Individual Embryos Research Articles

Establishment of cell lines from individual zebrafish embryos.

With the increasing use of fish as model species for research, cell cultures derived from caudal fin explants as well as pre-hatching stage embryos have provided powerful in vitro tools that can complement or serve as an ethically more acceptable alternative to live animal experiments. The widely-used protocols to establish these lines require, as a starting point, homogeneous pools of embryos or viable adult fish which are large enough for collecting sufficient fin tissue. This excludes the use of fish lines with adverse phenotypes or lines that exhibit mortality at early developmental stages and so can only be propagated as heterozygotes. Specifically, when no visually overt mutant phenotype is detectable for identifying homozygous mutants at early embryonic stages, it is then impossible to sort pools of embryos with the same genotypes to generate cell lines from the progeny of a heterozygote in-cross. Here, we describe a simple protocol to generate cell lines on a large scale starting from individual early embryos that can subsequently be genotyped by polymerase chain reaction. This protocol should help to establish fish cell culture models as a routine approach for the functional characterization of genetic changes in fish models such as the zebrafish. Furthermore, it should contribute to a reduction of experiments which are ethically discouraged to avoid pain and distress.

Tritiated Thymidine Internalization in Zebrafish Early Life Stages: Joint Use of Experimental Procedures and Microdosimetry.

Tritium is found in the environment under three forms: free in the water, gaseous, and bound to organic matter. Once internalized in living organisms, it can be found in two forms: tissue free water tritium (TFWT) and organically bound tritium (OBT). This study aims to better understand OBT internalization in living organisms and to show the complementarity between experimental procedures and microdosimetry simulations that have often been used to obtain more information on imparted energy to cell nuclei. To do so, tritiated thymidine, an organic form of tritium, was chosen and zebrafish embryos [3.5 h post fertilization (hpf)] were exposed to a range of activity concentrations (2.21 × 103 to 5.95 × 105 Bq/mL). First, individual zebrafish embryos were sampled after different exposure times (1 to 96 h) to qualify the internalization kinetics. Then, the barrier role of the chorion was assessed after 2 days of exposure. Lastly, individual zebrafish embryos were sampled after 1 and 4 days of exposure to measure the internalization in the whole fish and its DNA, but also to highlight a possible link between the internal dose rate and the external activity concentration. Microdosimetry simulations were also made to quantify the imparted energy that could occur in the zebrafish cells after exposure to tritium. Results showed that when bound to thymidine, tritium rapidly incorporates in zebrafish early life stages, with the internalization being almost complete after 24 h. Results also showed that while the chorion acted as a barrier to prevent thymidine from entering the embryos, significant levels could still be measured in the whole organisms as well as in DNA. This study also highlighted that when the external activity concentration increased, the internal dose rate increased as well, following a sigmoidal trend. Microdosimetry simulations highlighted that the size and shape of the cell matters, and that the smallest cells seem to be at the greater risk, with only low-energy electrons inducing energy depositions. A linear fit was also found between the mean energy deposited and the logarithm of the radius of the cell, thus showing that the quantity of deposited energy is proportional to the radius of the cell. While this study highlighted important internalization pattern, it will also be used as the starting point of a study focusing on the toxic effects of tritiated thymidine on zebrafish in its early life stages.

Oocyte developmental capacity is influenced by intrinsic ovarian factors in a bovine model for individual embryo production

The ovary and its hormones may have major effects on the in vitro developmental capacity of the oocytes it contains. We related intrinsic ovarian factors namely the presence of corpus luteum (CL) and/or dominant follicle (>8 mm) and the follicular count to cumulus expansion (CE), embryo development, and blastocyst quality in a bovine model. Cumulus-oocyte-complexes (COCs) were aspirated from follicles between 4 and 8 mm in diameter. In vitro embryo production was performed in a fully individual production system. The follicular fluid from which COCs were collected was pooled (per ovary) to evaluate the estrogen, progesterone, and insulin-like growth factor-1 (IGF-1) concentrations. Cumulus oocyte complexes collected from ovaries without a CL presented a greater CE than COCs derived from ovaries bearing CL. The absence of ovarian structures increased the blastocyst rate when compared to oocytes derived from ovaries with a CL, a dominant follicle, or both. Blastocysts derived from ovaries without a dominant follicle presented higher total cell numbers and a lower proportion of apoptosis than blastocysts derived from ovaries containing a dominant follicle. Cumulus oocyte complexes collected from ovaries with high follicular count resulted in higher cleavage than from ovaries with low follicular count, but the blastocyst rate was similar between groups. Ovaries bearing a CL had greater progesterone and IGF-1 follicular fluid concentrations in neighboring follicles than ovaries without a CL. Selection for bovine ovaries without CL or dominant follicle can have positive effects on CE, embryo development, and blastocyst quality in an individual embryo production system set-up.

Whole-embryonic identification of maternal microchimeric cell types in mouse using single-cell RNA sequencing

Even though the mother and the fetus of placental mammals are immunologically non-self with respect to one other, mutual exchange of small numbers of cells between them is known to occur. Maternal cells entering the fetus, called maternal microchimeric cells (MMc cells), are thought to be involved in different physiological phenomena, such as establishing immune tolerance, tissue repair, and the pathogenesis or deterioration of some inflammatory diseases and congenital malformations. While specific MMc cell types have been reported as associated with these phenomena, the contribution of MMc cells to these different outcomes remains unknown. As one possibility, we hypothesized that different embryos have differing repertoires of MMc cell types, leading to or biasing embryos toward different fates. To date, no studies have succeeded in identifying the MMc cell type repertoire of a single embryo. Accordingly, here, we isolated MMc cells from whole mouse embryos, determined their types, and analyzed their MMc cell type variability. By combining our previously established, whole-embryonic MMc isolation method with single-cell RNA sequencing, we successfully estimated the cell type repertoires of MMc cells isolated from 26 mouse embryos. The majority of MMc cells were immune-related cells, such as myeloid cells and granulocytes. We also detected stem cell-like MMc cells expressing proliferation marker genes and terminally differentiated cells. As hypothesized, we noted statistically significant inter-individual variation in the proportion of immune-related cells in the different embryos. We here successfully estimated MMc cell types in individual whole mouse embryos. The proportion of immune-related cells significantly differed among the individual embryos, suggesting that the variations are one of the potential mechanisms underlying the differing MMc-related physiological phenomena in offspring. These findings provide insight into cell-level epigenetics by maternal cells.

Ontogeny of risk assessment and escape-hatching performance by red-eyed treefrog embryos in two threat contexts.

Arboreal embryos of red-eyed treefrogs, Agalychnis callidryas, hatch prematurely in response to hypoxia when flooded and to mechanosensory cues in snake attacks, but hatching later improves tadpole survival. We studied ontogenetic changes in risk assessment and hatching performance of embryos in response to flooding and physical disturbance. We hypothesized that risk assessment decreases as hatchling survival improves and hatching performance increases as embryos develop. Because snakes eat faster than embryos asphyxiate, we hypothesized that embryos decide to hatch sooner and hatch faster in response to mechanosensory cues. We video-recorded individual embryos hatching in response to each cue type, then compared the incidence and timing of a series of events and behaviors from cue onset to complete hatching across ages and stimuli. Latency from cue to hatching decreased developmentally in both contexts and was shorter with mechanosensory cues, but the elements contributing to those changes differed. Hypoxia assessment involved position changes, which decreased developmentally along with assessment time. Mechanosensory cue assessment occurred more rapidly, without movement, and decreased with age. The first stages of hatching, membrane rupture and head emergence, were surprisingly age independent but faster with mechanosensory cues, congruent with greater effort under more immediate risk. In contrast, body emergence and compression showed ontogenetic improvement consistent with morphological constraints but no cue effect. Both appropriate timing and effective performance of hatching are necessary for continued development. Different stages of the process vary with development and environmental context, suggesting combinations of adaptive context- and stage-dependent behavior, cue-related constraints on information acquisition, and ontogenetic constraints on elements of performance.

Individual or group embryo culture? A pilot study and subsequent prospective randomized trial to evaluate blastocyst formation rate, quality and reproductive outcomes

Background and Aim: Embryo culture until the blastocyst stage is a common practice in most assisted reproduction laboratories. There are two different strategies for embryo culture, individually or in groups. Until now, it is accepted that embryos cultured in groups can interact each other, improving embryo quality. However, other studies did not find differences in embryo development when using either method. The aim of this study was to determine which culture method is better in terms of quality embryo rate, blastocyst formation rate, usable embryo rate, implantation and pregnancy rates.

A NON-COMMERCIAL WITNESSING SYSTEM A JOURNEY TO CREATE A BAR CODE SYSTEM FROM DISH LABELS TO MICROSCOPIC BAR CODES NEXT TO THE EMBRYO

The complexity of the procedures in the IVF laboratory requires a witnessing system to decrease the risk of human errors. Current quality control practices include a second person in a double witnessing protocol in gamete collection, insemination, and embryo transfer procedures. Commercial electronic witnessing systems have provided an alternative option for increasing the security level of matching samples in the laboratory; however, blastocyst biopsies, cell loading, and individual embryo freezing have not been covered entirely due to the complexity of the microscopic level.

Initiative on Avian Primordial Germ Cell Cryobanking in Thailand.

Background: Biobanking the reproductive tissues or cells of animals preserves the genetic and reproductive ability of the species in long-term storage and promotes sharing of reproductive materials. In avian species, the primordial germ cell (PGC) is one of the most promising reproductive cells to be preserved in biobanks, due to self-renewal properties and direct access to the germ line mediated by PGC transfer. Methods: To conserve the genetic resource of local chicken breeds that are of conservation importance, we systematically isolated two types of pregonadal PGCs from chicken embryos-circulating and tissue PGCs. PGCs of individual embryos were separately isolated, cultured, and cryopreserved. Characteristics of cultured PGCs are described and evaluated. Results: The efficiency of PGC isolation from individual embryos was 98.9% (660/667). In most cases, both matching circulating and tissue PGC lines were isolated from the same embryo (68.2%, 450/660), whereas the remaining lines were from a single source, being either tissue (30.6%, 202/660) or circulating (1.2%, 8/660). Efficient PGC isolation and proliferation can be expected in cultures of circulating PGCs (68.7% and 64.3%, respectively) and tissue PGCs (97.8% and 80.7%, respectively). Following cryopreservation, recovered cells sustained PGC identities including expression of chicken vasa homolog and deleted in azoospermia-like proteins and migration ability to recipient embryonic gonads. Culture conditions equally supported proliferation of circulating and tissue PGCs from both sexes. Combining tissue PGC culture in the regimen prevented 30.3% loss of PGC cultures in the case where circulating PGC culture was ineffective. Cultured circulating and tissue PGCs were similar in morphology, but optimal culture characteristics were different. Conclusion: We applied the approach of PGC isolation from blood and tissue origins on a wide scale and demonstrated its efficiency for biobanking chicken PGCs. The workflow can be operated effectively almost year-round in a tropical climate. It was also described in ample and practical details, which are suitable for adoption or optimization in other conditions.

Effects of dietary daidzein supplementation on reproductive performance, immunity, and antioxidative capacity of New Zealand White does

This study was conducted to explore the effects of dietary daidzein (DA) supplementation on reproductive functions in rabbit does. A total of 200 New Zealand White does were randomly assigned to four groups and fed a basal diet with no DA (control, CON) or the basal diet supplemented with 85 (DA85), 170 (DA170), and 340 (DA340) mg DA/kg throughout the gestation. Results showed that DA elevated individual embryo weight at 19 days of gestation. DA supplementation at 170 mg/kg also elevated the individual and litter birth weight at parturition (P < 0.05). DA supplementation at 170 and 340 mg/kg elevated the serum concentrations of estrogen and IGF-I at 19 days of gestation (P < 0.05), and elevated the concentrations of leptin and high-density lipoprotein cholesterol (HDL-C) at parturition (P < 0.05). DA increased the serum concentration of IgG, but decreased the concentrations of tumour necrosis factor-α (TNF-α) and malonic dialdehyde (MDA) (P < 0.05). DA supplementation at 170 mg/kg increased the serum activities of glutathione peroxidase (GSH-Px) and catalase (CAT) at parturition (P < 0.05). Moreover, DA elevated the expression levels of critical genes related to reproductive functions such as the progesterone receptor (PGR), estrogen receptor-β (ESRRB), and transforming growth factor-α (TGF-α) in the uterus (P < 0.05). DA supplementation at a moderate dose (170 mg/kg) also elevated the expression levels of epidermal growth factor receptor (EGFR) and nuclear receptor subfamily 5 group A member 2 (NR5A2) in the placenta (P < 0.05). These results suggested that DA can improve the reproductive performance of does, which was associated with elevated serum hormones, immunity and antioxidant capacity, as well as the expression levels of critical genes related to reproductive functions.

Real-time computer assisted measurement of oocyte and embryo volume for assessment of transport parameters

Cryopreservation of gametes has revolutionized both animal agriculture and human reproductive medicine. Although many new technologies have tremendously improved the cryopreservation of oocytes and embryos, osmotic stress encountered during the equilibration process can cause their loss of function. Rational cryoprotective agent (CPA) equilibration strategies can be used to minimize this stress but require trained personnel to monitor the process in individual oocytes or embryos or require the use of suboptimal average transport parameter values in mathematically guided protocols. To enable individually optimized equilibration of CPAs in individual cells, here we establish experimental and computational techniques to track the osmotic behavior of individual bovine oocytes and embryos during CPA equilibration in real time. We designed a microfluidic device to provide a controlled flow of CPA and modified standard image analysis techniques to estimate real-time cell volume changes. In particular, we used a level-set method to define a boundary within a contour plot which could automate the image analysis process. A colour based level set algorithm coupled with contour smoothing not only provided the best fit but also reduced the segmentation time to well under a second per image. The accuracy of the automated method was comparable to human segmented images for both oocytes and embryos. This technology should enable both rapid evaluation of key biophysical parameters in oocytes and embryos undergoing CPA equilibration and the development of real-time feedback-control of CPA equilibration, enabling individual oocyte- and embryo-specific optimal protocols.

Higher Doses of FSH Used for Superovulation Do Not Adversely Affect Embryonic Ploidy: A Randomised Controlled Trial (Stimulation Resulting in Embryonic Aneuploidy Using Menopur (STREAM) Trial)

Background: High dose gonadotropin stimulation protocols may increase costs, patient discomfort, likelihood of ovarian hyper stimulation syndrome (OHSS), ovarian torsion and increased bleeding after multiple ovarian punctures. In addition, it is possible that high dose stimulation may result in impaired oocyte “quality” with adverse effect of embryonic ploidy. This may negate any benefits seen from a higher oocyte yield after high dose stimulation. Aim: To determine the proportion of euploid blastocysts resulting from higher versus lower dose ovarian stimulation protocols. Method: Multicentre randomised controlled trial recruiting 57 women who were treated with ovarian stimulation using either 150 or 300 IU Menopur per day. In Vitro Fertilisation (IVF) or Intracytoplasmic Sperm Injection (ICSI) were performed according to individual unit protocol and embryos were cultured to blastocyst stage. A trophectoderm biopsy was performed for preimplant action genetic testing for aneuploidy. Results: The number of oocytes obtained from women randomised to 150 IU Menopur was between 3–17 (mean=9), whereas the number of oocytes obtained from women randomised to 300 IU Menopur was between 3–24 (mean=11). There was a positive linear relationship between serum AMH concentration and oocyte yield in both the 150 &amp; 300 IU Menopur groups (R=0.3359, R2=0.1129 &amp; R=0.3741, R2=0.1399). 63% of blastocysts were euploid in the 150 IU Menopur group and 75% in the 300 IU Menopur group (p=0.17). Conclusion: The higher dose ovarian stimulation protocol did not significantly increase the number of oocytes retrieved, nor did the higher dose protocol reduce the proportion of euploid embryos created. This study does not support the hypothesis that use of higher doses of gonadotropin for ovarian stimulation results in a reduction in the proportion of euploid embryos obtained after IVF.

AI, Time-lapse and Biomarkers: What Can We Expect in the Next Decade?

For 40 years we have relied upon morphological assessment of the human embryo for its selection for transfer. With the advent of time-lapse microscopy (TLM), we have been able examine the timings of key developmental events and create algorithms to further advance embryo selection. However, such algorithms do not utilise all available data. Artificial intelligence (AI) can utilise the hundreds of successive images of each embryo as it develops to produce highly predictive rankings. This approach increases the speed of assessment, while decreasing variation typically associated with the subjective analysis of individual embryologists. Therefore, AI represents a means to increase both accuracy and standardisation of embryo selection. In the IVF laboratory, beyond its current function in embryo selection, AI will be used for gamete selection prior to fertilisation, and to increase laboratory function by its integration into Quality Control and Management systems. Although TLM combined with AI holds great potential, it does not quantitate the physiological status of the embryo. Analysis of spent culture medium has revealed that metabolism (glucose uptake and the utilisation of amino acids) of the human blastocyst is related to pregnancy. Currently, analysis of metabolic function by individual embryos requires highly specialised technologies, but with the advent of microfluidics and microfabrication we are entering an era of novel benchtop technologies capable of rapid single embryo analysis. Subsequently, it is envisaged that AI will be used in combination with TLM and the analysis of spent culture medium (to quantitate metabolism and plausibly cell-free DNA), to identify non-invasively the healthiest embryo for transfer.

Preparation of Intact Nuclei for Single-Nucleus Omics Using Frozen Cell Suspensions from Mutant Embryos of Xenopus tropicalis.

Single-cell omics such as single-cell RNA-sequencing (RNA-seq) have been used extensively to obtain single-cell genome-wide expression data. This technique can be used to compare mutant and wild-type embryos at predifferentiation stages when individual tissues are not yet formed (therefore requiring genotyping to distinguish among embryos), for example, to determine effects of mutations on developmental trajectories or congenital disease phenotypes. It is, however, hard to use single cells for this technique, because such embryos or cells would need to be frozen until genotyping is complete to capture a given developmental stage precisely, but intact cells cannot be isolated from frozen samples. We developed a protocol in which high-quality nuclei are isolated from frozen cell suspensions, allowing for genotyping individual embryos based on a small fraction of a single embryo suspension. The remaining suspension is frozen. After genotyping is complete, nuclei are isolated from embryo suspensions with the desired genotype and encapsulated in 10× Genomics barcoded gel beads for single-nucleus RNA-seq. We provide a step-by-step protocol that can be used for single transcriptomic analysis as well as single-nucleus chromatin accessibility assays such as ATAC-seq. This technique allows for high-quality high-throughput single-nucleus analysis of gene expression in genotyped embryos. This approach may also be valuable for collection of wild-type embryonic material, for example, when collecting tissue from a particular developmental stage. In addition, freezing of tissue suspensions allows precise staging of collected embryos or tissue that may be difficult to manage when collecting and processing cells from living embryos for single-cell RNA-seq.

Significance of the association between early embryonic development and endocytosis.

Fertilization triggers a process called maternal-to-zygotic transition, in which the oocyte undergoes oocyte-to-embryo transition, leading to massive intracellular remodeling toward early embryogenesis. This transition requires the degradation of oocyte-derived components; however, the significance and mechanism of degradation of cell surface components remain unknown. In this review, we focused on the dynamics of plasma membrane proteins and investigated the relationship between embryonic development and endocytosis. Our survey of the extant literature on the topic led to the conclusion that clathrin-mediated endocytosis is essential for the progression of early embryogenesis and selective degradation of oocyte-derived plasma membrane proteins in mouse embryos, as reported by studies analyzing maternal cellular surface proteins, including a glycine transporter, GlyT1a. Evaluation of such endocytic activity in individual embryos may allow the selection of embryos with higher viability in assisted reproductive technologies, and it is important to select viable embryos to increase the rates of successful pregnancy and live birth. Although the early embryonic developmental abnormalities are mainly accompanied with chromosomal aneuploidy, other causes and mechanisms remain unclear. This review summarizes molecular biological approaches to early embryonic developmental abnormalities and their future prospects.

Genome-wide profiling of histone H3K4me3 and H3K27me3 modifications in individual blastocysts by CUT&Tag without a solid support (NON-TiE-UP CUT&Tag)

Individual analysis of the epigenome of preimplantation embryos is useful for characterizing each embryo and for investigating the effects of environmental factors on their epigenome. However, it is difficult to analyze genome-wide epigenetic modifications, especially histone modifications, in a large number of single embryos due to the small number of cells and the complexity of the analysis methods. To solve this problem, we further modified the CUT&Tag method, which can analyze histone modifications in a small number of cells, such that the embryo is handled as a cell mass in the reaction solutions in the absence of the solid-phase magnetic beads that are used for antibody and enzyme reactions in the conventional method (NON-TiE-UP CUT&Tag; NTU-CAT). By using bovine blastocysts as a model, we showed that genome-wide profiles of representative histone modifications, H3K4me3 and H3K27me3, could be obtained by NTU-CAT that are in overall agreement with the conventional chromatin immunoprecipitation-sequencing (ChIP-seq) method, even from single embryos. However, this new approach has limitations that require attention, including false positive and negative peaks and lower resolution for broad modifications. Despite these limitations, we consider NTU-CAT a promising replacement for ChIP-seq with the great advantage of being able to analyze individual embryos.

P-780 Ten years electronic witnessing in the IVF laboratory

Abstract Study question What did we learn after 10 years of electronic witnessing? Summary answer Only when applied correctly, electronic witnessing can prevent sample mix-up. What is known already In the early days of IVF, manual double witnessing was used to reduce the misidentification of gametes for insemination. However, risk for human errors due to fatigue, automatism, distraction or work load make this a mistake-prone approach. Hence, there is increasing interest in using electronic witnessing systems for patient and sample identification. Although not always mandatory, such systems have been implemented in many fertility laboratories to avoid identification errors. A mismatch is generated when non-matching samples are simultaneously present in a single workstation. Yet, when the system is incorrectly used, corrective intervention by an administrator (administrator assign) may be required. Study design, size, duration This evaluation investigates the mismatch (MM) rate and administrator assigns (AA) over a 10-year period (March 2011- December 2021) with the use of RI Witness (RIW) (CooperSurgical). Radio frequency identification tags (RFID) were used for patient and sample identification. Conventional IVF and ICSI cycles, and frozen embryo cycles (FET) were included since 10 years, IUI cycles since 8 years. Participants/materials, setting, methods The total number of tags and witness points (WP) were recorded. Witness points represent all the actions in RIW that have been performed during the entire process. MM and AA were collected and stratified by procedure (sperm preparation, oocyte retrieval, IVF/ICSI, (embryo)biopsy, vitrification/warming, embryo transfer, medium changeover and IUI). Critical MM (such as mis-labeling or non-matching samples within one work area) and critical AA (such as non-RIW-identified samples and unconfirmed WP) were selected. Main results and the role of chance A total of 109,655 cycles were included: 53,023 IVF/ICSI, 36,347 FET, and 20,285 IUI cycles. The 724,096 used tags, led to a total of 849,650 WP. The overall MM rate per WP was 0.25% (2,132/849,650), 1.9% per cycle, of which 144 critical mismatches occurred in the different procedures: sperm preparation: 66, oocyte retrieval: 5, IVF/ICSI: 33, (embryo)biopsy: 1, vitrification/warming: 26, embryo transfer: 8, medium changeover: 3 and IUI: 2. The mean critical MM rate per WP per year was 0.017% ± 0.007% and 0.13 ± 0.05% per cycle. The overall AA rate per WP was 0.11% (940/849,650), 0.86% per cycle, including 320 critical assigns: sperm preparation: 83, oocyte retrieval: 34, IVF/ICSI: 173, (embryo)biopsy: 3, vitrification/warming: 6, embryo transfer: 0, medium changeover: 21. Critical AA rate per year was on average 0.041%/WP ± 0.01% and 0.30 ± 0.07% per cycle. All MM and AA rates remained stable during this 10-year period, except for sperm preparation, where the removal of a single witness point led to a huge increase in AA. RIW should still be used in combination with the manual labelling of bottom and lid of dishes and tubes to guarantee correct assignment in case of RFID malfunction or AA. Limitations, reasons for caution The procedures and method of integration of RIW may vary from one laboratory to another and result in differences in the potential risks. RIW cannot (yet) identify individual embryos, making double manual witnessing indispensable at certain critical steps, where potential errors are not recorded. Wider implications of the findings Using an electronic witnessing is considered to be the ultimate tool to safeguard correct identification of gametes and embryos. But this is only possible when used correctly and requires proper training and attention of the staff. It may also induce new risks, i.e. blind witnessing of samples by the operator. Trial registration number not applicable

P-190 The first cleavage plane in human embryos is dictated by the topology of pronuclei, not by the polar body position

Abstract Study question Is the first cleavage plane pre-determined in human embryos? Summary answer Unlike in mice, the human zygotes tend to divide at the right angle to the longitudinal pronuclear axis, irrespective of polar body position. What is known already In lower species, the orientation of the first cleavage plane defines the development of individual blastomeres and the three-dimensional (3D) body plan. The existence of embryonic pre-patterning in mammals remains controversial. Mouse studies suggested that the juxtaposed pronuclei (PN) are aligned with the polar body (PB) and cleavage furrow bisects the zygote along the polar axis thus ensuring symmetrical distribution of maternal resources to arising blastomeres. However, whether the same spatial control applies to humans is unclear. Study design, size, duration Retrospective observational analysis of high-resolution footage from 300 human embryos that gave rise to live births after a single embryo transfer. Participants/materials, setting, methods Embryo monitoring has been performed by the GERI time-lapse incubator. Image acquisition of individual embryo development lasted 6 days, starting within 10 minutes after intracytoplasmic sperm injection (ICSI). Transmitted light images have been automatically captured in 5-minute intervals and 11 focal planes covering the whole embryo volume. Analysis of 3D image data was performed at the time frame corresponding to the onset of the first mitosis. Main results and the role of chance The systematic analysis of the zygotic division pattern showed that the PN axis (defined by apposed pronuclei) was not stereotypically aligned with the animal-vegetal (AV) axis (with animal pole defined by the PB). Instead, PN/AV arrangement categories were equally represented in our cohort (PN/AV angle 0-30° - 34.7% vs. 30-60° - 36.0% and 60-90° - 29.3%; chi-square test p=.91351). Remarkably, we observed that the direction of the first cleavage was preferentially perpendicular to the PN axis (65.3%), while only 2.7% of embryos divided along the longitudinal PN axis. The remaining 32% embryos showed intermediated phenotype with cleavage plane/PN axis angle 30-60o. The results of this human embryo analysis contrast with the previous reports from mice. We hypothesize that this discrepancy might be attributed to the difference in centrosome inheritance and configuration of the zygotic spindle apparatus. Limitations, reasons for caution All analyzed embryos were produced by ICSI. Deposition of spermatozoa might affect the pronuclei positioning. Nevertheless, preliminary data from conventional IVF cycles are in agreement with the ICSI group results. Wider implications of the findings Our observations challenge the textbook view of the first division’s geometry, which has been derived from experiments in mice. The analysis of clinical time-lapse records improves our understanding of symmetry breaking during normal human development and creates scientific ground for evidence-based (de)selection of embryos for infertility treatment. Trial registration number not applicable

O-007 Simplifying the complexity of time-lapse decisions with AI: CHLOE (Fairtility) can automatically annotate morphokinetics and predict blastulation (at 30hpi), pregnancy and ongoing clinical pregnancy

Abstract Study question What is CHLOE’s (Fairtility) efficacy of prediction of blastulation (at 30hpi), pregnancy and ongoing clinical pregnancy following single embryo transfer (SET)? Summary answer CHLOE(Fairtility) algorithms are effective predictors of blastulation, ploidy, pregnancy, implantation and ongoing clinical pregnancy What is known already Time-lapse incubators have increased the amount of information available to the embryologist to help determine the fate of embryos. This has led to differences in clinical practice between clinics in how this information is prioritised. Moreover, inter-operator inconsistencies and the time-consuming nature of manually annotating time-lapse videos are challenges currently experienced by time-lapse users that can be relieved with Artificial Intelligence(AI) tools, such as CHLOE(Fairtility). CHLOE levergaes AI-based predictors to predict blastulation and implantation, whilst providing transparency to which biological characteristics have led to that determination. There is a need to validate AI tools before their incorporation into clinical practice. Study design, size, duration This was a single centre study that took place between 2017-2020, at Istanbul Memorial Sisli Hospital in Turkey, ART and Center. This was a retrospective cohort analysis that reviewed 6748 time-lapse videos containing 5392 cleaved embryos, 3763 blastocysts, 877 single embryo transfers(SET) with known ongoing pregnancy outcome (KOPO), 306 euploid SETs and 25 mosaic embryo SETs with KOPO. CHLOE blastocyst and implantation score efficacy of prediction of clinical outcomes was quantified using the metric AUC. Participants/materials, setting, methods Time-lapse videos were assessed using CHLOE(Fairtility), an AI based tool, to quantify quantitative and qualitative morphokinetics (including automated annotations of tPNa,tPNf,t2,t3,t4,t5,t6,t7,t8,t9,tM,tSB,tB,tEB), CHLOE implantation score and CHLOE blastocyst score (calculated at 30hpi) relative to laboratory (ploidy results, blastulation) and clinical outcomes (biochemical, clinical and ongoing pregnancy) following overall SET. Binary logistic regression was used to calculate area under the curve (AUC) as a measure of prediction efficacy. Main results and the role of chance Blastulation score assessment of cleaved embryos was predictive of blastulation (AUC=0.96, baseline=70% n = 5392, p &amp;lt; 0.001). Following PGT-A, implantation score was predictive of euploids (AUC=0.61, baseline=34%, n = 1456, p &amp;lt; 0.001), but not of embryos classified as mosaics (AUC=0.5, baseline=19%, n = 1456, p &amp;gt; 0.05). Following SET, implantation score was predictive of biochemical (AUC=0.71, baseline=49%, n = 866, p &amp;lt; 0.001), clinical and ongoing pregnancy rate (AUC=0.69, baseline=37%, n = 866, p &amp;lt; 0.001). Following SET of non-PGT-A embryos, implantation score decreased with increasing patient age (p &amp;lt; 0.001). The type of aneuploidy (such as monosomy, trisomy, segmental) did not affect implantation score or blastulation score (p &amp;gt; 0.05). Implantation score prediction of outcome was higher for non-PGT-A transfers than overall transfers for biochemical (Non-PGTA: AUC=0.73, baseline=33%, n = 535, p &amp;lt; 0.001; OVERALL: AUC=0.71, baseline=49%, n = 866, p &amp;lt; 0.001), clinical and ongoing pregnancy (Non-PGTA: AUC=0.76, baseline=24%, n = 535, p &amp;lt; 0.001; OVERALL: AUC=0.69, baseline=37%, n = 866, p &amp;lt; 0.001), despite lower baselines. Limitations, reasons for caution This is a single centre study, using retrospective data where embryos were selected for transfer by human embryologists. Despite the data has heterogeneity in terms of clinical features, the study is part of a larger framework for responsible incorporation of AI into clinical practice through robust validation. Wider implications of the findings AI-based tools have the potential of increasing consistency, efficiency and efficacy of embryo selection. The additional information on quantitative and qualitative morphokinetics that AI tools such as CHLOE provide, bring transparency to the prediction, allowing for improvement in personalisation of care down to each individual embryo. Trial registration number None

P-235 An analysis of qualitative and quantitative morphokinetic parameters automatically annotated using CHLOE (Fairtility), an AI-based tool, finds AI score predictive of blastulation and ploidy

Abstract Study question What is the relationship between qualitative and quantitative morphokinetic parameters automatically annotated using CHLOE(Fairtility), an AI-based tool? Summary answer CHLOE score is associated with ploidy. DUC embryos have lower blastulation, form fewer good blastocysts, have increased fragmentation, slower development, lower implantation than non-DUCs. What is known already The introduction of time-lapse technologies in IVF has led to the discovery of quantitiative and qualitative morphokinetic parameters which are predictive of embryo viability (ESHRE Workshop group, 2020). The challenges of annotating videos manually remain: (i)operator variation, (ii)time-consuming; (iii)complexity of how to prioritise numerous features when determining which embryos to transfer, freeze or discard. CHLOE (Fairtility) is an AI-based tool designed to automatically capture these parameters from the time-lapse videos, removing the “black box” associated with AI, and, instead, bringing transparency and support to the embryologist responsible for the decision, thus, enhancing personalisation of care down to each individual embryo. Study design, size, duration Prospective cohort analysis on time-lapse data retrospectively collected at a single private fertility clinic in Spain between 2018-2020. 693 videos were automatically annotated (without training) using the CHLOE Artificial Intelligence (AI) tool for the following quantitative features: tPNa,tPNf,t2,t3,t4,t5,t6,t7,t8,t9,tM,tsB,tB,teB, size of ICM; and the following qualitative parameters: number of pronucleates, morphological quality of Inner Cell Mass and Trophectoderm (CHLOE Morphological scoring), identification of unusual embryo cleavages i.e. Direct Uneven Cleavage (DUCs), amongst other features. Participants/materials, setting, methods All embryos were cultured using the Embryoscope (Vitrolife) incubator. Using a range of algorithms, CHLOE generated a prediction of blastulation (at 30hpi) and implantation which were compared to outcome (blastocysts vs non-blastocysts; euploids vs Aneuploids&amp;Mosaics; Mosaics vs euploids&amp;aneuploids). Embryos identified as DUCS by CHLOE were compared with non-DUCs in terms of outcomes and in terms of endpoints generated by CHLOE (parametric continuous data assessed using 2-tail t-test, categorical data using chi-square). Main results and the role of chance Within all cleaved embryos analysed (n = 693), 29% were DUCs. DUC embryos were less likely to blastulate (DUCvsNonDUCs: 25vs50%,p&amp;lt;0.001), had a higher proportion of embryos with severe fragmentation (26% vs 3%,p&amp;lt;0.001), less likely to be suitable for biopsy (23vs87%, p &amp;lt; 0.001) lower blastulation prediction score (0.53vs0.76,p&amp;lt;0.001), lower implantation prediction score (0.21vs0.48,p&amp;lt;0.001) and slower embryo development across the all morphokinetic time-points assessed(p &amp;lt; 0.001), except for t5 (NS); than non-DUCs. DUCs and non-DUCs had similar proportion of 1,2,3PNs(5,83,5%vs 7,84,3%, NS). Within embryos that blastulated (n = 581), 25% were DUCs. DUC blastocysts were less likely to have a good quality ICM (7vs33%,p&amp;lt;0.001) or a good quality trophectoderm (9vs35%,p&amp;lt;0.05), lower implantation score (0.29vs0.52,p&amp;lt;0.05) and slower embryo development across the following morphokinetics time-points than non-DUC blastocysts. DUCs (n = 38) and non-DUC (n = 292) blastocysts had similar euploidy rate (50vs43%,NS), mosaicism rate (8vs11%,NS), and similar ratio of Euploids:Aneuploid:Mosaics (19:16:3vs126:133:33, NS). One DUC embryo was transferred, leading to an ongoing clinical pregnancy. Blastulation score was predictive of blastulation (AUC of 0.91, p &amp;lt; 0.001). Mosaic embryos had similar implantation score to non-mosaics (0.61vs0.67, NS). Euploid embryos had a higher implantation score than aneuploid blastocysts (0.71bs0.62, p &amp;lt; 0.02), so implantation score was predictive of ploidy. Limitations, reasons for caution This study involved the validation of (i) a specific AI based tool which may not be generalised across other AI tools; (ii) in a single centre. Results obtained did not involve training, suggestive of CHLOE’s ability to generalise across clinics. Presenting a framework for responsibly incorporating AI into clinical practice. Wider implications of the findings CHLOE can simplify the processing of time-lapse data to effectively, consistently, and efficiently quantify parameters that can help explain a comprehensive prediction of embryo viability. This provides a useful tool which will ultimately assist clinicians with selecting the most optimal embryos for transfer and avoid wastage from discarding viable embryos. Trial registration number not applicable

O-063 Impact of group embryo culture vs individual embryo culture strategies on blastocyst rate and quality

Abstract Study question Does group embryo culture improve blastocyst formation compared to individual culture? Summary answer Individual culture provides an increased blastocyst formation rate. What is known already Embryo culture is one of the most important steps of an IVF treatment. Several studies pointed out that group embryo culture could help embryos with lower potential, through paracrine signaling, to achieve development and improve blastocyst formation rate. On the other hand, this issue continues to be controversial and other studies did not find differences in embryo development when using either method. Study design, size, duration A prospective randomized study was performed between January 2020 and December 2021. The data were obtained from 830 embryos that came from 103 egg donation treatments. We defined two groups: individual vs group culture. The number of embryos analyzed in the study was calculated to test a difference of 10% in the rate of blastocyst formation between the two groups. Participants/materials, setting, methods A total of 103 ICSI-egg donation cycles with normozoospermic males (fresh samples) were included. After fertilization assessment, zygotes were randomized into two groups; Group 1: individual culture (399 embryos), and Group 2: culture of 2-5 embryos per drop (431 embryos). The embryos were cultured in 35 µl drops of Global Total medium until day 5 when they were classified morphologically according to Gardner criteria (1998). The statistical analysis was performed using SPSS (version 20.0). Main results and the role of chance The rate of high-quality embryos on day 3 were similar in Group 1 compared to Group 2 (79.2% vs 81.4%, p = 0.432). However, we observed an increase in the blastocyst formation rate on day 5 (62.9% vs 56.1%, p = 0.048) and a trend for higher embryo quality (A/B blastocysts) (55.9% vs 52%, p = 0.266) in Group 1; compared to Group 2, respectively. Our data suggest that individual culture increases blastocyst formation rate and may benefit embryo quality on day 5. Our results challenge previous reports suggesting that culture in groups can improve embryo development. As hypothesis, we argue that some of the components that the embryos release to the culture environment during their division may affect the quality of the cohort potentially compromising further development up to the blastocyst stage. Limitations, reasons for caution The study is focused on laboratory outcomes until the blastocyst stage. Clinical outcomes were not analyzed. Wider implications of the findings Finding the most appropriate embryo culture conditions is essential to improve ART results. Time-lapse systems should be adapted to the use of both individual and group culture dishes. Larger randomized studies are needed in order to reach robust conclusions on the subject. Trial registration number NA