Molecular contribution to embryonic aneuploidy and karyotypic complexity in initial cleavage divisions of mammalian development.

Embryonic aneuploidy is highly complex, often leading to developmental arrest, implantation failure or spontaneous miscarriage in both natural and assisted reproduction. Despite our knowledge of mitotic mis-segregation in somatic cells, the molecular pathways regulating chromosome fidelity during the error-prone cleavage-stage of mammalian embryogenesis remain largely undefined. Using bovine embryos and live-cell fluorescent imaging, we observed frequent micro-/multi-nucleation of mis-segregated chromosomes in initial mitotic divisions that underwent unilateral inheritance, re-fused with the primary nucleus or formed a chromatin bridge with neighboring cells. A correlation between a lack of syngamy, multipolar divisions and asymmetric genome partitioning was also revealed, and single-cell DNA-seq showed propagation of primarily non-reciprocal mitotic errors. Depletion of the mitotic checkpoint protein BUB1B (also known as BUBR1) resulted in similarly abnormal nuclear structures and cell divisions, as well as chaotic aneuploidy and dysregulation of the kinase-substrate network that mediates mitotic progression, all before zygotic genome activation. This demonstrates that embryonic micronuclei sustain multiple fates, provides an explanation for blastomeres with uniparental origins, and substantiates defective checkpoints and likely other maternally derived factors as major contributors to the karyotypic complexity afflicting mammalian preimplantation development.

Assessing equine embryo developmental competency by time-lapse image analysis.

The timing of early mitotic events during preimplantation embryo development is important for subsequent embryogenesis in many mammalian species, including mouse and human, but, to date, no study has closely examined mitotic timing in equine embryos from oocytes obtained by ovum pick-up. Here, cumulus-oocyte complexes were collected by transvaginal follicular aspiration, matured invitro and fertilised via intracytoplasmic sperm injection. Each fertilised oocyte was cultured up to the blastocyst stage and monitored by time-lapse imaging for the measurement of cell cycle intervals and identification of morphological criteria indicative of developmental potential. Of the 56 fertilised oocytes, 35 initiated mitosis and 11 progressed to the blastocyst stage. Analysis of the first three mitotic divisions in embryos that formed blastocysts determined that typical blastocyst timing (median±IQR) is 30.0±17.5min, 8.8±1.7h and 0.6±1.4h respectively. Frequent cellular fragmentation, multipolar divisions and blastomere exclusion suggested that equine embryos likely contend with a high incidence of chromosomal missegregation. Indeed, chromosome-containing micronuclei and multinuclei with extensive DNA damage were observed throughout preimplantation embryogenesis. This indicates that time-lapse image analysis may be used as a non-invasive method to assess equine embryo quality in future studies.

Chronically elevated androgen and/or consumption of a Western-style diet impairs oocyte quality and granulosa cell function in the nonhuman primate periovulatory follicle.

PURPOSE: To investigate the impact of chronically elevated androgens in the presence and absence of an obesogenic diet on oocyte quality in the naturally selected primate periovulatory follicle. METHODS: Rhesus macaques were treated using a 2-by-2 factorial design (n = 10/treatment) near the onset of menarche with implants containing either cholesterol (C) or testosterone (T, 4-5-fold increase above C) and a standard or "Western-style" diet alone (WSD) or in combination (T+WSD). Following ~ 3.5 years of treatment, females underwent controlled ovulation (COv, n = 7-10/treatment) cycles, and contents of the naturally selected periovulatory follicle were aspirated. Follicular fluid (FF) was analyzed for cytokines, chemokines, growth factors, and steroids. RNA was extracted from luteinizing granulosa cells (LGCs) and assessed by RNA-seq. RESULTS: Only healthy, metaphase (M) I/II-stage oocytes (100%) were retrieved in the C group, whereas several degenerated oocytes were recovered in other groups (33-43% of T, WSD, and T+WSD samples). Levels of two chemokines and one growth factor were reduced (p < 0.04) in FF of follicles with a MI/MII oocyte in WSD+T (CCL11) or T and WSD+T groups (CCL2 and FGF2) compared to C and/or WSD. Intrafollicular cortisol was elevated in T compared to C follicles (p < 0.02). Changes in the expression pattern of 640+ gene products were detected in LGC samples from follicles with degenerated versus MI/MII-stage oocytes. Pathway analysis on RNAs altered by T and/or WSD found enrichment of genes mapping to steroidogenic and immune cell pathways. CONCLUSIONS: Female primates experiencing hyperandrogenemia and/or consuming a WSD exhibit an altered intrafollicular microenvironment and reduced oocyte quality/competency, despite displaying menstrual cyclicity.

Single-cell sequencing of primate preimplantation embryos reveals chromosome elimination via cellular fragmentation and blastomere exclusion.

Aneuploidy that arises during meiosis and/or mitosis is a major contributor to early embryo loss. We previously showed that human preimplantation embryos encapsulate missegregated chromosomes into micronuclei while undergoing cellular fragmentation and that fragments can contain chromosomal material, but the source of this DNA was unknown. Here, we leveraged the use of a nonhuman primate model and single-cell DNA-sequencing (scDNA-seq) to examine the chromosomal content of 471 individual samples comprising 254 blastomeres, 42 polar bodies, and 175 cellular fragments from a large number (N = 50) of disassembled rhesus cleavage-stage embryos. Our analysis revealed that the aneuploidy and micronucleation frequency is conserved between humans and macaques, and that fragments encapsulate whole and/or partial chromosomes lost from blastomeres. Single-cell/fragment genotyping showed that these chromosome-containing cellular fragments (CCFs) can be maternally or paternally derived and display double-stranded DNA breaks. DNA breakage was further indicated by reciprocal subchromosomal losses/gains between blastomeres and large segmental errors primarily detected at the terminal ends of chromosomes. By combining time-lapse imaging with scDNA-seq, we determined that multipolar divisions at the zygote or two-cell stage were associated with CCFs and generated a random mixture of chromosomally normal and abnormal blastomeres with uniparental or biparental origins. Despite frequent chromosome missegregation at the cleavage-stage, we show that CCFs and nondividing aneuploid blastomeres showing extensive DNA damage are prevented from incorporation into blastocysts. These findings suggest that embryos respond to chromosomal errors by encapsulation into micronuclei, elimination via cellular fragmentation, and selection against highly aneuploid blastomeres to overcome chromosome instability during preimplantation development.

Time-Lapse Imaging for the Detection of Chromosomal Abnormalities in Primate Preimplantation Embryos.

The use of time-lapse microscopic imaging has proven to be a powerful tool for the study of mitotic divisions and other cellular processes across diverse species and cell types. Although time-lapse monitoring (TLM) of human preimplantation development was first introduced to the in vitro fertilization (IVF) community several decades ago, it was not until relatively recently that TLM systems were commercialized for clinical embryology purposes. Traditionally, human IVF embryos are assessed by successful progression and morphology under a stereomicroscope at distinct time points prior to selection for transfer. Due to the high frequency of aneuploidy, embryos may also be biopsied at the cleavage or blastocyst stage for preimplantation genetic screening (PGS) of whole and/or partial chromosomal abnormalities. However, embryo biopsy is invasive and can hinder subsequent development, and there are additional concerns over chromosomal mosaicism and resolution with PGS. Moreover, embryos are typically outside of the incubator in suboptimal culture conditions for extended periods of time during these procedures. With TLM systems, embryos remain in the stable microenvironment of an incubator and are simultaneously imaged for noninvasive embryo evaluation using a fraction of the light exposure as compared to a stereomicroscope. Each image is then compiled into a time-lapse movie, the information from which can be extrapolated to correlate morphological, spatial, and temporal parameters with embryo quality and copy number status. Here, we describe the various TLM systems available for clinical and/or research use in detail and provide step-by-step instructions on how the measurement of specific timing intervals and certain morphological criteria can be implemented into IVF protocols to enhance embryo assessment and avoid the selection of aneuploid embryos. We also discuss the biological significance of processes unique to mitotically dividing embryos and the likelihood that complex chromosomal events such as chromothripsis occur during preimplantation development in humans and other mammals, particularly nonhuman primates.

Chromosomal instability in mammalian pre-implantation embryos: potential causes, detection methods, and clinical consequences.

Formation of a totipotent blastocyst capable of implantation is one of the first major milestones in early mammalian embryogenesis, but less than half of in vitro fertilized embryos from most mammals will progress to this stage of development. Whole chromosomal abnormalities, or aneuploidy, are key determinants of whether human embryos will arrest or reach the blastocyst stage. Depending on the type of chromosomal abnormality, however, certain embryos still form blastocysts and may be morphologically indistinguishable from chromosomally normal embryos. Despite the implementation of pre-implantation genetic screening and other advanced in vitro fertilization (IVF) techniques, the identification of aneuploid embryos remains complicated by high rates of mosaicism, atypical cell division, cellular fragmentation, sub-chromosomal instability, and micro-/multi-nucleation. Moreover, several of these processes occur in vivo following natural human conception, suggesting that they are not simply a consequence of culture conditions. Recent technological achievements in genetic, epigenetic, chromosomal, and non-invasive imaging have provided additional embryo assessment approaches, particularly at the single-cell level, and clinical trials investigating their efficacy are continuing to emerge. In this review, we summarize the potential mechanisms by which aneuploidy may arise, the various detection methods, and the technical advances (such as time-lapse imaging, "-omic" profiling, and next-generation sequencing) that have assisted in obtaining this data. We also discuss the possibility of aneuploidy resolution in embryos via various corrective mechanisms, including multi-polar divisions, fragment resorption, endoreduplication, and blastomere exclusion, and conclude by examining the potential implications of these findings for IVF success and human fecundity.