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. 2024 Sep;41(9):2405-2418.
doi: 10.1007/s10815-024-03173-y. Epub 2024 Jul 12.

Enrichment of cell cycle pathways in progesterone-treated endometrial organoids of infertile women compared to fertile women

B N Bui  1 , A I Ardisasmita  2 , F H van de Vliert  3 , M S Abendroth  3 , M van Hoesel  3 , S Mackens  4 , S A Fuchs  2 , E E S Nieuwenhuis  5   6 , F J M Broekmans  3   7 , G S Steba  3
Affiliations

Enrichment of cell cycle pathways in progesterone-treated endometrial organoids of infertile women compared to fertile women

B N Bui et al. J Assist Reprod Genet. 2024 Sep.

Abstract

Purpose: To investigate whether the transcriptome profile differs between progesterone-treated infertile and fertile endometrial organoids.

Methods: Endometrial biopsies were obtained from 14 infertile and seven fertile women, after which organoids were generated from isolated epithelial cells. To mimic the secretory phase, organoids were sequentially treated with 17β-estradiol (E2) and progesterone (P4) and subjected to RNA sequencing. Differentially expressed genes (DEGs) were identified using DESeq2 (lfcThreshold = 0, log2 Fold Change ≥ 1.0 or ≤ -1.0), and a principal component analysis (PCA) plot was generated. Functional enrichment analysis was performed by overrepresentation analysis and Gene Set Enrichment Analysis (GSEA). To functionally assess proliferation, OrganoSeg surface measurements were performed before (T0) and after (T1) differentiation of organoids, and T1/T0 ratios were calculated to determine the proliferation rate.

Results: Although the PCA plot did not show clear clustering of the fertile and infertile samples, 363 significant DEGs (129 upregulated and 234 downregulated) were detected in infertile compared to fertile organoids. Mainly cell cycle processes were highly enriched in infertile organoids. Thus, we hypothesised that proliferative activity during differentiation may be higher in infertile organoids compared to fertile organoids. However, this could not be validated by cell surface measurements.

Conclusions: This study revealed that cell cycle processes were enriched in E2/P4-treated infertile endometrial organoids as compared to fertile organoids. This could reflect persistently higher proliferative activity of the endometrial epithelial cells in differentiated infertile organoids compared to fertile organoids. To confirm this hypothesis, further studies are warranted.

Keywords: Endometrium; Fertility; Infertility; Organoids; RNA-sequencing; Transcriptome.

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Conflict of interest statement

The SCRATCH and SCRaTCH-OFO trials and the current endometrial organoid transcriptome study received funding from public sources and Merck, but none of these sources had input in the study design and analysis. In addition, several authors report having received personal fees for consultations, conference attendance, and travel as follows: BB from Gedeon Richter Benelux and Guerbet; GS from Guerbet; and SM from Abbott Pharmaceuticals, Ferring, Merck, Oxolife and IBSA. FB is a paid member of the advisory boards of Merck and Ferring and also received speakers’ fees from Besins Healthcare, Cooper Surgical and Merck. All of these personal fees were unrelated to the study reported in this manuscript. The remaining authors declare no conflicts of interest relevant to this study.

Figures

Fig. 1
Fig. 1
Proliferation assay of infertile and fertile endometrial organoids. a Hormonal treatment of endometrial organoids. T−1 represented the start of estradiol (E2) supplementation, T0 the start of progesterone (P4) supplementation for differentiation of the organoids and T1 after differentiation. Photos were taken at the three-time points for evaluation in OrganoSeg. Two organoid lines received a second medium refreshment with E2 medium on days 8–9 and therefore P4 supplementation started 2–3 days later in these lines. BM, base medium; E2, estradiol medium; P4, progesterone medium. b Analysis of the two-dimensional surfaces of endometrial organoids in OrganoSeg, using the brightfield images taken at T0 and T1. The left image shows the original image, the middle image highlights the organoid borders and the right image delineates the measured organoid surfaces
Fig. 2
Fig. 2
RNA-seq gene expression analysis of infertile vs fertile endometrial organoids. a Principal component analysis plot of infertile and fertile endometrial organoid samples after batch effect correction. b Volcano plot of differentially expressed genes in infertile organoids compared to fertile organoids. In red are displayed the genes that are significantly differentially expressed with a log2 fold change (FC) ≥ 1.0 or ≤  − 1.0 and an adjusted p-value (padj) of < 0.05; in green the genes with a log2FC ≥ 1.0 or ≤  − 1.0, but with padj ≥ 0.05; in blue the genes with − 1.0 < log2FC < 1.0 and padj < 0.05; and in grey the genes that are not significantly differentially expressed (i.e. − 1.0 < log2FC < 1.0 and padj ≥ 0.05). NS not significant, FC fold change
Fig. 3
Fig. 3
Enrichment analysis annotating significantly differentially expressed genes (DEGs) to top 15 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and Gene Ontology (GO) Biological Processes (BP). Pathways and BP are sorted by adjusted p-value. Terms with red bars have an adjusted p-value of < 0.1 a KEGG pathways annotated to significantly upregulated DEGs. b GO BP annotated to significantly upregulated DEGs. c KEGG pathways annotated to significantly downregulated DEGs. d GO BP annotated to significantly downregulated DEGs
Fig. 4
Fig. 4
Gene set enrichment analysis (GSEA) of Gene Ontology (GO) gene sets. Biological process (a), molecular function (b) and cellular component (c)
Fig. 5
Fig. 5
Proliferation assay in infertile vs fertile endometrial organoids. a RNA-seq principal component analysis plot with the subset of organoid lines used for the proliferation assay indicated by the numbers (n = 7 infertile, n = 5 fertile). b, c Mean proliferation rates in infertile and fertile endometrial organoids expressed as T1/T0 (b) and T0/T−1 (c) OrganoSeg surface measurements
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