Mitotic Errors Research Articles

Mitotic Errors Promote Genomic Instability and Leukemia in a Novel Mouse Model of Fanconi Anemia.

Fanconi anemia (FA) is a disease of genomic instability and cancer. In addition to DNA damage repair, FA pathway proteins are now known to be critical for maintaining faithful chromosome segregation during mitosis. While impaired DNA damage repair has been studied extensively in FA-associated carcinogenesis in vivo, the oncogenic contribution of mitotic abnormalities secondary to FA pathway deficiency remains incompletely understood. To examine the role of mitotic dysregulation in FA pathway deficient malignancies, we genetically exacerbated the baseline mitotic defect in Fancc-/- mice by introducing heterozygosity of the key spindle assembly checkpoint regulator Mad2. Fancc-/-;Mad2+/- mice were viable, but died from acute myeloid leukemia (AML), thus recapitulating the high risk of myeloid malignancies in FA patients better than Fancc-/-mice. We utilized hematopoietic stem cell transplantation to propagate Fancc-/-; Mad2+/- AML in irradiated healthy mice to model FANCC-deficient AMLs arising in the non-FA population. Compared to cells from Fancc-/- mice, those from Fancc-/-;Mad2+/- mice demonstrated an increase in mitotic errors but equivalent DNA cross-linker hypersensitivity, indicating that the cancer phenotype of Fancc-/-;Mad2+/- mice results from error-prone cell division and not exacerbation of the DNA damage repair defect. We found that FANCC enhances targeting of endogenous MAD2 to prometaphase kinetochores, suggesting a mechanism for how FANCC-dependent regulation of the spindle assembly checkpoint prevents chromosome mis-segregation. Whole-exome sequencing revealed similarities between human FA-associated myelodysplastic syndrome (MDS)/AML and the AML that developed in Fancc-/-; Mad2+/- mice. Together, these data illuminate the role of mitotic dysregulation in FA-pathway deficient malignancies in vivo, show how FANCC adjusts the spindle assembly checkpoint rheostat by regulating MAD2 kinetochore targeting in cell cycle-dependent manner, and establish two new mouse models for preclinical studies of AML.

At a Crossroads to Cancer: How p53-Induced Cell Fate Decisions Secure Genome Integrity.

P53 is known as the most critical tumor suppressor and is often referred to as the guardian of our genome. More than 40 years after its discovery, we are still struggling to understand all molecular details on how this transcription factor prevents oncogenesis or how to leverage current knowledge about its function to improve cancer treatment. Multiple cues, including DNA-damage or mitotic errors, can lead to the stabilization and nuclear translocation of p53, initiating the expression of multiple target genes. These transcriptional programs may be cell-type- and stimulus-specific, as is their outcome that ultimately imposes a barrier to cellular transformation. Cell cycle arrest and cell death are two well-studied consequences of p53 activation, but, while being considered critical, they do not fully explain the consequences of p53 loss-of-function phenotypes in cancer. Here, we discuss how mitotic errors alert the p53 network and give an overview of multiple ways that p53 can trigger cell death. We argue that a comparative analysis of different types of p53 responses, elicited by different triggers in a time-resolved manner in well-defined model systems, is critical to understand the cell-type-specific cell fate induced by p53 upon its activation in order to resolve the remaining mystery of its tumor-suppressive function.

Loss of polycomb repressive complex 1 activity and chromosomal instability drive uveal melanoma progression

Chromosomal instability (CIN) and epigenetic alterations have been implicated in tumor progression and metastasis; yet how these two hallmarks of cancer are related remains poorly understood. By integrating genetic, epigenetic, and functional analyses at the single cell level, we show that progression of uveal melanoma (UM), the most common intraocular primary cancer in adults, is driven by loss of Polycomb Repressive Complex 1 (PRC1) in a subpopulation of tumor cells. This leads to transcriptional de-repression of PRC1-target genes and mitotic chromosome segregation errors. Ensuing CIN leads to the formation of rupture-prone micronuclei, exposing genomic double-stranded DNA (dsDNA) to the cytosol. This provokes tumor cell-intrinsic inflammatory signaling, mediated by aberrant activation of the cGAS-STING pathway. PRC1 inhibition promotes nuclear enlargement, induces a transcriptional response that is associated with significantly worse patient survival and clinical outcomes, and enhances migration that is rescued upon pharmacologic inhibition of CIN or STING. Thus, deregulation of PRC1 can promote tumor progression by inducing CIN and represents an opportunity for early therapeutic intervention.

ACCURATE GENOMIC PREDICTION OF MOSAICISM THROUGH CELL DIVISION ORIGIN OF ANEUPLOIDY ANALYSIS IN THE PREIMPLANTATION EMBRYO

Simple intermediate copy number thresholds have proven to provide poor predictive value for diagnosing preimplantation mosaicism (both analytically and clinically). This study aims to validate a method to determine the cell division origin of aneuploidy from a single trophectoderm biopsy, and its ability to accurately predict mosaicism in the remaining embryo. A high throughput and PGT-validated DNA array platform was used to analyze over 800,000 SNPs within trophectoderm biopsies from a subset of PGT cases where parental DNA was available. Mendelian inheritance analyses were performed on embryonic genotypes within aneuploid chromosomes to predict their parental and cell division origins. Positive controls included embryos with a male karyotype (n=279) with known maternal origin of the X chromosome, and unbalanced embryos from PGT-SR cases (n=69) with known meiotic and parental origins of aneuploidy. The method was applied to clinical biopsies with aneuploidy and compared to rebiopsies (n=80) to evaluate the accuracy of predicting mosaicism (mitotic) and uniform (meiotic) aneuploidy. All samples were analyzed while blinded to prior results. Following validation, the incidence of mitotic origin of aneuploidy was investigated in 942 embryos. Blinded predictions of the X chromosome origin in male embryos, and parental and cell division origin of aneuploidy in PGT-SR derived unbalanced embryos were 100% concordant with expected results, demonstrating the validity of predicting parental and meiotic cell division origins of aneuploidy. Blinded analysis of rebiopsies showed that all embryos with mitotic origin of aneuploidy predicted in the original biopsy were confirmed as mosaic, and all embryos with meiotic origin were confirmed as uniform aneuploid, demonstrating the validity of predicting mitotic origins of aneuploidy and mosaicism. The overall incidence of mitotic whole chromosome aneuploidy (mosaicism) in embryos was 3%. The overall embryonic aneuploidy rate was 31% (mean maternal age 34.5+4). Aneuploid chromosomes primarily originated from the maternal genome (80%) and maternal whole chromosome aneuploidy primarily originated from meiotic errors (95%), while paternal aneuploidy more often originated from mitotic errors (35%)(p<0.05). These data indicate very high specificity in predicting mosaicism from a single aneuploid trophectoderm biopsy and a low prevalence of mosaicism (3%) in the human blastocyst. This is in stark contrast to existing methods of predicting mosaicism with intermediate copy number thresholds, where rebiopsy confirmation rates are ∼40%, and prevalence is estimated to be ∼20%. Improving the specificity of predicting mosaicism (reducing false positives) from a single biopsy may provide a unique opportunity to investigate the true clinical consequence of transferring bona fide mosaic embryos for the first time.

RELATIONSHIP BETWEEN LENGTH OF GONADOTROPIN STIMULATION FOR IN VITRO FERTILIZATION AND EMBRYONIC ANEUPLOIDY OF MATERNAL ORIGIN

There have been conflicting reports on the relationship between gonadotropin stimulation length and embryonic aneuploidy. We hypothesized that a prolonged stimulation could lead to an increase in meiotic and mitotic oocyte errors. This study aims to evaluate the relationship between length of gonadotropin stimulation for in vitro fertilization (IVF) and embryonic aneuploidy of maternal origin.

DOES THE MOSAIC EMBRYO RATIO OF PGT-A CYCLES IMPACT THE LIVE BIRTH CHANCE EVEN WHEN AN EUPLOID EMBRYO IS TRANSFERRED?

Preimplantation genetic testing for aneuploidy (PGT-A) has the potential to detect mosaicism in trophectoderm biopsies with Next-Generation Sequencing (NGS). Many couples who opted for PGT-A have both euploid and mosaic embryos in their cohort of tested embryos. However, little is known about how live birth outcomes are affected by mosaic embryo ratios of PGT-A tested embryos even though euploid embryos are transferred. This retrospective study was based on 636 PGT-A tested frozen-thawed single euploid embryo transfer cycles of which live birth outcome is known. The inclusion criteria were having at least one euploid and one mosaic embryos. Mosaicism was reported for PGT-A outcomes deviated for 20-80% from 2 chromosome copy number. Mosaic embryo ratio was defined as the number of mosaic embryos divided by the total number of blastocysts tested. This data included embryo transfers between January 2017 and March 2020 in a single ART clinic. PGT-A was done by NGS ReproSeq on IonTorrent S5 (Thermo Fisher) following trophectoderm biopsy. For the categorical data Chi-Square test was used. For continuous variables, Mann-Whitney Test was applied. Pearson correlation coefficient was used for testing how two variables are related to each other. The live birth rate of the study group was 55.8%. When data was evaluated according to the live birth status (+(n=355) or -(n=281)), no significant differences were observed for the number of oocytes collected, mature oocytes, blastocysts, biopsied blastocysts, number of euploid and mosaic embryos per cycle. Female age at the time of transfer and mosaic embryo ratio were statistically significant (p=0.015 and p=0.040, respectively). Female age for women who had reached live-birth was 0.9 years less and mosaic embryo ratio was 2.3% less when compared to women who did not reach live-birth. As only female age and mosaic embryo ratio were found to be statistically significant between the groups, their relationship with each other was tested and found to be inversely correlated, eg. with increasing female age, the mosaic embryo ratio was decreasing (r= -0.07, -0.14 to -0.01 95% CI, p=0.021). The median value for mosaic embryo ratio was 28.6%. When the study group was divided into two with 28.6% as a cut-off, the live birth rate was 10.1% higher for cycles with lower mosaic embryo ratio (62.4% vs 52.3%) (p=0.160). The live-birth rate for euploid FET with at least 1 mosaic embryo was not statistically different when compared to 1193 euploid FET of PGT-A cycles with no mosaic embryo (55.8% vs 55.3%, respectively) (p=0.839). Diverse molecular mechanisms including altered cell cycle checkpoints, aberrations of the centrosome and failed chromatid cohesion are known to contribute to mitotic errors (McCoy, 2017). Although euploid, some embryos from the same PGT-A tested cohort may be affected by the molecular pathways contributing to embryonic mosaicism.

Gene copy-number changes and chromosomal instability induced by aneuploidy confer resistance to chemotherapy.

Mitotic errors lead to aneuploidy, a condition of karyotype imbalance, frequently found in cancer cells. Alterations in chromosome copy number induce a wide variety of cellular stresses, including genome instability. Here, we show that cancer cells might exploit aneuploidy-induced genome instability and the resulting gene copy-number changes to survive under conditions of selective pressure, such as chemotherapy. Resistance to chemotherapeutic drugs was dictated by the acquisition of recurrent karyotypes, indicating that gene dosage might play a role in driving chemoresistance. Thus, our study establishes a causal link between aneuploidy-driven changes in gene copy number and chemoresistance and might explain why some chemotherapies fail to succeed.

PREVALENCE OF SPECIFIC CHROMOSOME-LEVEL ANOMALIES IN MOSAIC AND ANEUPLOID EMBRYOS

We have previously demonstrated that the frequency of individual chromosomal copy number variations identified in human embryos cultured in vitro mirrors those found following early pregnancy loss.1 It is unclear whether the same chromosomal abnormalities are identified in mosaic embryos. The aim of this study is to determine the frequency of individual chromosomal anomalies in aneuploid versus mosaic embryos. This study included patients who underwent IVF with preimplantation genetic testing for aneuploidy (PGT-A) at a single academic center from January 2020 through March 2021. Trophectoderm biopsies were analyzed using a modified FAST-SeqS NGS-based PGT method and bioinformatics pipeline, which detects whole chromosome and segmental aneuploidies (≥10Mb), most types of polyploidy, and many instances of single chromosome uniparental isodisomy through amplification of L1 sites. The reported chromosome complement for each embryo was analyzed. The prevalence of abnormalities for each chromosome among aneuploid and mosaic embryos was determined and compared using chi-square. Bonferroni correction was used to adjust for multiple comparisons with a corrected p value of .002. A total of 7,872 embryos from 1,371 patients were included in the study with a mean age of 35.9 years, including 2,763 aneuploid embryos (35.1%) and 1,035 mosaic embryos (13.1%). The chromosomes with the greatest prevalence of abnormalities among aneuploid embryos were 21 (20.6%) and 16 (17.4%) followed by 21 (12.5%) and 15 (11.0%). The lowest prevalences were seen in chromosomes 17 (2.9%) and 12 (2.8%). In mosaic embryos, in contrast, the variation in prevalence of anomalies per chromosome was lower, ranging from 2.4% in both chromosomes 19 and 20 to 7.1% in chromosome 6 and 8.2% in chromosome 2. When comparing the prevalence of abnormalities for each chromosome among aneuploid and mosaic embryos, significant differences were seen in chromosomes 1 (3.7% vs. 6.0%, p=.0015), 2 (4.3% vs. 8.2%, p=<.0001), 5 (3.9% vs. 6.6%, p=.0004), 6 (3.9% vs. 7.1%, p=<.0001), 15 (11.0% vs. 3.4%, p<.0001 ), 16 (17.4% vs. 3.9%, p=<.0001), 19 (6.0% vs. 2.4%, p<.0001), 20 (5.2% vs. 2.4%, p=.0002), 21 (12.5% vs. 3.6%, p<.0001), and 22 (20.6% vs. 5.7%, p<.0001). Specific chromosomes appear to have greater susceptibility to meiotic versus mitotic errors in the process of oocyte maturation and embryo development. These findings suggest that the differential mechanisms governing chromosome segregation during meiosis versus mitosis result in distinct differences in per chromosome copy number reports from embryo biopsies.

Augmin deficiency in neural stem cells causes p53-dependent apoptosis and aborts brain development

Microtubules that assemble the mitotic spindle are generated by centrosomal nucleation, chromatin-mediated nucleation, and nucleation from the surface of other microtubules mediated by the augmin complex. Impairment of centrosomal nucleation in apical progenitors of the developing mouse brain induces p53-dependent apoptosis and causes non-lethal microcephaly. Whether disruption of non-centrosomal nucleation has similar effects is unclear. Here, we show, using mouse embryos, that conditional knockout of the augmin subunit Haus6 in apical progenitors led to spindle defects and mitotic delay. This triggered massive apoptosis and abortion of brain development. Co-deletion of Trp53 rescued cell death, but surviving progenitors failed to organize a pseudostratified epithelium, and brain development still failed. This could be explained by exacerbated mitotic errors and resulting chromosomal defects including increased DNA damage. Thus, in contrast to centrosomes, augmin is crucial for apical progenitor mitosis, and, even in the absence of p53, for progression of brain development.

Beta-Genus Human Papillomavirus 8 E6 Destabilizes the Host Genome by Promoting p300 Degradation.

The beta genus of human papillomaviruses infects cutaneous keratinocytes. Their replication depends on actively proliferating cells and, thus, they conflict with the cellular response to the DNA damage frequently encountered by these cells. This review focus on one of these viruses (HPV8) that counters the cellular response to damaged DNA and mitotic errors by expressing a protein (HPV8 E6) that destabilizes a histone acetyltransferase, p300. The loss of p300 results in broad dysregulation of cell signaling that decreases genome stability. In addition to discussing phenotypes caused by p300 destabilization, the review contains a discussion of the extent to which E6 from other β-HPVs destabilizes p300, and provides a discussion on dissecting HPV8 E6 biology using mutants.

Processes shaping cancer genomes – From mitotic defects to chromosomal rearrangements

Sequencing of cancer genomes revealed a rich landscape of somatic single nucleotide variants, structural changes of chromosomes, as well as chromosomal copy number alterations. These chromosome changes are highly variable, and simple translocations, deletions or duplications have been identified, as well as complex events that likely arise through activity of several interconnected processes. Comparison of the cancer genome sequencing data with our knowledge about processes important for maintenance of genome stability, namely DNA replication, repair and chromosome segregation, provides insights into the mechanisms that may give rise to complex chromosomal patterns, such as chromothripsis, a complex form of multiple focal chromosome rearrangements. In addition, observations gained from model systems that recapitulate the rearrangements patterns under defined experimental conditions suggest that mitotic errors and defective DNA replication and repair contribute to their formation. Here, we review the molecular mechanisms that contribute to formation of chromosomal aberrations observed in cancer genomes.

P–526 Incidence of mosaic embryos in day–6 blastocysts, may late blastulation predispose to mosaicism?

Abstract Study question May the mosaicism ratio be influenced by the time of blastulation in preimplantation genetic testing for aneuploidies (PGT-A)? Summary answer The mosaicism ratio is significantly higher in day–6 blastocysts when compared to day–5 when transferable embryos are considered only (euploids and mosaics). What is known already Conventionally, PGT-A has classified preimplantation embryos as either euploid or aneuploid. Yet, a major improvement in PGT-A methodology, with the introduction of high sensitivity diagnostic Next Generation Sequencing (NGS) technology, has allowed the identification of a third embryo category: the mosaic (Coll et al., 2021). Embryonic mosaicism can be defined as the presence of karyotypically distinct cell lines within an embryo and can be detected by NGS at a rate between 20–80%. In the absence of euploid embryos, mosaic embryos, when transferred, have been shown to deliver healthy live births (PGDIS, 2019). Study design, size, duration This retrospective study was based on 9828 trophectoderm biopsies performed in a single ART clinic between January 2017 and October 2020 for PGT-A cycles with more than one blastocyst. PGT-A cycles with only one blastocyst were excluded because in these cycles’ day–5/day–6 biopsy percentage cannot be calculated. A total number of 8398 and 1430 blastocysts were biopsied on day–5 and day–6, respectively for PGT-A by ReproSeq on Ion Torrent S5 (Thermo Fisher Scientific). Participants/materials, setting, methods Three categories were defined in the PGT-A group with &amp;gt;1 blastocyst biopsied to compare the rate of mosaicism: C1:cycles in which blastocysts were only biopsied on day–5 (n = 1872), C2:99–60% of blastocysts were biopsied on day–5 (n = 483) and C3:0–60% of blastocysts biopsied on day–5 (n = 411). The mean female age (C1:36.0±5.2; C2:35.7±4.8; C3:37.1±4.9) and metaphase II oocytes punctured (C1:9.8±6.5; C2:10.4±5.2; C3:8.4±5.4) were similar and statistically non-significant in all groups. T-test and Chi-square tests were used where appropriate. Main results and the role of chance Overall, from the blastocysts biopsied on day–5 and 6, 35.4% and 25.5% were euploid, 13.7% and 14.7% were mosaic, 50.9% and 59.8% were aneuploid, respectively (p &amp;lt; 0.0001, p = 0.32, p &amp;lt; 0.0001), the mosaicism rate being not statistically different. However, when only transferable blastocysts (euploids and mosaics) were considered (aneuploids being discarded), the rate of mosaic embryos was significantly higher in day–6 when compared to day–5 blastocysts (36.6% vs. 28.0%; p &amp;lt; 0.0001). Morphological blastocyst grading was then investigated: from the day–5 and 6 blastocysts biopsied, 51.5% and 30.8% were of top-quality and 48.5% and 69.2% were of good-quality, respectively. Looking deeper into the categories defined, the euploidy rate was found to be higher in C1 (35.1%) when compared to C2 (34.9%) and C3 (27.7%) (p &amp;lt; 0.0001). The mosaic embryo rate was found to be non-significant when all blastocysts (euploids, aneuploids, mosaics) were considered (C1: 13.7%; C2: 14.1%; C3: 14.2%; p = 0.6492). However, when transferable blastocysts were considered (euploids and mosaics only), the mosaic embryo rate was significantly higher in C3 (33.9%) when compared to C1 (28.1%) and C2 (28.8%) (p = 0.02). For morphological blastocyst grading, regardless of blastulation day, mosaicism was higher for good-quality embryos both when all biopsies and only transferrable embryos were considered (p = 0.0018, p &amp;lt; 0.0001, respectively). Limitations, reasons for caution This study focused on blastocyst formation day and morphological blastocyst grading. Extrinsic factors have also been reported to induce mosaicism: ovarian stimulation, culture media, laboratory and culture conditions, technical issues during the biopsy and sample processing (Munné and Wells, 2017; Katz-Jaffe et al., 2018, Fragouli et al., 2010, 2019). Wider implications of the findings: Mitotic errors during cleavage stage causing mosaicism may lead to lower morphological grade and late blastulation as some cells in those embryos are not diploid thus leading to higher mosaicism for blastocysts that reach blastulation on day6 and/or yield only good-quality embryos. Trial registration number Not applicable

P–544 The results of embryo transfer cycles with low mosaic embryos: A case report

Abstract Study question What are the results of embryo transfer cycles with low mosaic embryos? Summary answer The clinical and ongoing pregnancy rates were 50%. The results of Amniocentesis were reported as normal karyotype for all the pregnant women. What is known already Many current studies, quote the rates of mosaicism in blastocyst biopsies to be higher at 20–30%. Embryonic mosaicism was found to result from mitotic errors occurring after fertilization, occasionally in the first cleavage but more commonly in the second or third cleavage. The increased reporting of mosaicism in embryos has given rise to new challenges in PGS results interpretation and patient counseling. Previously, embryos were diagnosed as either euploid or aneuploid, and in most cases only euploid embryos were considered for transfer. Now, if mosaic embryos are considered to represent a third category of results. Study design, size, duration Case Report for The results of 10 embryo Transfer Cycles with Low Mosaic Embryos Participants/materials, setting, methods: We are presenting the results of ten embryo transfer cycles with low mosaic embryos at Bahceci BIH IVF Center, Sarajevo BIH, between January 2019 – October 2020. All the patients have been informed by a written informed consent form, and had genetic counseling before embryo transfer. Amniocentesis has been performed for all the pregnant women between 14–18 weeks of the pregnancy as prenatal testing. Main results and the role of chance Out of the total ten patients, 5 had pregnancy, and the 5 other were not pregnant. The average age of the whole group was; 34.0 ± 5.49 years. The average age of the pregnant women was ; 30.6 ± 5.54 years, and 37.40 ± 2.88 years who were not pregnant Limitations, reasons for caution There are limited number of papers in literature about transferring of the low mosaic embryos. The strength of our presentation is, amniocentesis have been performed as prenatal testing for all the pregnancies. Wider implications of the findings: There were no correlation between the type of low mosaicism and pregnancy. Since all the pregnant women were 35 years of age or less, It was the only factor which is influencing the pregnancy.We hope that, the results of our cases will improve the management protocols for these cases. Trial registration number Not applicable

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

Abstract 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&amp;lt;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&amp;lt;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&amp;lt;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&amp;lt;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&amp;lt;0.01), DUC (N = 26/37,70.3% versus N = 83/373,22.3%, OR:8.3,95%CI:3.9–17.4,p&amp;lt;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&amp;lt;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&amp;lt;0.01), DUC (N = 27/126,21.4% versus N = 278/551,50.5%, OR:0.27,95%CI:0.17–0.42,p&amp;lt;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

O-202 Usage of laser during trophectoderm biopsy and its affect on PGT-A results and moscaism

Abstract Study question Does laser use during trophectoderm biopsy affect biopsy results on prehatching embryos with regard to mosaicism ? Summary answer According to our findings laser usage during trophectoderm biopsy increases mosaic results on next generation sequencing (NGS) irrespective of embryo quality. What is known already Chromosomal mosaicism, which is a result of mitotic errors after fertilization, is defined as the presence of karyotypically distinct cell lines within an embryo. The introduction of NGS made it possible to detect chromosomal mosaicism at levels as low as 20%. The incidence of mosaicism is highly variable between clinics which reported the incidences between 4-32%. Apart from the biological reasons, there are also various technical factors that may impact the incidence of mosaicism. One of the most emphasized factors is the trophoectoderm biopsy technique. Laser usage and number of laser pulses may cause excessive heat during the procedure Study design, size, duration The mosaicism ratio in embryos in which trophectoderm biopsy was performed with or without laser, between January 2017 December 2020 in Istanbul Memorial Hospital (IMH) were examined retrospectively. A total of 13002 embryos were analyzed. A subgroup analysis was also performed regarding mosaicism ratios in different embryo qualities. Blastocysts were classified according to Gardner’s classification and classified as follows: top quality-TQ (4AA,5AA,6AA), good quality-GQ (3AA, 4,5,6AB,BA) and moderate quality-MQ (3,4,5 BB). Participants/materials, setting, methods The biopsy samples of the cases who had PGT-A in IMH between 2017-2020 were evaluated by NGS method. This method enables the identification of embryos with 20% to 80% mosaicism. The study assessed whether there was an increase in the embryos with mosaic results due to the use of laser during biopsy. The effects of laser use among the TQ (4AA,5AA,6AA), GQ (3AA, 4,5,6AB,BA) and MQ (3,4,5 BB) groups according to Garder classification were analyzed. Main results and the role of chance Trophectoderm biopsy was applied on 13002 embryos within the specified period. During biopsy in 5088 embryos laser was used and in 7843 embryos laser was not used, and biopsy was performed mechanically (flicking method). After observing the biopsy results, 945/5088 (18.5%) of the laser applied embryos; and 1087/7914 (13.7%) of laser not applied embryos were defined as mosaic(p &amp;lt; 0.0001). When mosaicism rates were examined according to embryo qualities, the rate of mosaicism was 19.3%(469/2430), 18.2%(290/1591) and 13.2%( 380/2875), 13.5 (426/3141) respectively in embryos with and without laser in TQ and GQ groups. A statistically high level of significance (p &amp;lt; 0.0001) was observed between the embryos evaluated as top quality and good quality before biopsy. Regarding the evaluation in the moderate group embryos, although the mosaicism rates tended to increase on the laser applied group side 40/248(16.1%), no statistical difference was observed when compared to non-laser group 103/670(15,4%). (P &amp;gt; 0.05) Limitations, reasons for caution The retrospective nature of the data is the main limitation of the study. On the other hand, the large number of NGS based PGT-A tested TQ and GQ embryos from a single center and resuts from single laboratory. However, further studies are required to corroborate our findings. Wider implications of the findings Laser dependent heat effect may increase mosaicism. To reduce the cell damage, teasing of cells should be avoided and a minimum number of laser pulses should be used in order to avoid excessive heat and contact points should be preferably confined to cell junctions Trial registration number None

Identification of abscission checkpoint bodies as structures that regulate ESCRT factors to control abscission timing.

The abscission checkpoint regulates the ESCRT membrane fission machinery and thereby delays cytokinetic abscission to protect genomic integrity in response to residual mitotic errors. The checkpoint is maintained by Aurora B kinase, which phosphorylates multiple targets, including CHMP4C, a regulatory ESCRT-III subunit necessary for this checkpoint. We now report the discovery that cytoplasmic abscission checkpoint bodies (ACBs) containing phospho-Aurora B and tri-phospho-CHMP4C develop during an active checkpoint. ACBs are derived from mitotic interchromatin granules, transient mitotic structures whose components are housed in splicing-related nuclear speckles during interphase. ACB formation requires CHMP4C, and the ESCRT factor ALIX also contributes. ACB formation is conserved across cell types and under multiple circumstances that activate the checkpoint. Finally, ACBs retain a population of ALIX, and their presence correlates with delayed abscission and delayed recruitment of ALIX to the midbody where it would normally promote abscission. Thus, a cytoplasmic mechanism helps regulate midbody machinery to delay abscission.

Suv420 enrichment at the centromere limits Aurora B localization and function.

Centromere structure and function are defined by the epigenetic modification of histones at centromeric and pericentromeric chromatin. The constitutive heterochromatin found at pericentromeric regions is highly enriched for H3K9me3 and H4K20me3. Although mis-expression of the methyltransferase enzymes that regulate these marks, Suv39 and Suv420, is common in disease, the consequences of such changes are not well understood. Our data show that increased centromere localization of Suv39 and Suv420 suppresses centromere transcription and compromises localization of the mitotic kinase Aurora B, decreasing microtubule dynamics and compromising chromosome alignment and segregation. We find that inhibition of Suv420 methyltransferase activity partially restores Aurora B localization to centromeres and that restoration of the Aurora B-containing chromosomal passenger complex to the centromere is sufficient to suppress mitotic errors that result when Suv420 and H4K20me3 is enriched at centromeres. Consistent with a role for Suv39 and Suv420 in negatively regulating Aurora B, high expression of these enzymes corresponds with increased sensitivity to Aurora kinase inhibition in human cancer cells, suggesting that increased H3K9 and H4K20 methylation may be an underappreciated source of chromosome mis-segregation in cancer. This article has an associated First Person interview with the first author of the paper.

Human adipose-derived mesenchymal stromal cells from face and abdomen undergo replicative senescence and loss of genetic integrity after long-term culture

Body fat depots are heterogeneous concerning their embryonic origin, structure, exposure to environmental stressors, and availability. Thus, investigating adipose-derived mesenchymal stromal cells (ASCs) from different sources is essential to standardization for future therapies. In vitro amplification is also critical because it may predispose cell senescence and mutations, reducing regenerative properties and safety. Here, we evaluated long-term culture of human facial ASCs (fASCs) and abdominal ASCs (aASCs) and showed that both met the criteria for MSCs characterization but presented differences in their immunophenotypic profile, and differentiation and clonogenic potentials. The abdominal tissue yielded more ASCs, and these had higher proliferative potential, but facial cells displayed fewer mitotic errors at higher passages. However, both cell types reduced clonal efficiency over time and entered replicative senescence around P12, as evaluated by progressive morphological alterations, reduced proliferative capacity, and SA-β-galactosidase expression. Loss of genetic integrity was detected by a higher proportion of cells showing nuclear alterations and γ-H2AX expression. Our findings indicate that the source of ASCs can substantially influence their phenotype and therefore should be carefully considered in future cell therapies, avoiding, however, long-term culture to ensure genetic stability.

Plasticity of the human preimplantation embryo: developmental dogmas, variations on themes and self-correction.

IVF for the treatment of infertility offers unique opportunities to observe human preimplantation development. Progress in time-lapse technology (TLT) and preimplantation genetic testing (PGT) has greatly expanded our knowledge of developmental patterns leading to a healthy pregnancy or developmental failure. These technologies have also revealed unsuspected plastic properties of the preimplantation embryo, at macromolecular, cellular and multicellular levels. This review focuses on the emerging concept of plasticity of the human embryo as revealed by recent evidence derived from TLT and PGT, calling for an updated and more precise redefinition of the boundaries between normal and abnormal development. PubMed was used to search the MEDLINE database for peer-reviewed English-language original articles and reviews concerning human preimplantation development. Cross-searches were performed by adopting 'fertilisation', 'pronucleus', 'cleavage', 'multinucleation', 'compaction', 'embryo', 'preimplantation genetic testing', 'aneuploidy', mosaicism', 'micromanipulation', 'time-lapse microscopy' and 'IVF/assisted reproduction' as main terms. The most relevant publications, i.e. those concerning major phenomena occurring during normal and abnormal development-with a focus on the human species-were assessed and discussed critically. Advances in TLT and PGT have revealed an astonishing plasticity and self-correction ability of the human preimplantation embryo in vitro. At fertilisation, an abnormal number of pronuclei do not always result in the formation of an aneuploid blastocyst. Animal studies and preliminary human observations indicate that combining of parental genomes may occur at the early cleavage stage, if not at fertilisation. Multinucleation occurs with much higher prevalence than previously thought and may be corrected at later cleavage stages. Irregular cleavage (multichotomous, direct, rapid and reverse cleavages) can generate chromosome segregation abnormalities that often lead to developmental arrest, but that sporadically may be confined to cells excluded from the blastocyst, and may sometimes result in viable pregnancy. Mitotic errors can generate mosaic blastocysts, but alternatively normal embryos may form from selective death or clonal depletion of aneuploid cells. Deviations from developmental dogmas and the increasing evidence of plasticity of the human embryo challenge current embryological notions and suggest the need to write new rules governing cell cycle, cell determination and chromosome segregation during preimplantation development.

Identifying New Cofactors for Cytokinetic Abscission

A single cell division in the presence of incompletely segregated chromatin can result in chromosome beaks, triggering a chain of events that culminate in chromosomal damage and rapid genome evolution, which are classic hallmarks of cancer. In order to maintain genomic integrity, the cell has evolved a cell cycle checkpoint known as the abscission checkpoint. This checkpoint detects persistent mitotic errors such as incompletely segregated chromosomes, misformed nuclear pores, under-replicated DNA and tension at the intercellular bridge. Once detected, these errors trigger an abscission arrest to allow cells a chance to correct and prevent their transmission to daughter cells. How the cell integrates these diverse signals into a concerted response to delay abscission is not well understood. However, it is well-known that progression through abscission is regulated in part through the sequential action of several key kinases including Aurora B, that inhibit the membrane fission activity of ESCRT-III proteins, which localize to the intercellular bridge connecting daughter cells and are required for completing cell division. In addition to providing the mechanical force necessary for membrane constriction and fission, ESCRT-III proteins recruit essential cofactors via a complex set of interactions between their C-terminal MIT-Interacting Motifs (MIMs) and proteins that contain Microtubule Interacting and Trafficking (MIT) domains. In order to discover new activities required to correctly perform abscission, we mapped the complete network of ESCRT-III binding partners for ~20 human MIT domain-containing proteins. We then focused on three of these MIT proteins: a protease, Calpain-7, and two microtubule severing AAA-ATPases, Katanin, and Spastin. For each protein, we mapped the binding site for their respective ESCRT-III binding partners and tested whether they function in abscission or the abscission checkpoint. Our studies reveal Spastin and Calpain-7 are required for maintaining the abscission checkpoint, with Calpain-7 having an additional role in abscission. These studies biochemically describe how ESCRT-III proteins can recruit multiple different proteins to the intercellular bridge and highlight the importance of these interactions in supplying additional enzymatic activities that promote faithful abscission.