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Metabolic and epigenetic dysfunctions underlie the arrest of in vitro fertilized human embryos in a senescent-like state [1]

['Yang Yang', 'Center For Reproductive Medicine', 'Shuguang Hospital Affiliated To Shanghai University Of Traditional Chinese Medicine', 'Shanghai', 'Liyang Shi', 'Shenzhen Key Laboratory Of Gene Regulation', 'Systems Biology', 'Department Of Biology', 'School Of Life Sciences', 'Southern University Of Science']

Date: 2022-07

Around 60% of in vitro fertilized (IVF) human embryos irreversibly arrest before compaction between the 3- to 8-cell stage, posing a significant clinical problem. The mechanisms behind this arrest are unclear. Here, we show that the arrested embryos enter a senescent-like state, marked by cell cycle arrest, the down-regulation of ribosomes and histones and down-regulation of MYC and p53 activity. The arrested embryos can be divided into 3 types. Type I embryos fail to complete the maternal-zygotic transition, and Type II/III embryos have low levels of glycolysis and either high (Type II) or low (Type III) levels of oxidative phosphorylation. Treatment with the SIRT agonist resveratrol or nicotinamide riboside (NR) can partially rescue the arrested phenotype, which is accompanied by changes in metabolic activity. Overall, our data suggests metabolic and epigenetic dysfunctions underlie the arrest of human embryos.

Funding: This work was supported by the National Key R&D Program of China (2018YFC1704300 to Y.J.W.), the National Natural Science Foundation of China (81070494 and 81170571 to G.Q.T, 81571442 to W.Z., and 31970589 to A.P.H.), the Shenzhen Innovation Committee of Science and Technology (JCYJ20200109141018712 to A.P.H. and ZDSYS20200811144002008 to the Shenzhen Key Laboratory of Gene Regulation and Systems Biology and to A.P.H.), and the Stable Support Plan Program of the Shenzhen Natural Science Fund (20200925153035002 to A.P.H.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. The datasets supporting the conclusions of this article are available in the GSA (Genome Sequence Archive): HRA001406 under controlled access for human samples. The normalized gene expression matrix for all samples and genes/TEs used in the study and the raw tag count matrix of all samples (excluding resveratrol) used in this study for CytoTRACE analysis are available at https://figshare.com/articles/dataset/Human_embryo_normalized_gene_expression_data/19775992 .

In this study, we explored the transcriptomic basis behind the arrest of human embryos. A subset of the arrested embryos enter into a senescent-like state characterized by the up-regulation of p53, MYC, FOXO1, and the widespread down-regulation of ribosomes, histones, and translation initiation factors. We show that this senescent phenotype can be partially overcome using the antioxidant resveratrol and nicotinamide riboside (NR), and our data suggest that these 2 molecules activate the sirtuin family of acetyltransferases (SIRTs) to modulate metabolism. Modulation of SIRT activity leads to a reactivation of the arrested embryos and progression to a morula and early blastocyst.

In vitro developmental models of embryogenesis in other organisms has not brought clarity to this problem, as preimplantation development is divergent between species [ 8 ]. For example, ZGA mainly occurs at the 2-cell stage in mice, but in humans, there are 2 waves, a minor ZGA at the 2-cell stage and the major ZGA at the 8-cell stage [ 9 ]. Relatedly, in contrast to humans, some species have good in vitro developmental potential. For example, approximately 90% of mouse, approximately 80% of (monospermic) pig, approximately 70% of cat, and approximately 60% of Macaca mulatta embryos successfully develop to the blastocyst stage [ 10 – 12 ]. Conversely, humans are not the only species with poor in vitro embryonic developmental potential, only 25% to 30% of cattle and horse embryos will develop to a blastocyst [ 6 , 13 , 14 ]. However, it is unclear if the same mechanisms are active in other species. Human embryonic stem cells (ESCs) can be manipulated to form artificial blastocyst-like “blastoids” that mimic natural blastocysts [ 15 , 16 ]. Interestingly, blastoids are generated at low efficiency, which may reflect developmental problems inherent to natural blastocysts. However, blastoids cannot address pre-morula developmental arrest, as they model a later developmental stage, and it is unclear if the problems seen in blastoids are the same as preimplantation embryos. Ultimately, to investigate the arrest of human embryos, it is necessary to assay the problems directly.

In vitro fertilization (IVF) has revolutionized the treatment of human fertility problems. However, a large number of human embryos fail to develop in vitro, and typically, only 30% of human embryos will progress to the blastocyst stage [ 1 , 2 ]. Human preimplantation embryos can arrest at all stages between the zygote and the blastocyst, and a large fraction irreversibly arrest between the 2-cell and 8-cell stages and remain un-compacted [ 2 ]. Several cellular mechanisms have been proposed to explain this arrest, specifically: failed zygotic genome activation (ZGA) [ 3 ], delayed maternal RNA clearance [ 4 ], reactive oxygen species causing endoplasmic reticulum stress [ 5 , 6 ], and aneuploidy [ 7 ]. Computational machine learning techniques can detect morphological patterns in microscope images of otherwise normal-appearing embryos that will later go on to arrest [ 1 ], suggesting the arrest mechanisms are active before they manifest. However, the cellular mechanism remains unclear.

Results

Arrested embryos do not show excessive aneuploidy We next looked at the karyotype of the arrested embryos. Normal human embryos have surprisingly high levels of aneuploidy, compared to other species [27], and this may be a contributing factor to arrest during IVF, particularly in embryos from women of advanced maternal age [28]. Aneuploidy mainly takes 2 forms [29]: full embryo aneuploidy due to meiotic errors in the oocyte/zygote or mosaic aneuploidy due to mitotic errors after fertilization [3,30]. We used the RNA-seq data to estimate the karyotype, as we could then correlate it with the arrest type in the same embryo. We used the method described in [31] to estimate aneuploidy (S2 Data). Around 30% of cells were predicted to be aneuploid (S2A and S2B Fig and S2 Data), which agrees with previous data that aneuploidy is common in human embryos [32]. We notice that chromosomal defects become particularly evident at the 8-cell stage, and reach around 30% at the morula/E3 stage (14/48 cells, 29%) and persist to the E7-stage (late blastocyst) at similar rates (116/321 cells, 36%) (S2C Fig). Of the arrested embryos, 6/23 (26%) had a predicted aneuploidy (S2A and S2B Fig). This number is not substantially different from normal embryos and is in line with the typical levels of aneuploidy seen in human embryos. This computational approach cannot easily detect the difference between meiotic and mitotic aneuploidies, but assuming most of the aneuploidies we see are due to mitotic errors, there was no overall bias in the gain or loss of specific chromosomes (S2C and S2D Fig). Ultimately, these data suggest that aneuploidy is not a specific feature of arrested embryos. In a study of aneuploidy in embryos from women of advanced maternal age, 50% of embryos still developed to the blastocyst stage, despite 84% of the embryos having at least 1 chromosomal abnormality [28]. Similarly, there is evidence that mosaic aneuploidies are common and may not be detrimental to development [30,33], at least to the blastocyst stage. Finally, meiotic aneuploidies can develop to the blastocyst stage, although they have severe consequences for further development [34]. Hence, we argue that while aneuploidy is an important problem in postimplantation development, it is not responsible for developmental arrest pre-compaction or to reach the blastocyst.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3001682

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