STUDY QUESTION What are the factors associated with human blastocyst spontaneous collapse and the consequences of this event? SUMMARY ANSWER Approximately 50% of blastocysts collapsed, especially when non-viable, morphologically poor and/or aneuploid. WHAT IS KNOWN ALREADY Time-lapse microscopy (TLM) is a powerful tool to observe preimplantation development dynamics. Lately, artificial intelligence (AI) has been harnessed to automate and standardize such observations. Here, we adopted AI to comprehensively portray blastocyst spontaneous collapse, namely the phenomenon of reduction in size of the embryo accompanied by efflux of blastocoel fluid and the detachment of the trophectoderm (TE) from the zona pellucida (ZP). Although the underlying causes are unknown, blastocyst spontaneous collapse deserves attention as a possible marker of reduced competence. STUDY DESIGN, SIZE, DURATION An observational study was carried out, including 2348 TLM videos recorded during preimplantation genetic testing for aneuploidies (PGT-A, n = 720) cycles performed between January 2013 and December 2020. All embryos in the analysis at least reached the time of starting blastulation (tSB), 1943 of them reached full expansion, and were biopsied and then vitrified. PARTICIPANTS/MATERIALS, SETTING, METHODS ICSI, blastocyst culture, TE biopsy without Day 3 ZP drilling, comprehensive chromosome testing and vitrification were performed. The AI software automatically registered tSB and time of expanding blastocyst (tEB), start and end time of each collapse, time between consecutive collapses, embryo proper area, percentage of shrinkage, embryo:ZP ratio at embryo collapse, time of biopsy (t-biopsy) and related area of the fully (re-)expanded blastocyst before biopsy, time between the last collapse and biopsy. Blastocyst morphological quality was defined according to both Gardner’s criteria and an AI-generated implantation score. Euploidy rate per biopsied blastocyst and live birth rate (LBR) per euploid single embryo transfer (SET) were the main outcomes. All significant associations were confirmed through regression analyses. All couple, cycle and embryo main features were also investigated for possible associations with blastocyst spontaneous collapse. MAIN RESULTS AND THE ROLE OF CHANCE At least one collapsing embryo (either viable or subsequently undergoing degeneration) was recorded in 559 cycles (77.6%) and in 498 cycles (69.2%) if considering only viable blastocysts. The prevalence of blastocyst spontaneous collapse after the tSB, but before the achievement of full expansion, was 50% (N = 1168/2348), irrespective of cycle and/or couple characteristics. Blastocyst degeneration was 13% among non-collapsing embryos, while it was 18%, 20%, 26% and 39% among embryos collapsing once, twice, three times or ≥4 times, respectively. The results showed that 47.3% (N = 918/1943) of the viable blastocysts experienced at least one spontaneous collapse (ranging from 1 up to 9). Although starting from similar tSB, the number of spontaneous collapses was associated with a delay in both tEB and time of biopsy. Of note, the worse the quality of a blastocyst, the more and the longer its spontaneous collapses. Blastocyst spontaneous collapse was significantly associated with lower euploidy rates (47% in non-collapsing and 38%, 32%, 31% and 20% in blastocysts collapsing once, twice, three times or ≥4 times, respectively; multivariate odds ratio 0.78, 95%CI 0.62-0.98, adjusted P = 0.03). The difference in the LBR after euploid vitrified-warmed SET was not significant (46% and 39% in non-collapsing and collapsing blastocysts, respectively). LIMITATIONS, REASONS FOR CAUTION An association between chromosomal mosaicism and blastocyst collapse cannot be reliably assessed on a single TE biopsy. Gestational and perinatal outcomes were not evaluated. Other culture strategies and media should be tested for their association with blastocyst spontaneous collapse. Future studies with a larger sample size are needed to investigate putative impacts on clinical outcomes after euploid transfers. WIDER IMPLICATIONS OF THE FINDINGS These results demonstrate the synergistic power of TLM and AI to increase the throughput of embryo preimplantation development observation. They also highlight the transition from compaction to full blastocyst as a delicate morphogenetic process. Blastocyst spontaneous collapse is common and associates with inherently lower competence, but additional data are required to deepen our knowledge on its causes and consequences. STUDY FUNDING/COMPETING INTEREST(S) There is no external funding to report. I.E., A.B.-M., I.H.-V. and B.K. are Fairtility employees. I.E. and B.K. also have stock or stock options of Fairtility. TRIAL REGISTRATION NUMBER N/A.
Preimplantation genetic testing for aneuploidies (PGT-A) is arguably the most effective embryo selection strategy. Nevertheless, it requires greater workload, costs, and expertise. Therefore, a quest towards user-friendly, non-invasive strategies is ongoing. Although insufficient to replace PGT-A, embryo morphological evaluation is significantly associated with embryonic competence, but scarcely reproducible. Recently, artificial intelligence-powered analyses have been proposed to objectify and automate image evaluations. iDAScore v1.0 is a deep-learning model based on a 3D convolutional neural network trained on time-lapse videos from implanted and non-implanted blastocysts. It is a decision support system for ranking blastocysts without manual input. This retrospective, pre-clinical, external validation included 3604 blastocysts and 808 euploid transfers from 1232 cycles. All blastocysts were retrospectively assessed through the iDAScore v1.0; therefore, it did not influence embryologists’ decision-making process. iDAScore v1.0 was significantly associated with embryo morphology and competence, although AUCs for euploidy and live-birth prediction were 0.60 and 0.66, respectively, which is rather comparable to embryologists’ performance. Nevertheless, iDAScore v1.0 is objective and reproducible, while embryologists’ evaluations are not. In a retrospective simulation, iDAScore v1.0 would have ranked euploid blastocysts as top quality in 63% of cases with one or more euploid and aneuploid blastocysts, and it would have questioned embryologists’ ranking in 48% of cases with two or more euploid blastocysts and one or more live birth. Therefore, iDAScore v1.0 may objectify embryologists’ evaluations, but randomized controlled trials are required to assess its clinical value.
Study question What are the prevalence and implications of cytoplasmic strings (Cyt-S) in preimplantation development after blastulation? Summary answer Cyt-S are common in human embryos and are associated with faster blastocyst development, larger expansion, and better morphological quality. What is known already Cyt-S are dynamic cellular projections connecting inner-cell-mass (ICM) and trophectoderm during blastocyst expansion. Their prevalence in human embryos in vitro has been estimated 44-93%. Currently, their role is unknown. Studies in animal models suggested they might be involved in ICM-to-trophectoderm communication or cell fate-related events. Nevertheless, comprehensive descriptions and clear definitions, evidence-based hypotheses about their function, and data about their clinical implications are lacking, limited or controversial. Here we leveraged time-lapse microscopy, artificial intelligence, and chromosomal testing to comprehensively portray these common features of blastulation and expansion processes. Study design, size, duration Observational study involving 124 PGT-A cycles in EmbryoScope (Vitrolife) with ≥1 blastocyst (N = 315) between May-2013 and November-2014. Timings from tSB to biopsy (t-biopsy, i.e., blastocyst full-expansion) in hours-post-insemination (hpi) and embryo area (including zona-pellucida in µm2) were automatically annotated through an AI-based software (CHLOE™, Fairtility). One senior embryologist annotated Cyt-S presence, number, timings, and type (thick cell-to-cell connections and/or threads), and blastocyst collapses (i.e., reduction of embryo area preventing the discrimination of ICM from trophectoderm). Participants/materials, setting, methods ICSI, continuous blastocyst culture (day 5-7) in time-lapse incubators, trophectoderm biopsy without zona-pellucida drilling on fully-expanded blastocysts, and qPCR to assess non-mosaic full-chromosome aneuploidies were all conducted. Blastocyst morphological quality was defined according to Gardner’s schemes as excellent (AA), good (AB,BA), average (BB,AC,CA) and poor (CC,BC,CB). Only vitrified-warmed euploid single-embryo-transfers were performed. Along with developmental timings, extent of expansion and morphological quality, we also assessed euploidy and live-birth rates in blastocysts with and without Cyt-S. Main results and the role of chance 94.4% of the patients (N = 117/124) had ≥1 embryo with Cyt-S (mean:2.2±1.6, range:0-9). 86% of all blastocysts analyzed (N = 271/315) had ≥1 Cyt-S (3.5±2.1, 1-13; duration of the longest Cyt-S: 5.8±3.4 hours, 0.5-20.9). Overall, we analyzed 937 Cyt-S detected at 117.3±11.8 hpi (85.7-160.5) and lasting 3.8±2.7 hours (0.2-20.9). The first Cyt-S of each embryo lasted longer (4.4±3.2 hours) compared to the following (≈3 hours or less). Cyt-S were mostly threads (N = 133/271, 54.2%) or thick cell-to-cell connections becoming threads (N = 382/937, 40.8%) than connections of constant diameter (N = 47/937, 5%). Of the 271 embryos with ≥1 Cyt-S, 71.9% showed these projections only during early expansion processes after tSB (N = 195; 3.0±1.6, 1-10), 7.7% only after collapses (N = 21; 2.6±1.5, 1-6), and 20.3% both in early expansion and after collapses (N = 55; 2.3±1.3 and 3.2±2.4, 1-11 respectively). Poor- versus excellent-quality blastocysts (N = 54/75, 72% versus N = 120/129, 93%, multivariate-OR=0.32, 95%CI 0.11-0.92, adjusted-p=0.03), t-biopsy (multivariate-OR=0.97, 95%CI 0.95-0.99, p = 0.05) and blastocyst area at t-biopsy (multivariate-OR=1.1, 95%CI 1.01-1.18, p = 0.02) were all associated with the prevalence of embryos with Cyt-S. Lastly, while euploidy rates were comparable between blastocysts with and without Cyt-S, an association instead was shown with live-birth-rates per vitrified-warmed euploid transfers (N = 34/77, 44.2% versus N = 0/10, 0%; p < 0.01), although these data are very preliminary. Limitations, reasons for caution Some Cyt-S after collapses might correspond to connections already present before these events. Larger datasets are required to confirm putative associations between Cyt-S and clinical outcomes. Only associations could be reported here, but not causations/consequences. Specific basic research studies are required to this end Wider implications of the findings Embryos with ≥1 Cyt-S were faster in reaching all blastocyst timings (tSB to t-biopsy) and to expand, achieved a larger area at t-biopsy and had a better morphology. These dynamic features of blastocyst expansion might be physiological and represent biomarkers of embryo quality. Nevertheless, their function is yet unknown Trial registration number not applicable
Study question What are the causes and consequences of blastocyst collapse? Summary answer ∼50% of blastocysts collapsed, especially if they are aneuploid and/or morphologically-poor. Yet, no impact on the live-birth-rate (LBR) per vitrified-warmed euploid single-embryo-transfer (SET) was reported. What is known already Time-lapse-microscopy (TLM) is a powerful tool to describe the peculiar dynamics of preimplantation development. Lately, artificial intelligence (AI) has been also implemented to automatize and standardize such description. Here, we adopted AI to comprehensively portray blastocyst collapse, namely the phenomenon of embryo contraction with an efflux of blastocoel fluid and the detachment of the trophectoderm (TE) from the Zona Pellucida (ZP). Although, the causes of this event are still undetermined, small blastocyst contractions have been reported beneficial for the hatching process, while a full collapse has been associated with lower competence. Study design, size, duration Observational study including 1943 blastocysts from 643 couples cultured in the Embryoscope between January-2013 and December-2020. TE biopsy without day3 ZP drilling and comprehensive-chromosome-testing were performed. The Fairtility® software automatically registered: (i)time of starting-blastulation (tSB), (ii)starting and ending time of each collapse (tSC and tEC), (iii)blastocysts’ areas, (iv)shrinkage% [(area at SC – area at EC)/area at SC)], (v)embryo:ZP ratio at EC (area of the collapsed embryo/area of the ZP), and (vi)time of biopsy (t-biopsy). Participants/materials, setting, methods Blastocyst quality was defined according to Istanbul Consensus (11, excellent; 12-21, good; 22-13-31, average; 33-23-32, poor) and with the Fairtility implantation score (IS) as well, i.e., a continuous variable from 0 to 1 generated by the KID+ software based on the TLM videos of preimplantation development. The main outcome was the LBR per euploid SET adjusted for confounders through logistic regressions. All couple and embryo features were also investigated for their association with blastocyst collapse. Main results and the role of chance 47.3% of the blastocysts collapsed 1- to 9-times (interval between collapses: 4-8hr), and 73% of the couples had ≥1 collapsed blastocyst (1.8±1.1, range:1-8). No couple feature, though, was associated with blastocyst collapse. The longest collapses lasted 1.5±1.1 (0.13-5.1)hr, while the largest shrinkage% and embryo:ZP ratio at EC were 35±14% (10-78%) and 81±9% (33-90%), respectively. In ∼50-60% of collapses a 20-40% blastocyst volume reduction was registered, 40-60% or 20-40% in ∼15-30%, 60-80% in 0-4%. In case of multiple collapses, the first three involved smaller shrinkages. Blastocysts undergoing ≥1 collapse showed similar tSB as not-collapsing blastocysts, but progressively longer tEB and t-biopsy. The earlier the first event, the more the consecutive collapses. Notably, the poorer the morphology, the higher the risk (excellent, good, average, and poor not-collapsing blastocysts were 64%,50%,44% and 37%), number (e.g.,≥4 collapses were 0.4%,2%,4% and 8%) and duration (1.2±1.0,1.4±1.0,1.6±1.1 and 1.9±1.3hr) of blastocyst collapse. Collapsing blastocysts were significantly less euploid than non-collapsing (35% vs 47%; multivariate-OR:0.75,95%CI 0.6-0.92,p<0.01); conversely, their LBR per euploid SET (39% vs 46%) and miscarriage rate per clinical pregnancy (17% vs 11%), were not significantly different (adjusted-OR:1.0,95%CI 0.69-1.48,p=0.96 and adjusted-OR:1.65,95%CI 0.79-3.42,p=0.18, respectively). All data were confirmed also by defining blastocyst quality through the Fairtility IS. Limitations, reasons for caution Gestational and perinatal outcomes were not assessed. Other culture strategies and media shall be assessed for their association with blastocyst collapse. Perhaps, future studies from other groups and with a larger sample size might unveil a significant impact on the clinical outcomes. Wider implications of the findings Collapse is common and delays blastocyst full-expansion. Moreover, poor morphology and aneuploidies involve a higher risk of collapse(s); however, no impact was reported on the clinical outcomes after euploid SET. AI appears to increase the throughput of the analysis, but additional data are required to research the causes of collapse. Trial registration number Not applicable
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