Transcriptomics of receptive endometrium in women with sonographic features of adenomyosis

Abstract Background Women with uterine adenomyosis seeking assisted reproduction have been associated with compromised endometrial receptivity to embryo implantation. To understand the mechanisms involved in this process, we aimed to compare endometrial transcriptome profiles during the window of implantation (WOI) between women with and without adenomyosis. Methods We obtained endometrial biopsies LH-timed to the WOI from women with sonographic features of adenomyosis (n=10) and controls (n=10). Isolated RNA samples were subjected to RNA sequencing (RNA-seq) by the Illumina NovaSeq 6000 platform and endometrial receptivity classification with a molecular tool for menstrual cycle phase dating (beREADY®, CCHT). The program language R and Bioconductor packages were applied to analyse RNA-seq data in the setting of the result of accurate endometrial dating. To suggest robust candidate pathways, the identified differentially expressed genes (DEGs) associated with the adenomyosis group in the receptive phase were further integrated with 151, 173 and 42 extracted genes from published studies that were related to endometrial receptivity in healthy uterus, endometriosis and adenomyosis, respectively. Enrichment analyses were performed using Cytoscape ClueGO and CluePedia apps. Results Out of 20 endometrial samples, 2 were dated to the early receptive phase, 13 to the receptive phase and 5 to the late receptive phase. Comparison of the transcriptomics data from all 20 samples provided 909 DEGs (p<0.05; nonsignificant after adjusted p value) in the adenomyosis group but only 4 enriched pathways (Bonferroni p value < 0.05). The analysis of 13 samples only dated to the receptive phase provided suggestive 382 DEGs (p<0.05; nonsignificant after adjusted p value) in the adenomyosis group, leading to 33 enriched pathways (Bonferroni p value < 0.05). These included pathways were already associated with endometrial biology, such as “Expression of interferon (IFN)-induced genes” and “Response to IFN-alpha”. Data integration revealed pathways indicating a unique effect of adenomyosis on endometrial molecular organization (e.g., “Expression of IFN-induced genes”) and its interference with endometrial receptivity establishment (e.g., “Extracellular matrix organization” and “Tumour necrosis factor production”). Conclusions Accurate endometrial dating and RNA-seq analysis resulted in the identification of altered response to IFN signalling as the most promising candidate of impaired uterine receptivity in adenomyosis.

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A Two-Cohort RNA-seq Study Reveals Changes in Endometrial and Blood miRNome in Fertile and Infertile Women

Genes ◽

10.3390/genes9120574 ◽

2018 ◽

Vol 9 (12) ◽

pp. 574 ◽

Cited By ~ 5

Author(s):

Kadri Rekker ◽

Signe Altmäe ◽

Marina Suhorutshenko ◽

Maire Peters ◽

Juan F. Martinez-Blanch ◽

...

Keyword(s):

Embryo Implantation ◽

Endometrial Receptivity ◽

Small Rna Sequencing ◽

Rna Seq ◽

Recurrent Implantation Failure ◽

Secretory Phase ◽

Blood Samples ◽

Infertile Women ◽

Secretory Endometrium ◽

Fertile Women

The endometrium undergoes extensive changes to prepare for embryo implantation and microRNAs (miRNAs) have been described as playing a significant role in the regulation of endometrial receptivity. However, there is no consensus about the miRNAs involved in mid-secretory endometrial functions. We analysed the complete endometrial miRNome from early secretory (pre-receptive) and mid-secretory (receptive) phases from fertile women and from patients with recurrent implantation failure (RIF) to reveal differentially expressed (DE) miRNAs in the mid-secretory endometrium. Furthermore, we investigated whether the overall changes during early to mid-secretory phase transition and with RIF condition could be reflected in blood miRNA profiles. In total, 116 endometrial and 114 matched blood samples collected from two different population cohorts were subjected to small RNA sequencing. Among fertile women, 91 DE miRNAs were identified in the mid-secretory vs. early secretory endometrium, while no differences were found in the corresponding blood samples. The comparison of mid-secretory phase samples between fertile and infertile women revealed 21 DE miRNAs from the endometrium and one from blood samples. Among discovered novel miRNAs, chr2_4401 was validated and showed up-regulation in the mid-secretory endometrium. Besides novel findings, we confirmed the involvement of miR-30 and miR-200 family members in mid-secretory endometrial functions.

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Uterine Epithelial Progesterone Receptor Governs Uterine Receptivity Through Epithelial Cell Differentiation

Endocrinology ◽

10.1210/endocr/bqaa195 ◽

2020 ◽

Vol 161 (12) ◽

Author(s):

Mona Gebril ◽

Yasushi Hirota ◽

Shizu Aikawa ◽

Yamato Fukui ◽

Tetsuaki Kaku ◽

...

Keyword(s):

Cell Differentiation ◽

Epithelial Cell ◽

Progesterone Receptor ◽

Embryo Implantation ◽

Rna Seq ◽

Uterine Receptivity ◽

Epithelial Cell Differentiation ◽

Matrix Cell ◽

Laser Capture ◽

Embryo Attachment

Abstract Progesterone receptor (PGR) is indispensable for pregnancy in mammals. Uterine PGR responds to the heightened levels of ovarian progesterone (P4) after ovulation and regulates uterine gene transcription for successful embryo implantation. Although epithelial and stromal P4-PGR signaling may interact with each other to form appropriate endometrial milieu for uterine receptivity and the subsequent embryo attachment, it remains unclear what the specific roles of epithelial P4-PGR signaling in the adult uterus are. Here we generated mice with epithelial deletion of Pgr in the adult uterus (Pgrfl/flLtfCre/+ mice) by crossing Pgr-floxed and Ltf-Cre mice. Pgrfl/flLtfCre/+ mice are infertile due to the impairment of embryo attachment. Pgrfl/flLtfCre/+ uteri did not exhibit epithelial growth arrest, suggesting compromised uterine receptivity. Both epithelial and stromal expressions of P4-responsive genes decreased in Pgrfl/flLtfCre/+ mice during the peri-implantation period, indicating that epithelial Pgr deletion affects not only epithelial but stromal P4 responsiveness. In addition, uterine LIF, an inducer of embryo attachment, was decreased in Pgrfl/flLtfCre/+ mice. The RNA-seq analysis using luminal epithelial specimens dissected out by laser capture microdissection revealed that the signaling pathways related to extracellular matrix, cell adhesion, and cell proliferation are altered in Pgr fl/flLtf Cre/+ mice. These findings suggest that epithelial PGR controls both epithelial and stromal P4 responsiveness and epithelial cell differentiation, which provides normal uterine receptivity and subsequent embryo attachment.

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Endometrial receptivity and embryo implantation in carnivores—commonalities and differences with other mammalian species

Biology of Reproduction ◽

10.1093/biolre/ioab001 ◽

2021 ◽

Author(s):

Erika Elinor Paulson ◽

Pierre Comizzoli

Keyword(s):

Mammalian Species ◽

Embryo Implantation ◽

Research Priorities ◽

Germinal Vesicle ◽

Reproductive Traits ◽

Current Data ◽

Endometrial Receptivity ◽

Ovarian Activity ◽

Uterine Receptivity ◽

Preimplantation Embryo Development

Abstract Endometrial receptivity and embryo implantation processes are a major point of pregnancy failure in many mammalian species, including humans. Although reproductive biology in many carnivore species remains enigmatic, the few that have been studied so far are invaluable comparative models. The goals of this review are to (1) summarize current data on the mechanisms involved in uterine receptivity and embryo implantation in carnivores, including commonalities and differences with other mammalian species and (2) identify research priorities to better understand a key phenomenon in a critical group of mammals. Besides unique reproductive traits in some carnivores (induced vs. spontaneous ovulation in cats, ovulation at the germinal vesicle stage in dogs), preimplantation embryo development is comparable with other orders. However, the timing of implantation varies, especially in species having an embryonic diapause. Mechanisms involved in endometrial receptivity and decidualization still remain to be fully understood, but specific markers have already been identified. Importantly, the use of endogenous hormones to control the ovarian activity may impact endometrial receptivity and subsequent embryo implantation. Next, research efforts should take advantage of advanced technologies to further study embryo implantation in carnivores and to provide more relevant models to reproductive medicine or for the conservation of rare and endangered species.

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Influence of hyaluronan on endometrial receptivity and embryo attachment in sheep

Reproduction Fertility and Development ◽

10.1071/rd16232 ◽

2017 ◽

Vol 29 (9) ◽

pp. 1763 ◽

Cited By ~ 3

Author(s):

Waleed F. A. Marei ◽

D. Claire Wathes ◽

Kabir A. Raheem ◽

Omnia Mohey-Elsaeed ◽

Fataneh Ghafari ◽

...

Keyword(s):

Embryo Implantation ◽

Endometrial Receptivity ◽

Uterine Receptivity ◽

Female Reproduction ◽

Mucin 1 ◽

Synthesis Inhibitor ◽

The Impact ◽

Hyaluronidase 2 ◽

Embryo Attachment

An increasing number of reports suggests a role of hyaluronan (HA) in female reproduction and interest in its application in assisted reproduction is rising. However, there are contrasting data about the effectiveness of adding HA to the embryo-transfer medium on improving pregnancy rates. Using sheep as an experimental model, the studies reported here analysed the impact of HA infusion into the uterus on embryo attachment to uterine luminal epithelium (LE) and expression of selected markers of uterine receptivity. On Day 14 after natural mating (pre-attachment), uterine horns were infused with either (n = 4 each): PBS (control), HA (1 mg mL–1), HA + hyaluronidase 2 (Hyal2; 300 IU mL–1) or 4-methyl-umbelliferone (HA-synthesis inhibitor; 4MU, 1 mM). HA immunostaining on uterine sections collected on Day 17 was negative in the 4MU group and weak in the HA+Hyal2 group. In contrast to 4MU, which resulted in 100% attachment, HA infusion blocked embryo attachment in all treated animals. This was accompanied by the disappearance of mucin 1 and increased expression of osteopontin and CD44v6 in the LE of uteri with attached embryos. In conclusion, the presence of HA at the embryo–maternal interface during embryo implantation resulted in reduced endometrial receptivity and inhibited the interaction of trophoblasts with the LE, whereas clearance of HA favoured embryo attachment.

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miR-183-5p regulates uterine receptivity and enhances embryo implantation

Journal of Molecular Endocrinology ◽

10.1530/jme-19-0184 ◽

2020 ◽

Vol 64 (1) ◽

pp. 43-52 ◽

Cited By ~ 2

Author(s):

Rubab Akbar ◽

Kamran Ullah ◽

Tanzil Ur Rahman ◽

Yi Cheng ◽

Hai-Yan Pang ◽

...

Keyword(s):

Embryo Implantation ◽

Endometrial Receptivity ◽

Luciferase Reporter ◽

Uterine Receptivity ◽

Positive Role ◽

Endometrial Cells ◽

Estrogenic Effects ◽

Reporter Assays ◽

Downstream Analysis

Receptive endometrium is a prerequisite for successful embryo implantation, and it follows that poor endometrial receptivity is a leading cause of implantation failure. miRNAs play important roles as epigenetic regulators of endometrial receptivity and embryo implantation through post-transcriptional modifications. However, the mechanisms of action of many miRNAs are poorly understood. In this study, we investigated the role of the miR-183 family, comprising three miRNAs (miR-183-5p, miR-182-5p, and miR-96-5p) in endometrial receptivity and embryo implantation. The miR-183 family shows estrogen-dependent upregulation in endometrial Ishikawa (IK) cells. The miR-183 family also has a positive role in migration and proliferation of IK cells. Furthermore, JAr spheroid attachment experiments show that attachment rates were significantly decreased after treatment of IK cells with inhibitors for miR-183-5p and miR-182-5p and increased after treatment with miR-183-5p-mimic and miR-96-5p-mimic, respectively. The downstream analysis shows that catenin alpha 2 (CTNNA2) is a potential target gene for miR-183-5p, and this was confirmed in luciferase reporter assays. An in vivo mouse pregnancy model shows that inhibition of miR-183-5p significantly decreases embryo implantation rates and increases CTNNA2 expression. Downregulation of CTNNA2 in endometrial cells by miR-183-5p may be significant in mediating estrogenic effects on endometrial receptivity. In conclusion, miR-183-5p and the CTNNA2 gene may be potential biomarkers for endometrial receptivity and may be useful diagnostic and therapeutic targets for successful embryo implantation.

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P–292 Transcriptome signature of receptive endometrium is not affected by the presence of mild adenomyosis

Human Reproduction ◽

10.1093/humrep/deab130.291 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

E Prasnikar ◽

T Kunej ◽

M Gorenjak ◽

P Uroš ◽

B Kovačič ◽

...

Keyword(s):

Gene Expression ◽

Significant Influence ◽

Gene Expression Signature ◽

Study Groups ◽

Endometrial Receptivity ◽

Functional Enrichment ◽

Control Group ◽

P Value ◽

Rna Seq ◽

Expression Signature

Abstract Study question Does the presence of mild adenomyosis, a common acquired uterine anomaly, affect the endometrial gene expression levels during window of receptivity? Summary answer Mild adenomyosis has no significant influence on gene expression signature in the window of implantation (WOI). What is known already The improvements in imaging techniques have led to frequent detection of adenomyosis in women undergoing investigations for infertility. Although the data are conflicting, some clinical studies have shown that the presence of adenomyosis may interfere with embryo implantation and lead to poor pregnancy outcomes. The knowledge of molecular background that would lead to the phenomenon of altered endometrial receptivity in women with adenomyosis is limited and mainly demonstrated by selected candidate genes. Next-generation sequencing platforms enable genome-wide transcriptomic profiling of desired tissue samples and present a powerful tool to identify differentially expressed genes (DEGs) between women with adenomyosis and controls. Study design, size, duration We designed a prospective case-control study comparing women with sonographic evidence of mild adenomyosis (n = 10) and women with normal uteri seeking assisted reproduction due to male factor infertility as the control group (n = 10). All eligible women underwent infertility treatment at the Department of Reproductive Medicine and Gynaecological Endocrinology, University Medical Centre Maribor, Slovenia between years 2018 and 2020. For the present study, they were scheduled for cycle monitoring by urinary luteinizing hormone (LH) tests. Participants/materials, setting, methods Each endometrial biopsy was obtained in the presumed window of implantation (WOI) on days LH + 7 to LH + 9 after LH surge (LH + 0). Isolated total RNA was applied for mRNA + lncRNA sequencing (RNA-seq) by Illumina Novaseq 6000. An aliquot of RNA samples was used to verify the WOI by the endometrial receptivity test “beREADY” (CCHT, Estonia). Gene Ontology and Reactome pathway enrichment analyses were conducted in ClueGO bioinformatics tool to study biological role behind obtained DEGs. Main results and the role of chance The R program language and Bioconductor packages were used to align generated RNA-seq reads on the human reference genome assembly (hg19) and to calculate gene expression differences between study groups using normalized counts per million (CPM)>10 in at least 10 samples. A total 233 DEGs (p < 0.05) was identified of which 126 genes were up- and 107 were down-regulated in adenomyosis compared to the control group. However, there was no significantly DEG according to the adjusted p-value. According to the beREADY test, all 20 samples were in receptive phase, however two samples were early-receptive and five were late-receptive. In a sensitivity analysis, all border receptive samples were removed and RNA-seq data sets were re-analysed only by 8 adenomyosis cases and 5 controls. A total of 382 DEGs (p < 0.05) were detected in adenomyosis group (216 up- and 166 down-regulated genes), again with no statistical difference between both groups after adjustment. Functional enrichment analyses of 233 and 382 DEGs identified pathways (adjusted p-value< 0.05) associated with positive regulation of exosomal secretion and expression of IFN-induced genes, respectively. The comparison of 233 and 382 DEGs revealed 28 common genes that may present stronger candidate of adenomyosis-related markers associated with endometrial receptivity. Limitations, reasons for caution Only mild adenomyosis was considered in this study, which is most commonly detected in women. The results could differ in women in severe cases of adenomyosis. Multicellular whole-tissue endometrial samples that were used for RNA isolation could mask gene expression differences of specific cell types between study groups. Wider implications of the findings: According to our results of transcriptome analysis, the presence of mild adenomyosis has no significant influence on the gene expression signature during endometrial receptivity in natural menstrual cycle. Women being investigated for infertility can be reassured that this common acquired anomaly does not significantly influence the chances of successful conception. Trial registration number 0120–259/2018/16

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Advancing clinical genomics and precision medicine with GVViZ: FAIR bioinformatics platform for variable gene-disease annotation, visualization, and expression analysis

Human Genomics ◽

10.1186/s40246-021-00336-1 ◽

2021 ◽

Vol 15 (1) ◽

Author(s):

Zeeshan Ahmed ◽

Eduard Gibert Renart ◽

Saman Zeeshan ◽

XinQi Dong

Keyword(s):

Data Analysis ◽

Patient Care ◽

Expression Analysis ◽

High Throughput ◽

Gene Annotation ◽

Next Generation Sequencing Data ◽

Rna Seq ◽

Sequencing Data ◽

Complex Disorders ◽

Transcriptomics Data

Abstract Background Genetic disposition is considered critical for identifying subjects at high risk for disease development. Investigating disease-causing and high and low expressed genes can support finding the root causes of uncertainties in patient care. However, independent and timely high-throughput next-generation sequencing data analysis is still a challenge for non-computational biologists and geneticists. Results In this manuscript, we present a findable, accessible, interactive, and reusable (FAIR) bioinformatics platform, i.e., GVViZ (visualizing genes with disease-causing variants). GVViZ is a user-friendly, cross-platform, and database application for RNA-seq-driven variable and complex gene-disease data annotation and expression analysis with a dynamic heat map visualization. GVViZ has the potential to find patterns across millions of features and extract actionable information, which can support the early detection of complex disorders and the development of new therapies for personalized patient care. The execution of GVViZ is based on a set of simple instructions that users without a computational background can follow to design and perform customized data analysis. It can assimilate patients’ transcriptomics data with the public, proprietary, and our in-house developed gene-disease databases to query, easily explore, and access information on gene annotation and classified disease phenotypes with greater visibility and customization. To test its performance and understand the clinical and scientific impact of GVViZ, we present GVViZ analysis for different chronic diseases and conditions, including Alzheimer’s disease, arthritis, asthma, diabetes mellitus, heart failure, hypertension, obesity, osteoporosis, and multiple cancer disorders. The results are visualized using GVViZ and can be exported as image (PNF/TIFF) and text (CSV) files that include gene names, Ensembl (ENSG) IDs, quantified abundances, expressed transcript lengths, and annotated oncology and non-oncology diseases. Conclusions We emphasize that automated and interactive visualization should be an indispensable component of modern RNA-seq analysis, which is currently not the case. However, experts in clinics and researchers in life sciences can use GVViZ to visualize and interpret the transcriptomics data, making it a powerful tool to study the dynamics of gene expression and regulation. Furthermore, with successful deployment in clinical settings, GVViZ has the potential to enable high-throughput correlations between patient diagnoses based on clinical and transcriptomics data.

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Endometrial Perivascular Progenitor Cells and Uterus Regeneration

Journal of Personalized Medicine ◽

10.3390/jpm11060477 ◽

2021 ◽

Vol 11 (6) ◽

pp. 477

Author(s):

Shiyuan Li ◽

Lijun Ding

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Embryo Implantation ◽

Endometrial Receptivity ◽

Perivascular Cells ◽

Asherman’S Syndrome ◽

Outermost Layer ◽

Adventitial Cells ◽

Structure And Functions ◽

Large Vessels

Ovarian steroid-regulated cyclical regeneration of the endometrium is crucial for endometrial receptivity and embryo implantation, and it is dependent on the dynamic remodeling of the endometrial vasculature. Perivascular cells, including pericytes surrounding capillaries and microvessels and adventitial cells located in the outermost layer of large vessels, show properties of mesenchymal stem cells, and they are thus promising candidates for uterine regeneration. In this review, we discuss the structure and functions of the endometrial blood vasculature and their roles in endometrial regeneration, the main biomarkers and characteristics of perivascular cells in the endometrium, and stem cell-based angiogenetic therapy for Asherman’s syndrome.

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Bu Shen Zhu Yun Decoction Improves Endometrial Receptivity via VEGFR-2-Mediated Angiogenesis

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2019/3949824 ◽

2019 ◽

Vol 2019 ◽

pp. 1-14 ◽

Cited By ~ 2

Author(s):

Li Li ◽

Huabo Jiang ◽

Xuecong Wei ◽

Dandan Geng ◽

Ming He ◽

...

Keyword(s):

Signaling Pathway ◽

Embryo Implantation ◽

Mapk Signaling ◽

Endometrial Receptivity ◽

Mapk Signaling Pathway ◽

Mitogen Activated Protein Kinase ◽

Cell Counting ◽

Nuclear Antigen ◽

Vitro Fertilization

Vascular endothelial growth factor receptor-2 (VEGFR-2) regulates the mitogen-activated protein kinase (MAPK) signaling pathway and plays an important role in angiogenesis. Bu Shen Zhu Yun decoction (BSZYD) can improve endometrial receptivity and embryo implantation rates in patients undergoing in vitro fertilization. However, whether BSZYD improves endometrial receptivity via angiogenesis remains unclear. Here, we investigated the effects of BSZYD on the proliferation, migration, and angiogenesis of human endometrial microvascular endothelial cells (HEMECs) and found that BSZYD upregulated the expression of cyclin D1, matrix metalloproteinase 9 (MMP9), and proliferating cell nuclear antigen (PCNA) in HEMECs. Cell Counting Kit 8 assay, scratch-wound assay, and Tube Formation Assay results showed that BSZYD promoted the proliferation, migration, and angiogenesis of HEMECs. Western blot analysis results revealed the activation of the MAPK signaling pathway by BSZYD through the upregulation of VEGF and VEGFR-2 expression. Together, these findings highlight the novel mechanism underlying BSZYD-mediated improvement in endometrial receptivity through the MAPK signaling pathway.

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Heparan Sulfate Proteoglycan Sulfation Regulates Uterine Differentiation and Signaling During Embryo Implantation

Endocrinology ◽

10.1210/en.2018-00105 ◽

2018 ◽

Vol 159 (6) ◽

pp. 2459-2472 ◽

Cited By ~ 2

Author(s):

Yan Yin ◽

Adam Wang ◽

Li Feng ◽

Yu Wang ◽

Hong Zhang ◽

...

Keyword(s):

Signal Transduction ◽

Heparan Sulfate ◽

Signaling Pathways ◽

Reproductive Tract ◽

Ovarian Function ◽

Embryo Implantation ◽

Uterine Epithelium ◽

Female Reproductive Tract ◽

Uterine Receptivity ◽

Mouse Uterus

Abstract To prepare for embryo implantation, the uterus must undergo a series of reciprocal interactions between the uterine epithelium and the underlying stroma, which are orchestrated by ovarian hormones. During this process, multiple signaling pathways are activated to direct cell proliferation and differentiation, which render the uterus receptive to the implanting blastocysts. One important modulator of these signaling pathways is the cell surface and extracellular matrix macromolecules, heparan sulfate proteoglycans (HSPGs). HSPGs play crucial roles in signal transduction by regulating morphogen transport and ligand binding. In this study, we examine the role of HSPG sulfation in regulating uterine receptivity by conditionally deleting the N-deacetylase/N-sulfotransferase (NDST) 1 gene (Ndst1) in the mouse uterus using the Pgr-Cre driver, on an Ndst2- and Ndst3-null genetic background. Although development of the female reproductive tract and subsequent ovarian function appear normal in Ndst triple-knockout females, they are infertile due to implantation defects. Embryo attachment appears to occur but the uterine epithelium at the site of implantation persists rather than disintegrates in the mutant. Uterine epithelial cells continued to proliferate past day 4 of pregnancy, accompanied by elevated Fgf2 and Fgf9 expression, whereas uterine stroma failed to undergo decidualization, as evidenced by lack of Bmp2 induction. Despite normal Indian hedgehog expression, transcripts of Ptch1 and Gli1, both components as well as targets of the hedgehog (Hh) pathway, were detected only in the subepithelial stroma, indicating altered Hh signaling in the mutant uterus. Taken together, these data implicate an essential role for HSPGs in modulating signal transduction during mouse implantation.

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