RNA-sequencing of trophectoderm cells reveals deregulation of DNA damage, p53, cell-cycle, autophagy and apoptosis pathways in chromosomally abnormal human embryos.

a. Human blastocysts 5 or 6 days post-fertilization (dpf) underwent Preimplantation Genetic Testing (PGT) for monogenic diseases and/or aneuploidy screening, followed by cryopreservation. Embryos that were not transferred were donated for research after written informed content. After warming and 2hrs recuperation, euploid and fully aneuploid research embryos underwent a second TE biopsy for RNA-sequencing. The remainder of the embryos was live-stained with either CASP3/7 or CASP8 for 30min and fixed for immunostaining for proteins of interest (see figure 3). b. 3dpf human embryos were warmed and treated with 0.5µM reversine for 24hrs then transferred to normal culture medium until 5dpf to undergo TE biopsy for RNA-sequencing. After 2hrs recuperation the embryos were live-stained with CASP8 for 30min and fixed for immunostaining for proteins of interest (see figure 3). c. Unsupervised hierarchical clustering of all coding genes. Color coding: beige = euploid, red = aneuploid and blue = reversine-treated. d. Comparison between the diagnosis of aneuploid embryos obtained after PGT (list on the left) and the results obtained after using InferCNV after RNA-sequencing (plot on the right). e,f. Volcano plots after differential gene expression analysis with a cutoff value of |log2 fold change| > 1 and −log10(p-value) < 0.05 for aneuploid versus euploid (e) and reversine versus euploid (f). g,h. Venn diagrams comparing the upregulated (g) and downregulated (h) genes in aneuploid versus reversine treated embryos. i,j. Supervised plot of pathway analysis using the Molecular Signatures Database (FDR < 0.05) based on the list of all differentially expressed genes between aneuploid or reversine versus euploid embryos. Pathways are ranked based on the total number of genes in overlap of both test sets, ranked from sets with the highest number of genes in overlap to sets with the lowest. Outputs of the Hallmark library and C5 ontology gene sets are shown in (i) and (j), respectively. k,l. Supervised plots after Ingenuity Pathway Analysis of upstream regulators with an activation z-score ≤ −2 and ≥ 2 and a p-value < 0.05 in the aneuploid (k) or reversine (l) versus euploid embryos. m,n. Supervised plots after Ingenuity Pathway Analysis of canonical pathways in aneuploid (m) or reversine (n) versus euploid embryos. Pathways are ranked by p-value from the most to the least significant, those with a p-value < 0.05 and with an activation z-score ≤ −1.5 and ≥ 1.5 were considered as inhibited or activated, respectively. Significant pathways with an activation z-score between −1.5 and 1.5 were considered as deregulated.

Human embryos with the highest number of genes with abnormal copy number show stronger p53 pathway and apoptosis response.

a. Supervised hierarchical clustering of the TOP 500 deregulated genes sorted by p-value. Color coding: blue = euploid, green = Low dosage and red = High dosage. b,c. Volcano plots after differential gene expression analysis with a cutoff value of |log2 fold change| > 1 and −log10(p-value) < 0.05 for low dosage versus euploid embryos (b) and high dosage versus euploid embryos (c). d,e, Venn diagrams comparing the upregulated (d) and downregulated (e) genes in low dosage versus high dosage embryos. f. Supervised plot of pathway analysis using the Hallmark library of the Molecular Signatures Database (FDR < 0.05) based on the list of the TOP 500 expressed genes by p-value between low or high dosage versus euploid embryos. Pathways are ranked based on FDR-q-values of both test sets, the most significant deregulated pathway ranked from top to least significant pathway. g,h. Dot plots of the genes in overlap with the Hallmark p53 pathway (g) and Hallmark apoptosis pathway (h) of both low and high dosage embryos sorted by log2 Fold Change from the highest to the lowest. Dots with an increased size and a darker blue represent a higher value of log2 Fold Change and lower p-value, respectively. i, Violin plots with box and whisker plots of the counts per million mapped reads of a supervised set of 10 genes (from Supplementary Table 2) that are part of apoptosis (BAD, BCL2L1, TNFRSF10B), p53 pathway (DRAM1, MDM2, CDKN1A/B, PURPL), growth arrest (GADD45A), and DNA-damage (DDB2). *p = 0.027, **p = 0.010, ***p = 0.004, ****p < 0.001 using the Jonkheere-Terpstra test. Box and whisker plots show median and minimum to maximum values.

Immunostaining reveals increased apoptosis, autophagy, proteotoxic stress and DNA-damage in chromosomally abnormal human embryos.

a,b. Same experimental set-up as described in Fig. 1 a,b, but focused on immunostaining analysis. c, Orthogonal projections after immunostaining of euploid and aneuploid embryos for DNA (white) and CASP3/7 (green). d, Orthogonal projections after immunostaining of euploid, aneuploid and reversine-treated embryos for DNA (white) and CASP8 (green). e, CASP3/7 mean intensity per cell. Euploid n=13, Aneuploid n=8 embryos. Mann-Whitney test, *p = 0.0199. f, CASP8 mean intensity per cell. Euploid n=6, Aneuploid n=9, Reversine-treated n=8 embryos. Kruskal-Wallis test, *p = 0.0394, **p = 0.0085. g, Number of nuclei per embryo. Euploid n=19, Aneuploid n=18, Reversine-treated n=8 embryos. Kruskal-Wallis test, ****p < 0.0001, *p = 0.0305. h, Orthogonal projections after immunostaining of euploid, aneuploid and reversine-treated embryos for DNA (white), LC3B (turquoise) and HSP70 (magenta). i, LC3B puncta per cell. Euploid n=19, Aneuploid n=22 embryos, Reversine-treated n=8 embryos. Kruskal-Wallis test, *p = 0.0189, **p = 0.0074. j, HSP70 mean intensity per cell. Euploid n=13, Aneuploid n=8, Reversine-treated n=8 embryos. Kruskal-Wallis test, **p = 0.0077, ***p = 0.0008. k, Orthogonal projections after immunostaining of euploid, aneuploid for DNA (white) and p62 (turquoise). l, p62 puncta per cell. Euploid n=6, Aneuploid n=9 embryos. Mann-Whitney test, **p = 0.0076.

Brightfield pictures were obtained during confocal imaging. All scale bars are 20 µm. Box and whisker plots show median and whiskers show minimum to maximum values. Bar plots show mean ± s.d. For all plots each dot represents a single embryo and ns = not significant.

Aneuploid human embryos show less cells in trophectoderm and OCT4-positive cells and impaired second lineage segregation

a, 5 or 6dpf post-PGT-A euploid and fully aneuploid human blastocysts were warmed and kept for 16hrs in culture to allow for the second lineage segregation. The blastocysts were live-stained with CASP3/7 for 30min and fixed for immunostaining proteins of interest. In a first scan we analyzed OCT4 (ICM), CASP3/7 (apoptosis), LC3B (autophagy) and S15p53 (DNA damage) in both lineages. Embryos were then re-stained with GATA4 as a marker for PrE. b, Orthogonal projections after immunostaining of euploid, aneuploid for DNA (white) and Serine (S) 15 p53 (turquoise). c, Percentage (%) of Serine 15 p53 positive cells per embryo. Euploid n = 3, Aneuploid n = 5. Mann-Whitney test, *p = 0.0357. d, Orthogonal projections after immunostaining of euploid and aneuploid embryos for DNA (white) and OCT4 (magenta). The first aneuploid panel (Trisomy 14 and 16) shows a similar number of ICM cells compared to the euploid embryo. The second aneuploid panel (Trisomy 14 and 22) shows an embryo without an ICM. e,f,g, Differences in number of nuclei per embryo between euploid and aneuploid embryos in the OCT4-negative cells (trophectoderm) (e) **p = 0.0092, OCT4-positive cells (ICM/EPI), *p = 0.0412 (f) and whole embryo **p = 0.0067 (g). Euploid n = 10, Aneuploid n = 14. Student’s t-test with Welch’s correction. h, Orthogonal projections after immunostaining of euploid and aneuploid embryos for DNA (white), CASP3/7 (green) and OCT4 (magenta). i, Percentage (%) of CASP3/7 positive (+) cells in the trophectoderm lineage (OCT4-negative cells). Euploid n = 9, Aneuploid n = 14. Student’s t-test with Welch’s correction, *p = 0.0453. j, CASP3/7 mean intensity per cell of whole embryos. Euploid n = 9, Aneuploid n = 14. Student’s t-test with Welch’s correction, *p = 0.0332. k, Orthogonal projections after immunostaining of euploid and aneuploid embryos for DNA (white), OCT4 (magenta) and GATA4 (green). The first aneuploid panel (trisomy 13 and 15) shows an embryo with presence of a GATA4 positive cell (PrE). The second aneuploid panel (Trisomy 7 and monosomies 5, 12 and 21) shows an embryo that did not contain GATA4 positive cells. l, Differences in the number of cells per embryo that were OCT4 positive and either GATA4 negative (red) or positive (green) between euploid and aneuploid embryos. In case GATA4-positive cells (PrE) were present we considered the GATA4-negative cells to be epiblast (EPI). All euploid embryos contained GATA4-positive cells, 7/14 aneuploid embryos had an ICM completely lacking GATA4-positive cells (Fisher-exact test, p = 0.0188). m, Differences in the PrE/EPI ratio between euploid and aneuploid embryos that had an ICM containing GATA4-positive cells. Euploid n = 9, Aneuploid n = 7. Note here that one euploid embryo (#6, l) was lost during re-staining for GATA4. Mann-Whitney test, **p = 0.0089. n, Orthogonal projections after immunostaining of euploid and aneuploid embryos for DNA (white), LC3B (turquoise) and OCT4 (magenta). Yellow circle indicates the ICM. The first aneuploid panel (Trisomy 1 and 20 and monosomy 18) shows OCT4-positive cells (ICM/EPI) with low levels of autophagy and, after re-staining for GATA4, without PrE (right panel, ICM zoom). The second aneuploid panel (Monosomy 14) shows an embryo with high levels of autophagy in the ICM that contains PrE cells (right panel, ICM zoom). o,p,q. Differences in LC3B puncta per cell between euploid and aneuploid embryos in the whole embryo,*p = 0.0317 (o),(OCT4-negative cells (trophectoderm),*p = 0.0317 (p) and OCT4-positive cells (ICM/EPI) (q). Euploid n = 4, Aneuploid n = 5. Mann-Whitney test. r, Violin plots with box plots of OCT4-positive-cells (ICM/EPI) per embryo (left, ns), OCT4-negative cells (trophectoderm) per embryo (center, *p = 0.043) and GATA4-positive-cells (PrE) per embryo (right, ****p < 0.001, Jonkheere-Terpstra test) depending on gene dosage. Euploid (light blue), Low dosage (middle blue) and High dosage (darkest blue).

Yellow arrows indicate presence of the signal and yellow arrow heads indicate absence of signal. Brightfield pictures were obtained during confocal imaging. All scale bars are 20 µm. Box and whisker plots show median and minimum to maximum values. Bar plots show mean ± s.d. For all plots each dot represents a single embryo; ns = not significant. T = Trisomy, M = Monosomy

Expanded RNA-sequencing analysis

a, Principal component analysis after RNA-sequencing of euploid (green) versus aneuploid (red) and reversine (blue) embryos. b, InferCNV analysis of individual euploid samples compared to the reference set. c, InferCNV analysis of individual reversine samples compared to the reference set. d, Venn Diagram of overlap between differentially expressed genes of this study with three studies that analyzed human embryos that contain euploid and diverse types of aneuploidy from cleavage stage embryos (Vera-Rodriguez et al. 2015), single cells from 4 and 7 dpf human embryos from TE, ICM, EPI and PrE lineages (Starostik et al. 2020) and ICM and TE samples of 5 or 6 dpf embryos containing low- and high levels of mosaicism (Martin et al., 2023). e, Venn Diagram of overlap between differentially expressed genes of this study with four studies that analyzed specific aneuploidies (Licciardi et al. 2018, Fuchs-Weizman et al. 2019, Sánchez-Ribas et al. 2019 and Maxwell et al. 2022).

S15-p53 expression in TE vs OCT4-positive cells (ICM/EPI) and increased micronuclei in aneuploid embryos

a, Orthogonal projections after immunostaining of euploid and aneuploid embryos for DNA (white), S15p53 (turquoise) and OCT4 (magenta). b, Percentages of nuclei positive for S15p53 per embryo in euploid (n = 3) and aneuploid (n = 4) embryos in the ICM. c, Percentages of nuclei positive for S15p53 per embryo in euploid (n = 3) and aneuploid (n = 5) embryos in the TE. d, Left panel: Orthogonal projections after immunostaining of euploid and aneuploid embryos for DNA (white) and CASP3/7 (green) showing micronuclei. Yellow box indicates zoomed-in region in the right panel. Yellow arrows indicate presence of micronuclei and overlap between DNA and CASP3/7. e, Number of micronuclei per cell in euploid and aneuploid whole embryos. Euploid n = 9, Aneuploid n = 14. Student’s t-test with Welch’s correction, *p = 0.0115.

Brightfield pictures were obtained during confocal imaging. All scale bars are 20 µm. Box and plots show median and minimum to maximum values. Bar plots show mean ± s.d. For all plots each dot represents a single embryo; ns = not significant. ROI = Region of interest.