Developmental capacity is unevenly distributed among single blastomeres of 2-cell and 4-cell stage mouse embryos

During preimplantation development, mammalian embryo cells (blastomeres) cleave, gradually losing their potencies and differentiating into three primary cell lineages: epiblast (EPI), trophectoderm (TE), and primitive endoderm (PE). The exact moment at which cells begin to vary in their potency for multilineage differentiation still remains unknown. We sought to answer the question of whether single cells isolated from 2- and 4-cell embryos differ in their ability to generate the progenitors and cells of blastocyst lineages. We revealed that twins were often able to develop into blastocysts containing inner cell masses (ICMs) with PE and EPI cells. Despite their capacity to create a blastocyst, the twins differed in their ability to produce EPI, PE, and TE cell lineages. In contrast, quadruplets rarely formed normal blastocysts, but instead developed into blastocysts with ICMs composed of only one cell lineage or completely devoid of an ICM altogether. We also showed that quadruplets have unequal capacities to differentiate into TE, PE, and EPI lineages. These findings could explain the difficulty of creating monozygotic twins and quadruplets from 2- and 4-cell stage mouse embryos.

P–210 Abnormal cleavage patterns during embryo preimplantation development and their effect on blastulation: an overview from IVF patients with multiple IVF cycles in a time-lapse incubator

Study question How common abnormal cleavage patterns (ACP) are in IVF and what are their consequences on embryo developmental competence? Summary answer ACP might affect up to 25% of the 2PN-zygotes, independently from patients’/cycles’ characteristics, and mostly cause embryo developmental arrest around the 4-to–8-cell transition. What is known already Since its implementation in IVF, time-lapse-microscopy (TLM) allowed the standardization of embryo culture within undisturbed incubators, but it has not improved embryo selection especially if blastocyst transfer is performed. Nevertheless, TLM holds the potential for boosting our knowledge of embryo preimplantation development. In particular, a continuous observation of embryo morpho-dynamics unveiled peculiar blastomere cleavage patterns previously unidentifiable with a static morphological assessment. These events are possibly associated with massive mitotic errors, affecting both chromosomes and cytoskeletal components, as well as downstream metabolic imbalances. Still, the causes of ACP and their consequences on embryo developmental/reproductive competence require further investigation. Study design, size, duration Observational study including 75 patients (age:38.6±3.7yr, FSH:8.8±3.6IU/l, AMH:1.7±1.3ng/ml; BMI:21.4±2.4) who conducted multiple IVF cycles (N = 160; 8.7±5.0 cumulus-oocyte-complexes and 6.3±3.6 metaphase-II collected; 201±245 days between first and second cycles) in a time-lapse incubator between 2014–2020. All annotations were performed blindly by two operators and confirmed by a third in case of discordance. The outcomes were the blastulation rate after any ACP, their association between each other and with patients’/cycles’ characteristics. Participants/materials, setting, methods We included only ICSI-cycles after ovarian-stimulation with blastocyst culture conducted in the Embryoscope. Overall, 981 metaphase-II were inseminated and 677 2PN-zygotes annotated. The ACP investigated were: (i)cytokinesis-failure, formation of cytoplasmic septa without cell division; (ii)Chaotic-cleavage, disordered and uneven cleavages; (iii)Direct-unequal-cleavage (DUC), cleavage of zygotes or single blastomeres directly into 3; (iv)Rapid-cleavage, t3-t2<5hr; (v)Reverse-cleavage, fusion of 2 blastomeres into 1; (vi)Fragmentation, presence of numerous non-nucleated fragments; (vii)Blastomeres’ exclusion/extrusion, nucleated cells excluded/extruded from the morula. Main results and the role of chance Among the 2PN-zygotes, the prevalence of cytokinesis-failure was 5.9% (N = 40/677), 15.7% for chaotic-cleavage (N = 106/677), 18.6% for DUC (N = 126/677), 4.1% for rapid-cleavage (N = 28/677), 3.5% for reverse-cleavage (N = 24/677) and 24.1% for fragmentation (N = 163/677). Among the morulae, the prevalence of blastomere exclusion/extrusion was 27% (N = 109/410;1.5±1.2 excluded/extruded cells,range:1–7). The risk for reverse-cleavage was higher among 2PN-zygotes facing failed-cytokinesis (N = 8/40,20% versus N = 16/637,2.5%, OR:9.7,95%CI:3.9–24.3,p<0.01). Fragmentation was instead higher among 2PN-zygotes undergoing chaotic cleavage (N = 47/106,44.3% versus N = 116/571,20.3%, OR:3.1,95%CI:2–4.8,p<0.01) or DUC (N = 46/126,36.5% versus N = 117/551,21.2%, OR:2.1,95%CI:1.4–3.2,p<0.01). Lastly, higher prevalence of blastomeres’ exclusion/extrusion were reported among morulae obtained after chaotic-cleavage (N = 17/29,58.6% versus N = 92/381,24.1%, OR:4.4,95%CI:2–9.7,p<0.01), DUC (N = 26/37,70.3% versus N = 83/373,22.3%, OR:8.3,95%CI:3.9–17.4,p<0.01) and in presence of fragmentation (N = 79/195,75.2% versus N = 30/305,9.8%, OR:27.8,95%CI:15.6–49.8,p<0.01); only a higher trend after rapid-/reverse-cleavage. No predictive factor of ACP was identified among patients’ and cycles’ characteristics, except for higher risks of fragmentation (OR:2.6,95%CI:1.1–6.3,p= 0.04) and blastomeres’ exclusion/extrusion (OR:2.7,95%CI:1.1–7.2,p=0.04) among patients with previous experience with these events. The viable-blastocyst rate per 2PN-zygote was 45.1% (N = 305/677). It was lower in case of failed-cytokinesis (N = 12/40,30% versus N = 293/637,46%, OR:0.5,95%CI:0.25–0.99,p=0.05), chaotic cleavage (N = 20/106,18.9% versus N = 285/571,49.9%, OR:0.23,95%CI:0.14–0.39,p<0.01), DUC (N = 27/126,21.4% versus N = 278/551,50.5%, OR:0.27,95%CI:0.17–0.42,p<0.01), rapid-cleavage (N = 6/22,21.4% versus N = 299/649,46.1%, OR:0.32,95%CI:0.13–0.8,p=0.02), and reverse-cleavage (N = 5/19,20.8% versus N = 300/653,45.9%, OR:0.31, 95%CI:0.11–0.84,p=0.02). No difference was instead shown in case of fragmentation and/or blastomeres’ exclusion/extrusion. Limitations, reasons for caution The patients included were poor-prognosis women undergoing ≥2 cycles. We are expanding the sample size to account for all cycles conducted in time-lapse incubators. Larger sample size will provide also statistical-power to investigate the effect of ACP on blastocysts’ chromosomal and implantation competence, and more visualizations of rapid-/reverse-cleavage events. Wider implications of the findings: After ACP,developmental-arrest mostly occurs around the 4-to–8-cell transition (50–70% versus ∼30%), when embryonic-genome-activation takes place. Surviving embryos often fragment and/or exclude/extrude blastomeres at morulation, without further impact on blastulation-rates. Moreover, ACP seem independent from patients’/cycles’ characteristics. These evidence incite future Research on the biological/genetic mechanisms triggering ACP and their consequences. Trial registration number None

Totipotency of mouse zygotes extends to single blastomeres of embryos at the four-cell stage

In multicellular organisms, oocytes and sperm undergo fusion during fertilization and the resulting zygote gives rise to a new individual. The ability of zygotes to produce a fully formed individual from a single cell when placed in a supportive environment is known as totipotency. Given that totipotent cells are the source of all multicellular organisms, a better understanding of totipotency may have a wide-ranging impact on biology. The precise delineation of totipotent cells in mammals has remained elusive, however, although zygotes and single blastomeres of embryos at the two-cell stage have been thought to be the only totipotent cells in mice. We now show that a single blastomere of two- or four-cell mouse embryos can give rise to a fertile adult when placed in a uterus, even though blastomere isolation disturbs the transcriptome of derived embryos. Single blastomeres isolated from embryos at the eight-cell or morula stages and cultured in vitro manifested pronounced defects in the formation of epiblast and primitive endoderm by the inner cell mass and in the development of blastocysts, respectively. Our results thus indicate that totipotency of mouse zygotes extends to single blastomeres of embryos at the four-cell stage.

The dawn of the future: 30 years from the first biopsy of a human embryo. The detailed history of an ongoing revolution

Following early studies showing no adverse effects, cleavage stage biopsy by zona drilling using acid Tyrode’s solution, and removal of single blastomeres for preimplantation genetic testing (PGT) and identification of sex in couples at risk of X-linked disease, was performed by Handyside and colleagues in late 1989, and pregnancies reported in 1990. This method was later used for specific diagnosis of monogenic conditions, and a few years later also for chromosomal structural and/or numerical impairments, thereby establishing a valuable alternative option to prenatal diagnosis. This revolutionary approach in clinical embryology spread worldwide, and several other embryo biopsy strategies developed over three decades in a process that is still ongoing. The rationale of this narrative review is to outline the different biopsy approaches implemented across the years in the workflow of the IVF clinics that provided PGT: their establishment, the first clinical experiences, their downsides, evolution, improvement and standardization. The history ends with a glimpse of the future: minimally/non-invasive PGT and experimental embryo micromanipulation protocols. This grand theme review outlines a timeline of the evolution of embryo biopsy protocols, whose implementation is increasing worldwide together with the increasing application of PGT techniques in IVF. It represents a vade mecum especially for the past, present and upcoming operators and experts in this field to (re)live this history from its dawn to its most likely future.

Assessing heterogeneity among single embryos and single blastomeres using open microfluidic design

The process by which a zygote develops from a single cell into a multicellular organism is poorly understood. Advances are hindered by detection specificity and sensitivity limitations of single-cell protein tools and by challenges in integrating multimodal data. We introduce an open microfluidic tool expressly designed for same-cell phenotypic, protein, and mRNA profiling. We examine difficult-to-study—yet critically important—murine preimplantation embryo stages. In blastomeres dissociated from less well-studied two-cell embryos, we observe no significant GADD45a protein expression heterogeneity, apparent at the four-cell stage. In oocytes, we detect differences in full-length versus truncated DICER-1 mRNA and protein, which are insignificant by the two-cell stage. Single-embryo analyses reveal intraembryonic heterogeneity, differences between embryos of the same fertilization event and between donors, and reductions in the burden of animal sacrifice. Open microfluidic design integrates with existing workflows and opens new avenues for assessing the cellular-to-molecular heterogeneity inherent to preimplantation embryo development.