The endometrium undergoes regular regeneration and stromal proliferation as part of the normal menstrual cycle. To better understand cellular interactions driving the mechanisms in endometrial regeneration we employed single-cell RNA sequencing. Endometrial biopsies were obtained during the proliferative phase of the menstrual cycle from healthy fertile women and processed to single-cell suspensions which were submitted for sequencing. In addition to known endometrial cell types, bioinformatic analysis revealed multiple stromal populations suggestive of specific stromal niches with the ability to control inflammation and extracellular matrix composition. Ten different stromal cells and two pericyte subsets were identified. Applying different R packages (Seurat, SingleR, Velocyto) we established cell cluster diversity and cell lineage/trajectory, while using external data to validate our findings. By understanding healthy regeneration in the described stromal compartments, we aim to identify points of further investigation and possible targets for novel therapy development for benign gynecological disorders affecting endometrial regeneration and proliferation such as endometriosis and Asherman’s syndrome.

Background: The period of time when the embryo and the endometrium undergo significant morphological alterations to facilitate a successful implantation—known as “window of implantation”—is a critical moment in human reproduction. Embryo and the endometrium communicate extensively during this period, and lipid bilayer bound nanoscale extracellular vesicles (EVs) are purported to be integral to this communication.Methods: To investigate the nature of the EV-mediated embryo-maternal communication, we have supplemented trophoblast analogue spheroid (JAr) derived EVs to an endometrial analogue (RL 95–2) cell layer and characterized the transcriptomic alterations using RNA sequencing. EVs derived from non-trophoblast cells (HEK293) were used as a negative control. The cargo of the EVs were also investigated through mRNA and miRNA sequencing.Results: Trophoblast spheroid derived EVs induced drastic transcriptomic alterations in the endometrial cells while the non-trophoblast cell derived EVs failed to induce such changes demonstrating functional specificity in terms of EV origin. Through gene set enrichment analysis (GSEA), we found that the response in endometrial cells was focused on extracellular matrix remodelling and G protein-coupled receptors’ signalling, both of which are of known functional relevance to endometrial receptivity. Approximately 9% of genes downregulated in endometrial cells were high-confidence predicted targets of miRNAs detected exclusively in trophoblast analogue-derived EVs, suggesting that only a small proportion of reduced expression in endometrial cells can be attributed directly to gene silencing by miRNAs carried as cargo in the EVs.Conclusion: Our study reveals that trophoblast derived EVs have the ability to modify the endometrial gene expression, potentially with functional importance for embryo-maternal communication during implantation, although the exact underlying signalling mechanisms remain to be elucidated.

Embryo implantation depends on endometrial receptivity (ER). To achieve ER, the preparation of the uterine lining requires controlled priming by ovarian hormones and the expression of numerous genes in the endometrial tissue. microRNAs (miRs) have emerged as critical genetic regulators of ER in fertility and of the diseases that are associated with infertility. With the rapid development of next-generation sequencing technologies, it has become clear that miR genes can produce canonical miRs and variants—isomiRs. Here, we describe miR/isomiR expression dynamics across the four time points of natural chorionic gonadotropin (hCG)-administered cycles. Sequencing of the small RNAs (sRNA-seq) revealed that the most significant expression changes during the transition from the pre-receptive to the receptive phase occurred in the isomiR families of miR-125a, miR-125b, miR-10a, miR-10b, miR-449c, miR-92a, miR-92b, and miR-99a. Pairing the analysis of the differentially expressed (DE) miRs/isomiRs and their predicted DE mRNA targets uncovered 280 negatively correlating pairs. In the receptive endometrium, the 5′3′-isomiRs of miR-449c, which were among the most highly up-regulated isomiRs, showed a negative correlation with their target, transcription factor (TF) MYCN, which was down-regulated. Joint analysis of the miR/isomiR and TF expression identified several regulatory interactions. Based on these data, a regulatory TF-miR/isomiR gene-target circuit including let7g-5p and miR-345; the isomiR families of miR-10a, miR-10b, miR-92a, and miR-449c; and MYCN and TWIST1 was proposed to play a key role in the establishment of ER. Our work uncovers the complexity and dynamics of the endometrial isomiRs that can act cooperatively with miRs to control the functionally important genes that are critical to ER. Further studies of miR/isomiR expression patterns that are paired with those of their target mRNAs may provide a more in-depth picture of the endometrial pathologies that are associated with implantation failure.

(1) Background: Endometriosis is a highly prevalent gynecological disease affecting 10% of women of reproductive age worldwide. miRNAs may play a role in endometriosis, though their exact function remains unclear. This study aimed to identify differentially expressed miRNAs in endometriosis and study their functions in the disease. (2) Methods: Endometrial tissue was collected from women with endometriosis (n = 15) and non-endometriosis controls (n = 17). Dysregulated miRNAs were identified through small RNA-sequencing, and their biological significance was explored by target gene prediction and pathway analysis. Selected miRNAs were examined in paired ectopic endometriomas and eutopic endometrium (n = 10) using qRT-PCR. Their roles in cell migration and proliferation were further examined in vitro using functional assays. To identify potential target genes, we performed mRNA sequencing on transfected cells and the endometrioma cohort. (3) Results: We identified 14 dysregulated miRNAs in the eutopic endometrium of women with endometriosis compared to endometrial tissue from women without endometriosis. Pathway analysis indicated enrichment in cell migration and proliferation-associated pathways. Further ex vivo studies of miR-193b-5p and miR-374b-5p showed that both miRNAs were upregulated in endometrioma. Overexpression of these two miRNAs in vitro inhibited cell migration, and mRNA sequencing revealed several migration-related genes that are targeted by these miRNAs. (4) Conclusions: Our study identified two key endometrial miRNAs that may be involved in the pathogenesis of endometriosis by regulating cell migration.

STUDY QUESTION: How does a single dose of mifepristone on Day 2 after the LH peak (LH + 2) affect the endometrial receptivity transcriptome as assessed by the receptive signature established by the endometrial receptivity analysis (ERA)? SUMMARY ANSWER: A single dose of mifepristone on day LH + 2 renders the endometrium non-receptive by altering the transcriptome associated with endometrial receptivity. WHAT IS KNOWN ALREADY: Mifepristone is a progesterone receptor modulator that has been shown to alter endometrial receptivity. The ERA is a computational predictor that utilizes gene expression data of 248 genes from next generation sequencing to identify endometrial receptivity status. STUDY DESIGN, SIZE, DURATION: Endometrial biopsies were collected on day LH + 7 from controls (n = 11) and from women treated with mifepristone (n = 7). For further comparative analysis, samples were also obtained from women in the proliferative phase (n = 7). PARTICIPANTS/MATERIALS, SETTING, METHODS: Mifepristone treatment consisted of 200 mg administered on day LH + 2. Endometrial biopsies were treated for RNA isolation and cDNA conversion and sequencing. Endometrial receptivity status was assessed by the ERA computational predictor. Differential gene expression between groups was also assessed. Ingenuity Pathway Analysis was used for network analysis. Validation of gene expression results was done by qPCR. MAIN RESULTS AND THE ROLE OF CHANCE: Control samples were all staged around 'receptive' as would be clinically expected for LH + 7. Treatment samples were all staged as non-receptive (all but one was classified as 'proliferative' and the last as 'pre-receptive'). Differential gene expression analysis yielded 60 differentially expressed genes between the control and treatment groups. Bioinformatic pathway analysis for differential expression showed inactivation of the progesterone and glucocorticoid receptors, consistent with mifepristone action. LIMITATIONS, REASONS FOR CAUTION: The primary limitations are the relative small number of subjects and the use of a limited gene panel. WIDER IMPLICATIONS OF THE FINDINGS: This study sheds further light on the endometrial receptivity altering effects of mifepristone and on progesterone action. It further shows the capacity of the ERA to identify pharmacologically induced non-receptive endometrium, which expands its possible use clinically and in research.

To study the role of estrogen receptor beta in follicle development and maturation and in the response to gonadotropin stimulation aiming at superovulation DESIGN: Experimental study and transcriptomic analysis. Healthy wild-type and estrogen receptor beta (ERβ) knockout female mice undergoing superovulation at 4-weeks, 7-weeks, and 6-months of age. Oocyte yield after superovulation, transcriptomic profiling of cumulus-granulosa cell complexes and oocytes, and immunohistochemical analyses. Superovulation of ERβ knockout (Esr2-KO) mice resulted in reduced oocyte yield at 6-months of age compared to wild-type (WT) mice, but younger mice had similar yields. RNA-seq analysis of cumulus cells from superovulated WT and Esr2-KO mice identified genes and pathways associated with among others adhesion, proliferation, Wnt-signaling, and placed ERβ in bipotential granulosa cell cluster. Loss of ERβ increased expression of the other estrogen receptors Esr1 and Gper1. Our results show that ERβ has an important role in regulating ovulation in response to exogenous gonadotropins in 6-month-old mice, but not in younger mice. Our transcriptomic and immunohistochemical observations suggest a dysregulation of the granulosa cell communication and lack of tight coordination between granulosa cell replication and antrum expansion. A significant upregulation of other estrogen receptors may support a compensatory mechanism sustaining fertility during younger age in Esr2-KO mice.