

To investigate whether morphokinetic parameters differ between male and female embryos in IVF embryos resulting in live births, a retrospective cohort study was undertaken. Files of all live births resulting from a single embryo transfer (SET) cultured in time-lapse incubators between 2013 and 2019 in two tertiary care centres were reviewed. The study group consisted of 187 SETs resulted in 187 live births, of which 100 were females (53.5%) and 87 were males (46.5%). Embryo selection for transfer was based on the known implantation data (KID) score provided by the Embryoscope and morphological assessment by experienced embryologists. Neonatal sex was confirmed through live birth documentation. Morphokinetic parameters and day 3 and day 5 KID scores of male and female embryos were compared. Maternal baseline and treatment characteristics were similar between groups. Morphokinetic time-lapse parameters of male and female embryos including: pronuclei fading; cleavage timings (t2-t9); second and third cell cycle durations; synchrony of the second and third cleavages; late morphokinetic parameters and KID scores did not differ between groups. In conclusion, time-lapse morphokinetic parameters and embryo selection methods do not seem to differ between male and female embryos, and their utilization does not bias towards any neonatal sex.

Optimal Embryo Selection: The Irreplaceable Role of the Embryologist in an Age of Advancing Technology.

Time-lapse incubators allow for ongoing evaluation of embryos without culture condition disruption. The use of time-lapse incubation has been shown to improve outcomes either by improving overall conditions or providing additional information to aid in embryo selection for transfer. Time-lapse incubators can also utilise morphokinetic models to rank embryos based on morphokinetic parameters. We sought to compare a morphokinetic model for embryo comparison to traditional morphologic evaluation. The aim of the study is to compare a morphokinetic model for embryo comparison to traditional morphologic evaluation. This is a retrospective cohort design. Embryos cultured in a time-lapse culture system that had traditional morphologic evaluation, morphokinetic modelling and known live birth outcomes were included in this study. Embryos with unknown competence were excluded, including when two embryos were transferred with a single live birth resulted. Receiver operating characteristic (ROC) curves were determined for both the morphologic analysis and the morphokinetic model on culture day 3 and day 5. Using the ROC-determined cutoff that optimised both sensitivity and specificity, a binary outcome for each test was analysed using agreement statistics to determine if one method of embryo evaluation was superior to the other. Morphological and morphokinetic grading were both predictive of embryo competence on days 3 and 5. However, on day 3, morphologic grading was superior to morphokinetic grading with area under the curve (AUC) of 0.66 (P < 0.001) and 0.58 (P = 0.009), respectively. Contrarily, on day 5, the morphokinetic model had a higher AUC of 0.65 (P = 0.03) compared to the morphologic grading, AUC 0.56 (P = 0.02). Traditional morphology was noted to be a better diagnostic tool (higher AUC) on culture day 3 while a morphokinetic model was superior on day 5.

Sperm Chromatin Condensation Defect Accelerates the Kinetics of Early Embryonic Development but Does Not Modify ICSI Outcome.

The origin and quality of gametes are likely to influence the kinetics of embryonic development. The purpose of the study was to assess the impact of sperm nuclear quality, and in particular sperm chromatin condensation, on the kinetics of early embryo development after intracytoplasmic sperm injection (ICSI). Our study included 157 couples who benefitted from ICSI for male factor infertility. Chromatin condensation and DNA fragmentation were assessed in spermatozoa prior to ICSI. Above the 20% threshold of sperm condensation defect, patients were included in the abnormal sperm chromatin condensation (ASCC) group; below the 20% threshold, patients were included in the normal sperm chromatin condensation (NSCC) group. After ICSI, the oocytes were placed in the time-lapse incubator. The kinetics of the cohort's embryonic development have been modeled. The fading times of pronuclei and the time to two blastomeres (t2, first cleavage) and four blastomeres (t4, third cleavage) differed significantly between the NSCC and ASCC groups, with earlier events occurring in the ASCC group. On the other hand, the state of sperm chromatin condensation did not seem to have an impact on live birth rates or the occurrence of miscarriages. The kinetics of early embryonic development was accelerated in males with a sperm chromatin condensation defect without compromising the chances of pregnancy or promoting miscarriage. However, our study highlights the paternal contribution to early embryonic events and potentially to the future health of the conceptus.

Embryo Blastomere Exclusion Identified in a Time-Lapse Culture System Is Associated with Embryo Ploidy.

This study examinedblastomere exclusion which is seen during embryo development and could represent imperfect cell division or a mechanism of aneuploidy correction. This was a retrospective cohort study which included embryos cultured in a time-lapse incubator undergoing preimplantation genetic testing for aneuploidy (PGT-A) with trophectoderm biopsy. Embryos were evaluated for blastomere exclusion early in development, late in development, both, or neither. Blastomere exclusion was compared to embryo ploidy. Embryos with no blastomere exclusion had an aneuploidy rate of 52.9%, while embryos displaying blastomere exclusion at any stage had an aneuploidy rate of 68.5% (p < .001). Early blastomere exclusion was not significantly associated with an increased aneuploidy risk (59.2% vs. 52.9% in no blastomere exclusions; p = 0.22). However, embryos with late blastomere exclusion were significantly more likely to be aneuploid, compared to embryos with no blastomere exclusions (77.5% vs. 52.9%; p < 0.001) as were embryos with both early + late blastomere exclusions (71.2% vs. 52.9%; p < 0.001). Upon restricting the analysis to aneuploid embryos, the presence of any blastomere exclusion was not significantly associated with complex aneuploidy, defined as 2 more affected chromosomes (43.9% vs. 38.7%; p = 0.28). However, the proportion with adverse embryo genetics significantly increased with the timing of blastomere exclusion (38.7%, 37%, 45.5%, and 50% for none, early, late, and early + late; p = 0.043). Late blastomere exclusion or a combination of both early + late blastomere exclusion was associated with an increased risk of aneuploid embryo genetics. Embryo selection using time-lapse culture systems should incorporate these findings when untested embryos are transferred.

Prediction of live birth - selection of embryos using morphokinetic parameters.

The goal of assisted reproduction is for a couple treated with IVF techniques to end the treatment by giving birth to a healthy baby. A neccessary presumption for success is the identification of the best embryo with high implantation and developmental potential. One option is to select an euploid embryo by invasive preimplantaion genetic testing for aneuploidy (PGT-A) or it is possible to select the best embryo by non-invasive time-lapse monitoring (TLM), specifically based on morphokinetic parameters and morphological markers that are able to identify an embryo with high developmental potential. The study involved a total of 1060 embryos (585 euploid and 475 aneuploid embryos after PGT-A) with good morphology from 329 patients in the period 01/2016-10/2021. All embryos were cultured in a time-lapse incubator, trophectoderm (TE) cells biopsies for PGT-A examination were performed on day 5 (D5) or day 6 (D6) of culture. During the study period, 225 frozen embryo transfers (FET) of one euploid embryo were performed. Based on the treatment outcome, the embryos were divided into 2 groups - euploid embryos, which led to the birth of a healthy child, and euploid embryos that did not show fetal heartbeat (FHB) after FET. Based on the statistical analysis of the embryos without implantation and the embryos with live birth, it is clear that the morphokinetic parameters t5 (time of division into 5 cells) and tSB (time of start of blastulation) are significantly different. The results suggest that of the morphokinetic parameters tSB and t5 are predictive indicators for selecting an embryo with high developmental potential and with a high probability of achieving the birth of a healthy child.