Unequal Cleavage Research Articles

P-214 Direct unequal cleavage-1 (DUC1) in IVF/ICSI cycles: relationship with patient characteristics and insemination method

Abstract Study question Does the incidence of DUC1 in IVF/ICSI cycles vary with the maternal profile and insemination method? Summary answer The incidence of DUC1 cycles is higher in patients with a more favourable prognosis. DUC1 embryos are more frequent in ICSI than in IVF cycles. What is known already The emergence of Time-Lapse Microscopy (TLM) has enabled a dynamic and thorough evaluation of in vitro embryo development, unveiling the occurrence of morphological phenomena unobserved with static observation, such as direct unequal cleavage (DUC), which is associated with a drastically reduced competence to implant. The incidence of DUC-1 (DUC immediately after syngamy) has been reported to vary from 8.3% to 26%, but the reasons underlying its occurrence and variability are unknown. There is evidence that the maternal profile and insemination method can impact embryo developmental competence, but whether they impact the incidence of DUC is also unknown. Study design, size, duration The study includes 2920 first IVF/ICSI cycles (2018 to 2022) providing 12492 embryos. Cycles presenting male infertility were excluded from the analysis. The percentages of DUC1 embryos in relation to total embryos and of cycles providing at least one DUC1 embryo in relation to total cycles were compared between AMA (advanced maternal age; ≥35) and pre-AMA (<35) cycles. Patient/cycle characteristics were compared between cycles with and without DUC1 occurrence in AMA and pre-AMA cycles, separately. Participants/materials, setting, methods Ovarian stimulation was induced with rFSH or with a combination of FSH and LH using classic antagonist or short agonist protocols. Oocytes were inseminated with either IVF or ICSI standard methodology. Embryo culture was performed in an integrated embryo culture Time-lapse System (EmbryoScope; Vitrolife). Differences between percentages were assessed by the Fisher’s exact test, while differences between continuous variables were assessed with the Wilcoxon sum rank test. Main results and the role of chance The percentage of DUC1 embryos in relation to total embryos did not vary between pre-AMA (5.4%) and AMA (5.2%; p > 0.05) cycles. Alternatively, the incidence of cycles providing at least one DUC1 embryo was higher in pre-AMA (21.7%) as compared with AMA patients (16.0%; p < 0.0001). In both pre-AMA and AMA cycles, DUC1 occurrence was associated with higher oocyte yield (13.5±7.7 vs. 10.6±6.7 and 11.0±6.3 vs. 7.5±5.5, respectively; p < 0.0001), higher AMH serum levels (4.4±4.7 vs. 3.4±3.2 and 3.0±3.6 vs. 2.1±2.1ng/mL, respectively; p < 0.004) and lower basal FSH serum levels (7.0±2.1 vs. 7.6±2.9 and 7.8±3.3 vs. 8.5±3.4mU/mL, respectively; p < 0.0001). Overall, the percentage of DUC1 embryos was higher in ICSI (5.6%) as compared to IVF (4.4%, p < 0.01) cycles. However, when the data was stratified by maternal age, DUC1 incidence was significantly higher in ICSI vs. IVF in AMA (5.6 vs 4.0%; p = 0.002) but not in pre-AMA (5.7 vs 5.0%; p = 0.19). All 21 DUC1 embryos transferred during the study failed to implant. Limitations, reasons for caution Our study is limited by its retrospective nature. The present conclusions must be confirmed in other patient populations with different race/genetics and habits. Wider implications of the findings The occurrence of cycles providing DUC1 embryos is paradoxically associated with a more favourable patient prognosis, and the percentage of DUC1 embryos is higher in ICSI as compared to IVF cycles in AMA patients. Our findings represent new valuable clues for the clarification of DUC1 underlying causes. Trial registration number NA

P-218 Should embryos with direct unequal cleavage be given a chance or directly deprioritized? A follow-up on blastocyst formation and ploidy

Abstract Study question Should embryos with direct unequal cleavage (DUC) be given a chance to develop to blastocyst for a quality and ploidy assessment or directly deprioritized? Summary answer Although DUC embryos are clearly associated with poorer outcomes, if DUC embryos develop to good quality blastocysts the euploidy rate is comparable to non-DUC embryos. What is known already DUCs were first defined by time-lapse imaging and their implantation rate was found to be significantly lower than embryos with a normal cleavage pattern (1.2% vs. 20.2%, respectively) leading to the suggestion that rejection of these embryos for transfer could improve implantation rates (Rubio et al., 2012). Another study concluded that blastocyst formation, implantation potential and euploidy rate are significantly reduced in DUC embryos and that they should be deselected for day 3 transfers but should be cultured to blastocyst stage for possible embryo transfer (Zhan et al., 2016). However, in practice, DUCs are deselected and are therefore rarely transferred. Study design, size, duration This retrospective study included 1191 DUC embryos and 5058 non-DUC embryos incubated in the Embryoscope in 2022. Twenty-eight DUC and 185 non-DUC embryos were transferred in a fresh cycle. Preimplantation genetic testing for aneuploidy (PGT-A) was performed on 128 DUC and 1477 non-DUC blastocysts. Five DUC embryos were transferred in a frozen-thawed cycle. Participants/materials, setting, methods A total of 2430 IVF patients treated in a single private clinic were included in this retrospective study. The mean female age of the cohort was 36.5. Some of the blastocysts were subjected to PGT-A mainly for advanced maternal age. CHLOE (Fairtility) software analyzed time-lapse videos and identified pronuclei, DUCs and blastulation. DUC embryo outcomes (ploidy, blastulation and blastocyst quality) were assessed. Direct cleavages from 2 to 5 cells were not included in the study. Main results and the role of chance The outcomes of DUC embryos (n = 1191) were compared to non-DUC embryos (n = 5058). The blastulation rate was 23.8% for DUC embryos and 50.2% for non-DUC embryos. The good-quality blastocyst rate was 15.1% and 42.5%, respectively. The top-quality blastocyst rate was 2.4% and 18.5%, respectively. PGT-A was performed for 128 DUC blastocysts; 52 were euploid (40%) and 14 were mosaic (11%). The euploidy rate for non-DUC blastocysts was 43% and the mosaicism rate was 9.4%. Thirty-three DUC embryos were transferred giving 6 live births (18%). Limitations, reasons for caution The discrimination between fragments and blastomeres within the 5 hours from the first division is challenging for AI algorithms. Therefore, some DUC embryos may have been misclassified. Wider implications of the findings In conclusion, although DUC embryos are not prioritized for transfer, our results show that, once achieving the blastocyst stage with a good quality, their euploidy rate is comparable to non-DUC blastocysts. Morphokinetic analysis may propose additional markers to identify higher implantation potential DUC blastocysts. Trial registration number N/A

P-226 Consistent quality in Duostim cycle embryos: Concordant assessment by embryologists and Artificial Intelligence

Abstract Study question Does the Duostim protocol, evaluated by embryologists and AI, yield similar embryo quality in both cycles, and what impact does it have on reproductive outcomes? Summary answer Duostim protocol, when assessed by both AI and embryologists, maintains consistent embryo quality, making it a promising therapeutic option in assisted reproductive technology. What is known already Duostim optimises ovarian cycles with consecutive stimulations in the follicular and luteal phases, particularly beneficial for poor prognosis and oncological patients, aiming to boost oocyte production in a short period. It’s crucial to evaluate its impact on embryo quality. AI tools like CHLOE-EQ assist embryologists by automatically generating an embryo quality score (EQ Score) from time-lapse videos and morphokinetic annotations. This study compares the embryo quality from the initial and subsequent stimulations in a Duostim cycle, examining both AI-generated EQ Scores and assessments by embryologists. Study design, size, duration In a retrospective study spanning from February to December 2022, 234 embryos resulting from Duostim protocols were analyzed. The embryos were divided into “first” (n = 112) and “second” (n = 122) stimulations. Evaluation of embryo quality utilized CHLOE-EQ scores (Fairtility Ltd), morphokinetic events (tPNa-tEB), and Direct Unequal Cleavage (DUC) automatically annotated by AI. Manual annotations by embryologists included parameters like euploidy, utilization, and fragmentation. Participants/materials, setting, methods The study included embryos from Duostim protocols. Embryo quality of each stimulation group was evaluated using EQ score and embryo development pace (tPNa-tEB), measured by 2-sample t-test. Additional analysis compared euploidy, utilization, and abnormalities (DUCs, fragmentation) between first and second stimulation and was evaluated using chi-squared tests. Main results and the role of chance Patient age averaged 40±3 years. EQ scores revealed comparable embryo quality between “first” and “second” stimulation cycles (3.2 ± 3.6 vs. 2.9 ± 3.7), with a similar distribution of low, medium, and high-quality embryos.Similarities were observed in development rates between embryos derived from the first and second stimulation; tPNA(9 ± 3 vs 9 ± 3); tPNf(22 ± 7 vs 22 ± 5); t2(27 ± 4 vs 27 ± 4); t3(37 ± 6 vs 37 ± 5); t4 (39 ± 6 vs 39 ± 6); t5(48 ± 9 vs 49 ± 8); t6(54 ± 9 vs 56 ± 10); t7(57 ± 10 vs 59 ± 10); t8(61 ± 12 vs 61 ± 12); t9(70 ± 12 vs 73 ± 12); tM(89 ± 12 vs 88 ± 14); tSB(106 ± 12 vs 107 ± 16); tB(113 ± 12 vs 116 ± 15); tEB(124 ± 11 vs 125 ± 14), p=NS. Both stimulation groups had similar utilization (39% vs. 33%), euploidy (54% vs. 45%), DUCs (19% vs. 17%), and fragmentation rates (≤25%: 95% vs. 96%; >26%: 5% vs. 4 The quantity of oocytes retrieved from the first and second stimulations (112 vs. 122) further supported these findings. Limitations, reasons for caution The sample, while substantial, may not represent diverse populations. Generalizability and potential confounding variables should be considered. To enhance applicability, assessments across diverse IVF centers with varied patient demographics are crucial. Wider implications of the findings Duostim, assessed by both AI and embryologists, emerges as a promising therapeutic option without compromising embryonic quality or euploidy. The study supports AI integration for assessing therapeutic protocols and their impact on embryonic quality, emphasizing its potential in enhancing reproductive medicine practices. Trial registration number NA

The people behind the papers - Thomas Mullan and Richard Poole.

Asymmetric cell divisions can produce daughter cells of different sizes, but it is unclear whether unequal cell cleavage is important for cell fate decisions. A new paper in Development explores the role of unequal cleavages in Caenorhabditis elegans embryos. To learn more about the story behind the paper, we caught up with first author Thomas Mullan and corresponding author Richard Poole, Associate Professor of Developmental Biology at University College London, UK.

Control of successive unequal cell divisions by neural cell fate regulators determines embryonic neuroblast cell size.

Asymmetric cell divisions often generate daughter cells of unequal size in addition to different fates. In some contexts, daughter cell size asymmetry is thought to be a key input to specific binary cell fate decisions. An alternative possibility is that unequal division is a mechanism by which a variety of cells of different sizes are generated during embryonic development. We show here that two unequal cell divisions precede neuroblast formation in the C lineage of Caenorhabditis elegans. The equalisation of these divisions in a pig-1/MELK mutant background has little effect on neuroblast specification. Instead, we demonstrate that let-19/MDT13 is a regulator of the proneural basic helix-loop-helix transcription factor hlh-14/ASCL1 and find that both are required to concomitantly regulate the acquisition of neuroblast identity and neuroblast cell size. Thus, embryonic neuroblast cell size in this lineage is progressively regulated in parallel with identity by key neural cell fate regulators. We propose that key cell fate determinants have a previously unappreciated function in regulating unequal cleavage, and therefore cell size, of the progenitor cells whose daughter cell fates they then go on to specify.

P-308 Abnormal Oocytes are more likely to lead to abnormal embryo divisions (Direct Unequal Cleavage,DUC), but do not compromise embryo quality as assessed using CHLOE-EQ score

Abstract Study question Do oocyte dysmorphisms lead to abnormal embryo divisions and compromised embryo quality? Summary answer Oocyte cytoplasmic abnormalities (granularity, Smooth Endoplasmic Reticulum,SER) did not affect CHLOE-EQ score; whilst zona abnormalities (thickness and unevenness) and SER tend to lead to DUCs. What is known already Oocyte dysmorphisms include extracytoplasmic [zona pellucida (ZP) evenness and thickness] and cytoplasmic abnormalities [SERs, inclusions, darkness, granularity]. The impact of these abnormalities on embryo development and viability as reported in the literature is contradictory. CHLOE-EQ score is an Artificial Intelligence (AI) based algorithm designed to support embryologists in assessing embryo viability, and has previously been demonstrated to automatically detect embryo development anomalies (such as DUCs), to be predictive of blastulation, utilisation, selection for transfer, ploidy, implantation and live birth. Therefore, CHLOE-EQ is a metric of embryo viability. The impact of oocyte dysmorphisms on CHLOE-EQ and DUCs is poorly understood. Study design, size, duration Retrospective cohort analysis of 742 embryo time-lapse videos, cultured at a private fertility clinic between June and July 2022. Participants/materials, setting, methods The clinic provided annotations on extracytoplasmic abnormalities (ZP thickness and uniformity) and cytoplasmic abnormalities [SERs, inclusions, darkness, granularity]. CHLOE-EQ (Fairtility) automatically annotated morphokinetics and DUCs and further quantified embryo viability scores (CHLOE-EQ and Blast Score). Main results and the role of chance CHLOE-EQ score was not affected by the oocyte having cytoplasmic abnormalities (no vs yes: 4.2 ± 4, n = 122vs4.1 ± 4, n = 359, NS): dark (4.1 ± 4, n = 476vs3.1 ± 4, n = 5, NS), granular (4.1 ± 4, n = 179vs4.1 ± 4, n = 302, NS), SER (4.1 ± 4, n = 455 vs 3.8 ± 4, n = 26, NS), inclusion (4.1 ± 4, n = 430vs4.1 ± 4, n = 51, NS); or ZP abnormalities [overall (4.2 ± 4, n = 379vs3.7 ± 4, n = 102, NS), non-uniformity (4.1 ± 4, n = 409vs4.2 ± 4, n = 72, NS), thick ZP (4.2 ± 4, n = 457vs2.8 ± 4, n = 24, NS), thin ZP(4.1 ± 4, n = 475vs2.1 ± 4, n = 6, NS]. DUC embryos were two times more likely to be derived from oocytes with thick ZP (9/98, 9%oocytes) than oocytes without thick ZP (15/383, 3.9%, p = 0.03). DUCs were more likely to have a non-uniform ZP compared to non-DUCs [DUCs: 7%(7/98) vs Non-DUCs: 17%(65/383), p = 0.015]. DUCs were not associated with the following oocyte cytoplasmic dysmorphias: SER (DUC vs Non-DUCs: 6/98,6% vs 20/388,5%,NS), dark (0/98 vs 5/383, NS), granular (61/98 vs 241vs383, NS), inclusions (11/98 vs 40/383, NS). DUCs had lower blastulation rate than non-DUCs [DUC:1.8%(2/113) vs Non-DUCs:77%(298/389), p < 0.001]. DUCs were 4-fold less likely to be multinucleated at the 2 cell stage than non-DUCs [DUC: 7%(2/29)vsNon-DUCs: 30%(91/305), p = 0.03]. DUCs were 7-fold more likely to be multinucleated at the 4 cell stage than non-DUCs [DUC: 7%(2/29)vs Non-DUCs:1%(3/302), p = 0.06]. Patient age was not associated with DUCs (DUCs: 36.9 ± 4 vs non-DUCs:37.1 ± 4, NS). Limitations, reasons for caution This was a retrospective-single clinic study. Causality is not determined. Wider implications of the findings Given the growing evidence that DUCs have compromised viability, it is important to understand the biology of how DUCs are connected to oocyte quality. Using AI to detect DUCs to avoid critical information being missed during embryo assessment can assist embryologists in maximising their efficacy of embryo selection. Trial registration number NA

P-300 Not all DUCs are the same: Impact of DUC type on the blastulation, utilization and ploidy

Abstract Study question We have identified six categories of DUCs as identified automatically by CHLOE-EQ. Do these DUC categories differ in embryo competency? Summary answer DUC-1, Major chaotic DUC and Fragmented DUC have compromised blastulation, utilization and ploidy, compared to DUC2, minor chaotic DUC and not-direct DUC. What is known already CHLOE-EQ (Fairtility, an AI-based support tool) automatically annotates embryo morphokinetics and identifies embryo division anomalies, such as Direct Unequal Cleavage(DUCs). DUCs are defined as less than 5 hours from two cells to three cells. DUCs have been associated with being severely compromised, with lower chance of blastulating, being utilized, euploid, implanting or leading to live birth. In some clinics, DUCs are automatically discarded. Other clinics reported euploids and live births from DUC embryos, raising questions as to whether there are different types of DUCs with different competencies. In this study, we identified 6 types of DUCs and assessed their viability. Study design, size, duration Retrospective cohort study that took place between March to July 2022 at a private fertility clinic in Spain. This study included 1032 time-lapse videos of embryos with Direct unequal cleavage (DUCs) as identified by CHLOE-EQ. CHLOE-EQ defines DUCs as (t3-t2)<5 hours. DUCS annotated by CHLOE-EQ were classified into 6 types by embryologists. Participants/materials, setting, methods DUC1(direct division from the 1-cell to 3 or more, without a visible 2-cell stage); DUC2 (Direct Division from 2-cells where either cell divides directly from one to three cells); Minor Chaotic DUC (asynchronous irregular divisions: cells still countable); Major chaotic DUC (asynchronous irregular divisions: cells/fragments are too chaotic to count); Fragmented DUC1 (resembles a DUC1, but the third ‘cell’ is a fragment); Not direct DUC (quick division from 1 cell to 2cells to 3cells). Main results and the role of chance CHLOE-EQ correctly identified 97.4%(875/898) of 2PN DUCs, based on the definition of DUC (t3-t2<5). Most DUCs annotated by CHLOE-EQ, were classified by embryologists as minor chaotic [25%(231/921)], followed by DUC 1 [20.3%(187/921)], Not direct DUCs [18.1%(167/921)], major chaotic DUCs [14.6%(135/921)], DUC 2 [14%(129/921)], and lastly, fragmented DUC1 [7.8%(72/921)]. The average t3-t2 time did not differ between the six groups (1.6,1.5,1.5,1.6,1.5,1.6,respectively,p>0.05). Among DUC embryos, Minor chaotic DUCs [85%(110/129)] and Not direct DUCs [66.9%(109/163)] and DUC2 [57%(73/128)] had similar blastulation rates (p > 0.05) and utilization rates [DUC2:44.2% (57/129), Minor Chaotics: 37.7%(87/231), Not Direct DUCs: 56.3%(94/167), p > 0.05]. These three groups had a higher blastulation rate than DUC1 [20.1% (37/184)], Major chaotic [21.6% (29/134)], Fragmented DUC1 [19.7% (14/71), p < 0.05)] and a higher utilization rate than DUC1 [11.8% (22/187)], Major chaotic [14% (19/135)], Fragmented DUC1 [11.1%(8/72), p < 0.05)]. Type of DUC was not affected by Age(p > 0.05). Four live births from single embryo transfer of DUC embryos were recorded in this dataset. Limitations, reasons for caution DUCs were assessed by a single embryologist, further studies will assess intra and inter-operator variation in DUC classification across various clinics. It was particularly challenging to differentiate between fragments and cells. This study is ongoing to further understand implication of DUC types on clinical outcome. Wider implications of the findings Given that different DUC types have varied competency levels, the results of this study encourage embryologists not to discard DUC embryos simply because they are DUCs. It is important to assess the type of DUC when determining the fate of the embryo and when managing the expectation of affected patients. Trial registration number NOT APPLICABLE

P-146 Larger time intervals between pronuclear fading and the first mitosis are strongly associated with trichotomous mitoses and cleavage arrest in human embryos

Abstract Study question Does the duration of the interval between pronuclear fading and first cell division affect preimplantation embryo developmental competence? Summary answer Larger pronuclear-fading to first-division intervals (t2-tPNf) are associated with greater risk of direct unequal cleavage (DUC) and lower blastulation rate, but not of blastocyst aneuploidy. What is known already During the last decade, time lapse technology (TLT) has made possible detailed and dynamic observation of in vitro embryo development, offering a powerful non-invasive tool for improving embryo selection. Recent evidence suggests that the phase between pronuclear fading and the first cleavage is a perilous bridge between the zygote and the cleaving embryo. Following pronuclear breakdown, delayed progression through mitosis caused by incomplete DNA replication may result in chromosome lagging, whole and segmental segregation errors, and breakage. In this analysis, we specifically tested the hypothesis that delays in a short final phase of fertilization have an impact on embryo development. Study design, size, duration Retrospective study of 1316 zygotes cultured until day 5 using TLT and derived from first ICSI cycles (111 no PGT-A and 94 PGT-A) performed between 2018 and 2022. Testicular sperm or cryopreserved gametes cycles were excluded. DUC was defined as the cleavage of one zygote or blastomere into three daughter blastomeres or cell cycle interval shorter than five hours at the first (DUC1), second (DUC2) or third (DUC3) cell division cycle. Participants/materials, setting, methods t2-tPNf intervals were clustered into quartiles (Q1, <2hpi; Q2, 2–2.5hpi; Q3, 2.5–3hpi and Q4, > 3hpi), which were compared based on embryo development and live birth by linear and logistic regression. The Q1 cluster was set as control, while all other quartiles groups were considered as dummy variables. Multivariate analysis was conducted using the generalized estimating equations, to control for variable numbers of zygotes from each patient. The primary outcome was DUC rate. Main results and the role of chance Overall, rates of DUC1, DUC2 and DUC 3 rates were 10.3%, 8% and 2.9%, respectively. After adjusting for major confounders (maternal age, paternal age, maternal body mass index, cause of infertility, anti-Müllerian hormone, follicle stimulating hormone) we observed a higher DUC 1 and DUC 2 rate with increasing times of t2-tPNf. We observed abnormal distribution of DUC1 and DUC2 rate between quartiles groups. Specifically, 89% of DUC1 was shown among Q3 (21%) and Q4 (78%) and 61% of DUC2 among Q3 (22%) and Q4 (39%). The percentage of DUC1 was higher for Q3 (multivariate-OR = 3.29, 95%CI 0.99-10.85, adjusted-p = 0.01) and Q4 (multivariate-OR = 22.79, 95%CI 7.80-66.62, adjusted-p<0.01]. The percentage of DUC2 was higher only in Q4 (multivariate-OR = 2.58, 95%CI 1.13-5-57, adjusted-p = 0.01). Moreover, we observed a reduced blastulation rate in Q3 (multivariate-OR = 0.68, 95%CI 0.49-0.95, adjusted-p = 0.01) and Q4 (multivariate-OR = 0.27, 95%CI 0.19-0.38, adjusted-p<0.01). No significant differences were observed in DUC3 rate, top quality blastocyst, euploidy rate (PGT-A cycles analysis) and live birth rate. Limitations, reasons for caution The retrospective single-centre design and the limited sample size are limitations of the study Wider implications of the findings The study is consistent with the emerging view that the final phases of fertilization - although short - are crucial for development. Delays in progression towards mitosis probably express chromosome integrity loss and perturbances of chromosome segregation. In most embryos, such perturbances appear to cause trichotomous mitoses and cleavage arrest. Trial registration number Not applicable

Association between the morphokinetics of in-vitro-derived bovine embryos and the transcriptomic profile of the derived blastocysts.

The time-lapse system is a non-invasive method that enables a continuous evaluation through embryo development. Here, we examined the association between the morphokinetics of the developing embryo and the transcriptomic profile of the formed blastocysts. Bovine oocytes were matured and fertilized in vitro; then, the putative zygotes were cultured in an incubator equipped with a time-lapse system. Based on the first-cleavage pattern, embryos were categorized as normal or abnormal (68.5±2.2 and 31.6±2.3%, respectively; P<0.001). A cleaved embryo was defined as normal when it first cleaved into two equal blastomeres; it was classified as synchronous or asynchronous according to its subsequent cleavages. An abnormal pattern was defined as direct, unequal, or reverse cleavage. Direct cleavage was classified as division from one cell directly into three or more blastomeres; unequal cleavage was classified as division that resulted in asymmetrically sized blastomeres; and reverse cleavage of the first division was classified as reduced number of blastomeres from two to one. Of the normally cleaving embryos, 60.2±3.1% underwent synchronous cleavage into 4, 8, and 16 blastomeres, and 39.7±3.1% cleaved asynchronously (P<0.001). The blastocyte formation rate was lower for the synchronously vs. the asynchronously cleaved embryos (P<0.03). The abnormally cleaved embryos showed low competence to develop to blastocysts, relative to the normally cleaved embryos (P<0.001). Microarray analysis revealed 895 and 643 differentially expressed genes in blastocysts that developed from synchronously and asynchronously cleaved embryos, respectively, relative to those that developed from directly cleaved embryos. The genes were related to the cell cycle, cell differentiation, metabolism, and apoptosis. About 180 differentially expressed genes were found between the synchronously vs. the asynchronously cleaved embryos, related to metabolism and the apoptosis mechanism. We provide the first evidence indicating that an embryo’s morphokinetics is associated with the transcriptome profile of the derived blastocyst, which might be practically relevant for the embryo transfer program.

P-258 Impact of Direct Unequal Cleavage (DUC) on embryo development, blastocyst formation and ploidy - artificial intelligence (AI) analysis

Abstract Study question Do DUCs significantly impact embryo development? In particular, morphokinetics, grading, and Pre-implantation Genetic Testing for Aneuploidy (PGT-A) outcome? Is this analysis corroborated by artificial intelligence? Summary answer DUC embryos develop slower, have lower rates of blastulation and lower CHLOE (Fairtility) scores for blastulation and implantation. However, occasionally euploid blastocysts form from DUCs. What is known already Time-lapse technology enables the identification of DUCs during embryo development. Previous research associates DUCs with poorer blastulation, implantation, and ploidy outcomes. However, DUCs are not routinely annotated in all clinics. Some algorithms, deselect embryos with short second cell cycles; hence, DUCs are rarely transferred. Whether DUC embryos should be automatically discarded or deprioritised is an ongoing debate which leads to inconsistency in clinical practices across fertility centres. AI image processing algorithms may assist embryologists in the identification of DUCs. Study design, size, duration A retrospective single-centre study of normally-fertilised embryos cultured in time-lapse incubators throughout 2019--2021. We reviewed 9284 time-lapse videos using an AI image processing tool (CHLOE, Fairtility), and assessed DUC embryo outcomes (ploidy, blastulation, and blastocyst quality). Additionally, we analysed pronuclei data searching for possible causes of DUCs. Participants/materials, setting, methods CHLOE (Fairtility) software analysed time-lapse videos identifying pronuclei, DUCs, and blastulation; recording all morphokinetic time points (tPNa,tPNf,t2,t3,t4,t5,t6,t7,t8,t9,tM, tSB,tB,tEB), morphological grades for the inner cell mass (ICM) and trophectoderm, blastocyst size at 116hpi; and assessing the likelihood of blastulation (at 30hpi) and implantation. We evaluated the statistical significance for all variables using t-tests (continuous variables) and chi-squared tests (categorical variables). We quantified the two pronuclei (2PN) detection efficacy using four metrics: accuracy, sensitivity, specificity, and informedness. Main results and the role of chance Of all the embryos analysed (n = 9284), 35% showed DUCs (n = 3269). Blastulation was significantly higher in non-DUC versus DUC embryos (76% and 49%, p &amp;lt; 0.0001). Of the embryos that blastulated, ICM quality (A,B,C,D: 24%,13%,19%,21% and 3%,4%,16%,47%, p &amp;lt; 0.001) and trophectoderm quality (20%,21%,15%,23% and 2%,7%,14%,52%, p &amp;lt; 0.0001) were significantly higher in non-DUC than in DUC embryos. As defined, DUC embryos were significantly quicker at reaching t3 than non-DUC [Mean(SD): 34(15) and 39(10), p &amp;lt; 0.0001], with the minimum times being 4hpi and 13hpi respectively. Interestingly, there was no significant difference in achieving t5 [52(21) and 51(12), NS]. For all other morphokinetic milestones, DUC embryos were 6 hours slower than non-DUC embryos. DUC embryos had an euploidy rate of 27.2% (12/44). Only one DUC embryo was transferred in a double embryo transfer cycle leading to a negative outcome. Implantation score [0.14(0.24) and 0.46(0.36), p &amp;lt; 0.0001] and blastulation score [0.4(0.46) and 0.75(0.4), p &amp;lt; 0.0001] were lower for DUC embryos than for non-DUC embryos. CHLOE automatic PN assessment agreed with human annotation in 92% of cases (TP = 388,TN=5,FP=29,FN=7). CHLOE Blastocyst prediction at 30hpi had an AUC of 0.89. The embryologist agreed on 97% of all 483 embryos that CHLOE classified as DUC. Discrepancies arose from CHLOE misclassifying fragments as blastomeres. Further studies warranted. Limitations, reasons for caution Differentiating between fragments and blastomeres within the 5 hours from the first division proves challenging for embryologists and, especially, AI algorithms. Hence, some embryos’ DUC status may be misclassified. Additionally, our sample sizes are limited and larger sizes are needed to corroborate our findings, especially those pertaining to ploidy status. Wider implications of the findings DUC embryos are associated with poorer outcomes and DUC status should be integrated into embryo classification frameworks. Nevertheless, some DUC embryos prove to be euploid. Hence, DUC embryos should not excluded from culture at cleavage stage and instead be allowed to reach blastocyst stage before assessing their suitability for transfer/vitrification/PGT-A. Trial registration number IRB-001C01-01-22

O-100 Incidence, morphology, ploidy and developmental arrest in direct cleavage embryos: what are they teaching us? Morphokinetic analysis of 15.081 embryos

Abstract Study question What is the impact of direct cleavage (DC) occurrence during embryo/blastocyst development regarding morphology grade, embryo arrest and ploidy status? Summary answer Blastocyst formation and morphology grade are significantly reduced in DC when compared to non-DC embryos; however, there is no difference in blastocyst ploidy status. What is known already Chromosome instability leading to aneuploidy during early cleavage is well known in humans. DC is defined as the abrupt cleavage of one into three or more daughter blastomeres as the result from an irregular mitosis. Three or more mitosis poles can impair early embryo development; however, how some embryos with these irregular divisions might be able to develop into expanded blastocysts is still unknown. Time-lapse system provides an uninterrupted evaluation of embryo morphological and dynamic parameters, allowing our understanding in such events and if those embryos may be associated with a self-correction mechanism to reach blastocyst stage. Study design, size, duration Retrospective cohort study from 15.081 embryos, 3.962 cycles and 3.106 patients that were undergoing in vitro fertilization (IVF) treatment according to medical referral in a single private ART center from December 2018 to November 2021. Only two-pronuclear (2PN) embryos cultured in time-lapse incubators (EmbryoScope, Vitrolife, Sweden) were included in the study. Direct cleavage occurance was annotated when a single blastomere directly cleaved into three or more daughter blastomeres. Participants/materials, setting, methods DC embryos were also classified in DC 1-3: abnormal cleavage occurred in zygote (1-cell) resulting in 3–4 blastomeres and in DC 2+: abnormal cleavage occurred at the 2-cell stage resulting in 5 or 6 blastomeres. Embryo arrest, blastocyst formation rate and morphology grade (Gardner and Schoolcraft, 1999) were annotated. Blastocyst biopsy reports for ploidy status were also considered for analysis. Mann-Whitney, chi-squared and Fisher tests were applied for statistical analysis. p &amp;lt; 0,05 was considered significant. Main results and the role of chance There were 1168 DC embryos (7,7%) from 896 cycles/841 patients; 502 DC1-3 (3,3%) from 391 cycles/366 patients and 666 DC2+ embryos (4,4%) from 475 patients/505 cycles. In DC group, around 21% of patients have the incidence of more than 1 DC embryo per cycle. Maternal age was lower in DC groups: 38,0±3,61 (DC1-3) and 38,60±3,21 (DC2+) years old versus not-DC (39,02±3,01, p &amp;lt; 0,0001). Embryo arrest was higher (DC1-3:85,5% and DC2+:56,8% versus not-DC:35,4%, p &amp;lt; 0,0001) and blastocyst formation rate was lower (14,5% and 43,2% versus 64,6%, p &amp;lt; 0,0001) in both DC groups and also between DC groups (p &amp;lt; 0,0001). There were 4124 biopsied not-DC blastocysts, 24 in DC1-3 and 154 in DC2+. There was no difference in ploidy between not-DC (57,9%, 40,2%, 1,9%, aneuploid, euploid and mosaic respectively) and DC groups (54,2%, 41,7%, 4,2% and 61%, 37% and 1,9% respectively, p = 0.86) and neither between DC-groups (p = 0.69). Inner cell mass morphology grades were distinct in all groups (not-DC A:45,8%, B:45,2% C:9,0%, DC1-3 A:35,6%, B:33,9%, C:30,5% and DC2+ A:23,1%, B:57,0% C:19,9%, p &amp;lt; 0,0001) and between DC-groups (p &amp;lt; 0,0001). Trophoectoderm morphology grades were also higher for not-DC group (not-DC A:30,5%, B:48,9% C:20,6%, DC1-3 A:15,3%, B:47,5%, C:37,3% and DC2+ A:13,6%, B:44,4% C:42,0%, p &amp;lt; 0,0001), however there was no difference between DC-groups (p = 0.80). Limitations, reasons for caution Limited data are available on the implantation potential of embryos with DC, as they are not prioritize for transfer, although our study did not demonstrated a higher aneyploidy rate. Patient parameters, with the exception of maternal age, were not considered in this study. Wider implications of the findings Although the morphology grades are lower and embryo arrest is higher in DC embryos, probably due to unequal cleavage, if they reach blastocyst stage, the ploidy status of these embryos are similar to not-DC embryos, speculative for a self-correction mechanism. Future steps should focus on their performance in pregnancy/live-birth. Trial registration number Not Applicable

P-268 Can AI be used as a tool in the evaluation of the risk of pregnancy loss after euploid single embryo transfer?

Abstract Study question Can AI be used as a tool in the evaluation of the risk of pregnancy loss after euploid embryo transfer? Summary answer AI-annotated tSB (time to start blastulation) and tB (time to formation of full blastocyst) were predictive of miscarriage and live birth. What is known already Despite the advantage of PGT-A in preventing miscarriages, a pregnancy loss still can occur after the transfer of a chromosomally normal embryo. Unfortunately in the literature there are no clear criteria indicating which morphologically good euploid embryos may be at risk of resulting in pregnancy loss. Study design, size, duration Retrospective cohort analysis of 455 euploid embryos allowing for the analysis of a range of variables for prediction of live birth or miscarriage from ICSI cycles, that were cultured in the Embryoscope (Vitrolife) at a single clinic (Istanbul Memorial Hospital, ART and Reproductive Genetics Center), and transferred between 2017-2020. This is the largest reported AI study to date predicting outcome in euploid SETs. Participants/materials, setting, methods Patients were aged between 24-44 years. Each morphokinetic feature was annotated manually and by CHLOE-(Fairtility), and pregnancy outcomes were evaluated. Main results and the role of chance When annotated using AI, the average time (mean±standard deviation (SD)) for tSB (98±7 vs 97±7, p &amp;lt; 0.05) and tB (106±7 vs 105±, p = 0.02) were significantly longer in patients who miscarried compared to those that did not. Implantation and blastulation scores were not significantly predictive of clinical miscarriage (0.77±0.22 vs 0.74±0.24, NS and 0.97±0.15 vs 0.97±0.16, NS for live birth and miscarriage, respectively). Embryos that aborted and led to live birth had an equal proportion of direct unequal cleavage (DUC) (respectively, DUCs assessed by CHLOE was: 6.8% vs 6.8%, NS). There was no significant difference between the presence of DUC and the pregnancy outcome (miscarriage rate in the presence or absence of DUC was 16.1% vs 16.3%, NS). Limitations, reasons for caution Retrospective data using embryos selected for transfer Wider implications of the findings AI-annotated tSB and tB can be added to the already existing range of available evaluation methods for embryo viability and functions which can predict the risk of miscarriage after a euploid embryo transfer. Trial registration number NA

Morphological and morphokinetic associations with aneuploidy: a systematic review and meta-analysis.

A time lapse system (TLS) is utilized in some fertility clinics with the aim of predicting embryo viability and chance of live birth during IVF. It has been hypothesized that aneuploid embryos display altered morphokinetics as a consequence of their abnormal chromosome complement. Since aneuploidy is one of the fundamental reasons for IVF failure and miscarriage, attention has focused on utilizing morphokinetics to develop models to non-invasively risk stratify embryos for ploidy status. This could avoid or reduce the costs associated with pre-implantation genetic testing for aneuploidy (PGT-A). Furthermore, TLS have provided an understanding of the true prevalence of other dysmorphisms. Hypothetically, the incorporation of morphological features into a model could act synergistically, improving a model's discriminative ability to predict ploidy status. The aim of this systematic review and meta-analysis was to investigate associations between ploidy status and morphokinetic or morphological features commonly denoted on a TLS. This will determine the feasibility of a prediction model for euploidy and summarize the most useful prognostic markers to be included in model development. Five separate searches were conducted in Medline, Embase, PubMed and Cinahl from inception to 1 July 2021. Search terms and word variants included, among others, PGT-A, ploidy, morphokinetics and time lapse, and the latter were successively substituted for the following morphological parameters: fragmentation, multinucleation, abnormal cleavage and contraction. Studies were limited to human studies. Overall, 58 studies were included incorporating over 40000 embryos. All except one study had a moderate risk of bias in at least one domain when assessed by the quality in prognostic studies tool. Ten morphokinetic variables were significantly delayed in aneuploid embryos. When excluding studies using less reliable genetic technologies, the most notable variables were: time to eight cells (t8, 1.13 h, 95% CI: 0.21-2.05; three studies; n = 742; I2 = 0%), t9 (2.27 h, 95% CI: 0.5-4.03; two studies; n = 671; I2 = 33%), time to formation of a full blastocyst (tB, 1.99 h, 95% CI 0.15-3.81; four studies; n = 1640; I2 = 76%) and time to expanded blastocyst (tEB, 2.35 h, 95% CI: 0.06-4.63; four studies; n = 1640; I2 = 83%). There is potentially some prognostic potential in the degree of fragmentation, multinucleation persisting to the four-cell stage and frequency of embryo contractions. Reverse cleavage was associated with euploidy in this meta-analysis; however, this article argues that these are likely spurious results requiring further investigation. There was no association with direct unequal cleavage in an embryo that progressed to a blastocyst, or with multinucleation assessed on Day 2 or at the two-cell stage. However, owing to heterogeneous results and poor-quality evidence, associations between these morphological components needs to be investigated further before conclusions can be reliably drawn. This first systematic review and meta-analysis of morphological and morphokinetic associations with ploidy status demonstrates the most useful morphokinetic variables, namely t8, t9 and tEB to be included in future model development. There is considerable variability within aneuploid and euploid embryos making definitively classifying them impossible; however, it is feasible that embryos could be prioritized for biopsy. Furthermore, these results support the mechanism by which algorithms for live birth may have predictive ability, suggesting aneuploidy causes delayed cytokinesis. We highlight significant heterogeneity in our results secondary to local conditions and diverse patient populations, therefore calling for future models to be robustly developed and tested in-house. If successful, such a model would constitute a meaningful breakthrough when accessing PGT-A is unsuitable for couples.

Germline development during embryogenesis of the larvacean, Oikopleura dioica

Germ cells develop into eggs and sperms and represent a lineage that survives through multiple generations. Germ cell specification during embryogenesis proceeds through one of two basic modes: either the cell-autonomous mode or the inductive mode. In the cell-autonomous mode, specification of germ cell fate involves asymmetric partitioning of the specialized maternal cytoplasm, known as the germplasm. Oikopleura dioica is a larvacean (class Appendicularia) and a chordate. It is regarded as a promising animal model for studying chordate development because of its short life cycle (5 days) and small genome size (∼60 ​Mb). We show that their embryos possess germplasm, as observed in ascidians (class Ascidiacea). The vegetal cytoplasm shifted towards the future posterior pole before the first cleavage occurred. A bilateral pair of primordial germ cells (PGC, B11 ​cells) was formed at the posterior pole at the 32-cell stage through two rounds of unequal cleavage. These B11 ​cells did not undergo further division before hatching of the tadpole-shaped larvae. The centrosome-attracting body (CAB) is a subcellular structure that contains the germplasm and plays crucial roles in germ cell development in ascidians. The presence of CAB with germplasm was observed in the germline lineage cells of larvaceans via electron microscopy and using extracted embryos. The CAB appeared at the 8-cell stage and persisted until the middle stage of embryogenesis. The antigen for the phosphorylated histone 3 antibody was localized to the CAB and persisted in the PGC until hatching after the CAB disappeared. Maternal snail mRNA, which encodes a transcription factor, was co-localized with the antigen for the H3S28p antibody. Furthermore, we found a novel PGC-specific subcellular structure that we call the germ body (GB). This study thus highlights the conserved and non-conserved features of germline development between ascidians and larvaceans. The rapid development and short life cycle (five days) of O. dioica would open the way to genetically analyze germ cell development in the future.

High ovarian response to ovarian stimulation: effect on morphokinetic milestones and cycle outcomes.

To assess the effect of high ovarian response on oocyte quality and ovarian stimulation cycle outcomes. A retrospective cohort study conducted at three IVF units. The high ovarian response (HOR) and polycystic ovary syndrome (PCOS) with HOR (PCOS HOR) groups included 151 and 13 women who underwent controlled ovarian stimulation (COS) resulting in more than 15 retrieved oocytes, for a total of 1863 and 116 cultured embryos, respectively. The normal ovarian response (NOR) group comprised 741 women with 6-15 retrieved oocytes, resulting in 4907 cultured embryos. Data collected included fresh cycle data and pregnancy rates, in addition to annotation of morphokinetic events from time of pronuclei fading to time of initiation of blastocyst formation of embryos cultured in a time lapse incubator, including occurrence of direct unequal cleavage at first cleavage (DUC-1) (less than 5h from two to three blastomeres). Comparison was made between morphokinetic parameters between the 3 groups. Cycle outcomes were compared in the high vs. normal ovarian response groups. Oocyte maturation rate was significantly lower in the HOR vs. NOR groups (56.5% vs. 90.0%, p < 0.001), while the fertilization rates were similar (60.2% vs. 58.1%, p = 0.397). The prevalence of DUC-1 embryos was higher in the PCOS HOR and the HOR groups as compared to the NOR group (22.7% vs. 16.2% and 12.0%, respectively, p < 0.001). After exclusion of DUC-1 embryos, remaining embryos from the NOR and HOR groups reached the morphokinetic milestones at similar rates, with comparable implantation and clinical pregnancy rates, while the PCOS HOR showed shorter time to 5 blastomeres compared to the NOR and HOR groups. High ovarian response might be associated with decreased oocyte quality, manifested as a higher proportion of immature oocytes and higher rate of direct uneven cleavage embryos, while embryos exhibiting normal first cleavage have similar temporal milestones and implantation potential.

LIVE BIRTH RATES ARE SIGNIFICANTLY DECREASED FOLLOWING TRANSFER OF A EUPLOID BLASTOCYST WITH DIRECT UNEQUAL CLEAVAGE

Embryos can have direct unequal cleavage (DUC) during the first and/or second divisions. Although embryos with DUC have decreased blastulation rates, some develop into high quality euploid blastocysts. The objective of this study is to compare live birth (LB) rates following transfer of single frozen euploid blastocysts that either did or did not display DUC. This was a retrospective chart review of patients who underwent single frozen euploid blastocyst transfers from November 2018 to March 2020. Uninterrupted embryo culture was done in the Embryoscope up to 6 days post retrieval. Embryos were evaluated on day 2 for presence/absence of DUC and graded on day 5 or 6 according to the Gardner system. Blastocyst selection for frozen embryo transfer was based on euploidy status, developmental day, morphological score, and sex preference. Information regarding presence/absence of DUC was not accessed during blastocyst selection. Patient characteristics and LB rates were compared between the groups with and without DUC. Chi-square and Student’s t-tests were used for data analysis. A total of 114 single euploid blastocyst transfers were included, resulting in an overall LB rate per transfer of 63.2%. Twenty-two of the transferred blastocysts displayed DUC, whereas 92 did not. The overall LB rate in the DUC group was significantly lower than in the non-DUC group (31.8% vs 70.7%, p<0.01). Subgrouping blastocysts by developmental day and morphology showed significantly lower LB rates for day 6 and fair quality DUC blastocysts, compared with non-DUC blastocysts (18.2% vs 63.6%, p<0.05) (35.7% vs 70.6%, p<0.05). Live birth rates following single frozen euploid blastocyst transfer are significantly lower when blastocysts display DUC compared with blastocysts without DUC. When available, embryo division patterns should be considered when selecting a euploid blastocyst for frozen embryo transfer.

P–032 Assessment of embryonic developmental outcome of direct unequal cleavage in patients with non-obstructive azoospermia and/or obstructive azoospermia

Abstract Study question Does direct unequal cleavage (DC) affect embryonic development after ICSI with testicular sperm (TESE-ICSI) in patients with non-obstructive azoospermia (NOA) and/or obstructive azoospermia (OA)? Summary answer The incidence of DC at the first cleavage (DC1) was extremely high and DC1 negatively affected embryonic development in NOA patients. What is known already It has been reported that the blastocyst development of embryos with direct cleavage (DC) was significantly lower than that without DC, but the clinical pregnancy rate after blastocyst transfer was not different with or without DC. The incidence of DC has been reported to be significantly higher after ICSI with testicular sperm (TESE-ICSI) than ICSI with ejaculated sperm (Ej), but to our knowledge, there are few reports investigating that the embryos with DC after TESE-ICSI affect embryonic development. Study design, size, duration We conducted a retrospective cohort study using time-lapse incubators (Geri, Genea Biomedx, Australia) from September 2018 to November 2020. Of 1033 two-pronuclear (2PN) embryos from TESE-ICSI, 486 and 547 embryos were from OA (35.9±5.5 years) and NOA (33.7±5.2 years), respectively. As an age matched control, we chose 581 embryos from ICSI using Ej (36.5±4.4 years). Participants/materials, setting, methods DC embryos were classified as DC1 (DC at first cleavage), DC2 (DC at second cleavage), and non-DC (without DC). The incidences of DC1 or DC2 and blastocyst development rates were compared among OA, NOA and Ej groups. In TESE-ICSI group, we compared blastocyst development rates with or without DC between good and poor quality embryos on day 3. Good quality embryos were defined as 8 cells with G3 or more by the Veeck’s classification. Main results and the role of chance DC1 incidence was significantly higher in NOA (37.3%) than OA (27.8%) and Ej (22.7%) (P &amp;lt; 0.01), whereas DC2 incidence was not statistically different among three groups; NOA (15.7%), OA (15.0%) and Ej (13.4%). Blastocyst development rates in DC1 were 17.8%, 19.5% and 25.8% for NOA, OA and Ej, respectively, which were significantly lower compared to non-DC in corresponding three groups (65.1%, 67.7%, and 68.5%, respectively, P &amp;lt; 0.01). In TESE-ICSI group, good-quality embryo rate on day 3 was significantly lower in DC1 (34.5%, P &amp;lt; 0.01) than DC2 (60.9%) or non-DC (54.2%). Additionally, blastocyst development rates in DC1 and DC2 were significantly lower than non-DC regardless of embryonic grades on day 3 (35.1%, 51.0%, and 81.6% for good-quality embryos on day 3, 10.1%, 27.0%, and 49.1% for poor-quality embryos on day 3, respectively, P &amp;lt; 0.05). When immotile sperm was used for TESE-ICSI, DC1 incidence was 40.0% (6/15), which did not show statistically differences. When performing single frozen-thawed blastocyst transfers, no pregnancies resulted from either DC1 (n = 13) or DC2 (n = 3) embryos in TESE-ICSI group. Limitations, reasons for caution We had a few data about the pregnancy rates after blastocyst transfers with DC, because embryos with DC were seldom transferred due to those lower priority. Although DC might be influenced by the sperm, we did not analyze the incidence of DC by taking the semen factors into account. Wider implications of the findings: The incidence of DC1 was extremely high and DC1 negatively affected embryonic development in NOA patients. Therefore, it is important to observe embryos using time-lapse incubator in order to recognize embryos with/without pregnancy potential, especially for embryos with DC1 in NOA patients. Trial registration number Not applicable

Time-lapse imaging of human embryos fertilized with testicular sperm reveals an impact on the first embryonic cell cycle.

Testicular sperm is increasingly used during in vitro fertilization treatment. Testicular sperm has the ability to fertilize the oocyte after intracytoplasmic sperm injection (ICSI), but they have not undergone maturation during epididymal transport. Testicular sperm differs from ejaculated sperm in terms of chromatin maturity, incidence of DNA damage, and RNA content. It is not fully understood what the biological impact is of using testicular sperm, on fertilization, preimplantation embryo development, and postimplantation development. Our goal was to investigate differences in human preimplantation embryo development after ICSI using testicular sperm (TESE-ICSI) and ejaculated sperm. We used time-lapse embryo culture to study these possible differences. Embryos (n = 639) originating from 208 couples undergoing TESE-ICSI treatment were studied and compared to embryos (n = 866) originating from 243 couples undergoing ICSI treatment with ejaculated sperm. Using statistical analysis with linear mixed models, we observed that pronuclei appeared 0.55 h earlier in TESE-ICSI embryos, after which the pronuclear stage lasted 0.55 h longer. Also, significantly more TESE-ICSI embryos showed direct unequal cleavage from the 1-cell stage to the 3-cell stage. TESE-ICSI embryos proceeded faster through the cleavage divisions to the 5- and the 6-cell stage, but this effect disappeared when we adjusted our model for maternal factors. In conclusion, sperm origin affects embryo development during the first embryonic cell cycle, but not developmental kinetics to the 8-cell stage. Our results provide insight into the biological differences between testicular and ejaculated sperm and their impact during human fertilization.

Equalization of Cleavage Is Not Causally Responsible for Specification of Cell Lineage

An unequal cleavage gives rise to a dedicated population of larval skeletogenic cells in sea urchins. The timing of this unequal cleavage, associated localization of key lineage markers, and loss of this lineage when embryos are treated with cleavage-equalizing reagents have all suggested that the asymmetry of the daughter cells is causal to the specification of this cell lineage. However, the mechanism by which asymmetric cleavage specifies this cell type remains unidentified. I found that applying a classical cleavage-equalizing reagent (sodium dodecyl sulfate) to embryos of an equally cleaving urchin eliminates its larval skeleton. This result suggests that equalization of cleavage itself is not causally responsible for specification of this cell lineage but coincident.

Iterative and Complex Asymmetric Divisions Control Cell Volume Differences in Ciona Notochord Tapering.

The notochord of the invertebrate chordate Ciona forms a tapered rod at tailbud stages consisting of only 40 cylindrical cells in a single-file column. This tapered shape involves differences in notochord cell volume along the anterior-posterior axis. Here,we quantify sibling cell volume asymmetry throughout the developing notochord and find that there are distinctive patterns of unequal cleavage in all 4 bilateral pairs of A-line primary notochord founder cells and also in the B-line-derived secondary notochord founder cells. A quantitative model confirms that the observed patterns of unequal cleavage are sufficient to explain all the anterior-posterior variation in notochord cell volume. Many examples are known of cells that divide asymmetrically to give daughter cells of different size and fate. Here, by contrast, a series of subtle but iterative and finely patterned asymmetric divisions controls the shape of an entire organ. Quantitative 3D analysis of cell shape and spindle positioning allows us to infer multiple cellular mechanisms driving these unequal cleavages, including polarized displacements of the mitotic spindle, contributions from the shape of the mother cell, and late changes occurring between anaphase and abscission that potentially involve differential cortical contractility. We infer differential use of these mechanisms between different notochord blastomeres and also between different rounds of cell division. These results demonstrate a new role for asymmetric division in directly shaping a developing organ and point toward complex underlying mechanisms.