A Two-Cohort RNA-seq Study Reveals Changes in Endometrial and Blood miRNome in Fertile and Infertile 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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Decreased CD44v3 Expression Impairs Endometrial Stromal Cell Decidualization in Women With Recurrent Implantation Failure

10.21203/rs.3.rs-1043935/v1 ◽

2021 ◽

Author(s):

Xiaowei Zhou ◽

Yi Cao ◽

mingjuan Zhou ◽

Mi Han ◽

mengyu Liu ◽

...

Keyword(s):

Stromal Cells ◽

Culture System ◽

Embryo Implantation ◽

Group Identification ◽

Endometrial Receptivity ◽

Implantation Failure ◽

Recurrent Implantation Failure ◽

Secretory Phase ◽

Endometrial Stromal Cells ◽

Cd44 Isoforms

Abstract BackgroundThe precise pathogenesis of poor endometrial receptivity in recurrent implantation failure (RIF) still remains unclear. This study aims to explore the effects of different CD44 isoforms in the mid-secretory phase endometrium on endometrial receptivity in women with RIF.MethodsMid-secretory phase endometrial tissue samples were obtained from two groups of women who had undergone IVF: a) 24 patients with RIF, b) 18 patients with infertility due to tubal obstruction, who had achieved a successful clinical pregnancy after the first embryo transfer in IVF (control group). Identification of differentially expressed CD44 isoforms in endometrial tissues was assessed with immunohistochemistry, qPCR and western blotting. Effects of CD44v3 overexpression and knockdown on proliferation and decidualization of Immortalized human endometrial stromal cells (T-HESCs) and primary HESCs were investigated by qPCR and Western blot. A heterologous co-culture system of embryo implantation was constructed to mimics the process of trophoblast invasion during implantation.ResultsCD44v3 was significantly higher expressed in mid-secretory phase of endometrial stromal cells than proliferation phase, but was notably lower in RIF patients. The expression of decidualization markers, prolactin (PRL) and insulin like growth factor binding protein-1 (IGFBP1), was notably decreased following CD44v3 knockdown, whereas the expression levels of both PRL and IGFBP1 increased after CD44v3 overexpression in HESCs. Furthermore, the CD44v3-knockdown HESCs displayed a significantly deficiency in supporting trophoblast outgrowth through a co-culture system of embryo implantation; however, CD44v3 overexpression in HESCs promoted trophoblast outgrowth.ConclusionThe reduced expression of CD44v3 suppresses HESCs proliferation and decidualization, which might play a pivotal role in poor endometrial receptivity in women with RIF.

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Uterine fluid proteins for minimally invasive assessment of endometrial receptivity

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jcemdgz019 ◽

2019 ◽

Author(s):

Sergo Kasvandik ◽

Merilin Saarma ◽

Tanel Kaart ◽

Ilmatar Rooda ◽

Agne-Velthut Meikas ◽

...

Keyword(s):

Minimally Invasive ◽

Embryo Transfer ◽

Endometrial Receptivity ◽

Uterine Receptivity ◽

Recurrent Implantation Failure ◽

Secretory Phase ◽

Minimal Invasiveness ◽

Specificity And Sensitivity ◽

Uterine Fluid ◽

Secretory Endometrium

Abstract Context Clinically used endometrial (EM) receptivity assays are based on transcriptomic patterning of biopsies at mid-secretory endometrium (MSE) to identify the possible displacement or disruption of window of implantation (WOI) in patients with recurrent implantation failure (RIF). However, biopsies are invasive and cannot be performed in the same cycle with IVF embryo transfer, while uterine fluid (UF) analysis is considered minimally invasive and can immediately precede embryo transfer. Objective To determine whether UF proteome can be used for WOI monitoring and whether it would highlight the etiology of RIF. Patients Paired early secretory endometrial (ESE) and MSE UF samples from six fertile control women for discovery, and additional 11 paired ESE/MSE samples from controls and 29 MSE samples from RIF patients for validation. Results Using discovery mass spectrometry (MS) proteomics we detected 3,158 proteins from secretory phase UF of which 367 undergo significant (q < 0.05) proteomic changes while transitioning from ESE to MSE. 45 proteins were further validated with targeted MS, and 21 were found to display similar levels between control ESE and RIF MSE, indicating displacement of the WOI. Panel of PGR, NNMT, SLC26A2 and LCN2 demonstrated specificity and sensitivity of 91.7% for distinguishing MSE from ESE samples. The same panel distinguished control MSE samples from RIF MSE with a 91.7% specificity and 96.6% sensitivity. Conclusion UF proteins can be used for estimating uterine receptivity with minimal invasiveness. Women with RIF appear to have altered MSE UF profiles that may contribute to their low IVF success rate.

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Progesterone Receptor B (PGR-B) Is Partially Methylated in Eutopic Endometrium From Infertile Women With Endometriosis

Reproductive Sciences ◽

10.1177/1933719119828078 ◽

2019 ◽

Vol 26 (12) ◽

pp. 1568-1574 ◽

Cited By ~ 4

Author(s):

Carlos Valério Rocha-Junior ◽

Michele Gomes Da Broi ◽

Cristiana Libardi Miranda-Furtado ◽

Paula Andrea Navarro ◽

Rui Alberto Ferriani ◽

...

Keyword(s):

Dna Methylation ◽

Progesterone Receptor ◽

Embryo Implantation ◽

Methylation Pattern ◽

Endometrial Receptivity ◽

Control Group ◽

Secretory Phase ◽

Eutopic Endometrium ◽

Infertile Women ◽

Partially Methylated

Endometriosis is frequently related to infertility and little is known about the mechanisms underlying this association. Some studies point to an endometrial factor involved in this condition, which could compromise embryo implantation. Progesterone plays crucial role in endometrial receptivity by acting through progesterone receptor ( PGR) isoforms PR-A and PR-B whose expression is epigenetically regulated by DNA methylation, in a specific promoter region for each isoform. Epigenetic changes in PGR-A and PGR-B may be related to progesterone resistance of endometriosis-related infertility. In order to better understand the mechanisms involved in endometrial receptivity, this case–control study aimed to compare the methylation pattern of PGR-A and PGR-B in eutopic endometrium from infertile women with and without endometriosis during the secretory phase. Endometrial biopsies from 19 patients (10 infertile women with endometriosis and 9 infertile controls) with regular cycles were performed during the secretory phase and were dated according to Noyes’ criteria. The percentage of DNA methylation at PGR-A and PGR-B was carried out by high-resolution melting assay. The PGR-A gene showed 0% of DNA methylation (unmethylated) in both control and endometriosis groups. However, PGR-B gene showed a partially methylated pattern in majority of the patients (n = 7), with methylation percentage corresponding to 50%, while in the control group the percentage of methylation was 20% (hypomethylated; P = .04). The increased percentage of methylation at PGR-B may be related to reduced gene expression, which could compromise the endometrial receptivity in patients with endometriosis.

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Uterine fluid proteins for minimally invasive assessment of endometrial receptivity

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jcem/dgz019 ◽

2019 ◽

Cited By ~ 1

Author(s):

Sergo Kasvandik ◽

Merilin Saarma ◽

Tanel Kaart ◽

Ilmatar Rooda ◽

Agne-Velthut Meikas ◽

...

Keyword(s):

Minimally Invasive ◽

Embryo Transfer ◽

Endometrial Receptivity ◽

Uterine Receptivity ◽

Recurrent Implantation Failure ◽

Secretory Phase ◽

Minimal Invasiveness ◽

Specificity And Sensitivity ◽

Uterine Fluid ◽

Secretory Endometrium

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Increased METTL3-mediated m6A methylation inhibits embryo implantation by repressing HOXA10 expression in recurrent implantation failure

Reproductive Biology and Endocrinology ◽

10.1186/s12958-021-00872-4 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Pingping Xue ◽

Wenbo Zhou ◽

Wenqiang Fan ◽

Jianya Jiang ◽

Chengcai Kong ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Embryo Implantation ◽

Endometrial Receptivity ◽

Mrna Levels ◽

Implantation Failure ◽

Quantitative Real Time Pcr ◽

Recurrent Implantation Failure ◽

Ishikawa Cells ◽

Embryo Attachment

Abstract Background Recurrent implantation failure (RIF) is a major limitation of assisted reproductive technology, which is associated with impaired endometrial receptivity. Although N6-methyladenosine (m6A) has been demonstrated to be involved in various biological processes, its potential role in the endometrium of women with RIF has been poorly studied. Methods Global m6A levels and major m6A methyltransferases/demethylases mRNA levels in mid-secretory endometrium from normal and RIF women were examined by colorimetric m6A quantification strategy and quantitative real-time PCR, respectively. The effects of METTL3-mediated m6A modification on embryo attachment were evaluated by an vitro model of a confluent monolayer of Ishikawa cells co-cultured with BeWo spheroids, and the expression levels of homeo box A10 (HOXA10, a well-characterized marker of endometrial receptivity) and its downstream targets were evaluated by quantitative real-time PCR and Western blotting in METTL3-overexpressing Ishikawa cells. The molecular mechanism for METTL3 regulating HOXA10 expression was determined by methylated RNA immunoprecipitation assay and transcription inhibition assay. Results Global m6A methylation and METTL3 expression were significantly increased in the endometrial tissues from women with RIF compared with the controls. Overexpression of METTL3 in Ishikawa cells significantly decreased the ration of BeWo spheroid attachment, and inhibited HOXA10 expression with downstream decreased β3-integrin and increased empty spiracles homeobox 2 expression. METTL3 catalyzed the m6A methylation of HOXA10 mRNA and contributed to its decay with shortened half-life. Enforced expression of HOXA10 in Ishikawa cells effectively rescued the impairment of METTL3 on the embryo attachment in vitro. Conclusion Increased METTL3-mediated m6A modification represents an adverse impact on embryo implantation by inhibiting HOXA10 expression, contributing to the pathogenesis of RIF.

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ORIGINAL ARTICLE: High Expression of l-Selectin Ligand in Secretory Endometrium is Associated with Better Endometrial Receptivity and Facilitates Embryo Implantation in Human Being

American Journal of Reproductive Immunology ◽

10.1111/j.1600-0897.2008.00604.x ◽

2008 ◽

Vol 60 (2) ◽

pp. 127-134 ◽

Cited By ~ 26

Author(s):

Bo Wang ◽

Jian-Zhong Sheng ◽

Rong-Huan He ◽

Yu-Li Qian ◽

Fan Jin ◽

...

Keyword(s):

Embryo Implantation ◽

Endometrial Receptivity ◽

High Expression ◽

Human Being ◽

Selectin Ligand ◽

Secretory Endometrium

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hsa_circ_001946 elevates HOXA10 expression and promotes the development of endometrial receptivity via sponging miR-135b

Diagnostic Pathology ◽

10.1186/s13000-021-01104-4 ◽

2021 ◽

Vol 16 (1) ◽

Author(s):

Fang Zhao ◽

Yihong Guo ◽

Zhanrong Shi ◽

Menglan Wu ◽

Yuzhen Lv ◽

...

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Western Blotting ◽

Embryo Implantation ◽

Endometrial Receptivity ◽

Luciferase Reporter ◽

Implantation Failure ◽

Recurrent Implantation Failure ◽

Tissue Samples ◽

Qrt Pcr

Abstract Background Impaired endometrial receptivity is a major reason for embryo implantation failure. There’s a paucity of information regarding the role of circRNAs on endometrial receptivity. Here, we investigated the function of hsa_circ_001946 on endometrial receptivity and its mechanisms. Methods A total of 50 women composing 25 with recurrent implantation failure and 25 who conceived after their implantation were recruited in this study. Expression of hsa_circ_001946, miR-135b, and HOXA10 was evaluated by quantitative RT-PCR (qRT-PCR) in biopsied endometrial tissue samples. The levels of HOXA10, and cell cycle markers (CCNB1, CDK1, and CCND1) were determined by IHC and western blotting assays. Binding relationship among miR-135b, hsa_circ_001946 and HOXA10 were confirmed by dual luciferase reporter assays and western blotting. MTT assays and cell cycle assays by FACS were employed to evaluate the proliferation and cell cycle of cells. T-HESCs were cultured with 1 µM medroxyprogesterone acetate (MPA) and 0.5 mM 8-bromoadenosine 3’:5’-cyclic monophosphate (8-Br-cAMP) to induce decidualization. The mechanisms and functions of hsa_circ_001946 on decidualization were further assessed by qRT-PCR evaluating the expression of hsa_circ_001946, miR-135b, HOXA10 and decidual markers (PRL and IGFBP1) in T-HESCs. Results Endometrial tissues from patients with recurrent implantation failure had lower hsa_circ_001946 expression, higher miR-135b expression, and lower HOXA10 expression. Hsa_circ_001946 promoted HOXA10 expression by sponging miR-135b in T-HESCs. Overexpression of hsa_circ_001946 restored cell proliferation and cell cycle that were disrupted by miR-135b overexpression in T-HESCs. Decidualized T-HESCs had higher hsa_circ_001946 expression, lower miR-135b expression, and higher HOXA10 expression. Overexpression of hsa_circ_001946 reversed the expression of decidual markers (PRL and IGFBP1) that were suppressed by miR-135b overexpression in T-HESCs. Conclusions In conclusion, our findings suggest that hsa_circ_001946 promotes cell proliferation and cell cycle process and increases expression of decidualization markers to enhance endometrial receptivity progression via sponging miR-135b and elevating HOXA10.

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MicroRNA Biogenesis Machinery Is Dysregulated in the Endometrium of Infertile Women Suggesting a Role in Receptivity and Infertility

Journal of Histochemistry & Cytochemistry ◽

10.1369/0022155419854064 ◽

2019 ◽

Vol 67 (8) ◽

pp. 589-599 ◽

Cited By ~ 3

Author(s):

Hannah Loke ◽

Kate Rainczuk ◽

Evdokia Dimitriadis

Keyword(s):

Menstrual Cycle ◽

Staining Intensity ◽

Unexplained Infertility ◽

Rt Pcr ◽

Secretory Phase ◽

Infertile Women ◽

Normal Fertility ◽

Human Menstrual Cycle ◽

Late Secretory Phase ◽

Fertile Women

MicroRNAs (miRs) regulate endometrial function and their dysregulation could underlie unexplained infertility in women. Ribonucleases including DICER and DROSHA, and the proteins, ARGONAUTE 1 (AGO 1) and 2 (AGO 2) regulate the biogenesis/maturation of miRs. We aimed to elucidate the expression and localization of miR biogenesis machinery components during the human menstrual cycle and compare their levels in endometrium from women with normal fertility and primary unexplained infertility. miR biogenesis components were measured by quantitative-RT-PCR and immunohistochemistry. In the endometrium of women with normal fertility, DROSHA immunolocalized maximally to the epithelium during the early and mid-secretory phases compared with the proliferative and late-secretory phases. Stromal DICER immunostaining intensity was higher in the late-secretory phase compared with all other phases in fertile women. DROSHA mRNA was reduced in the mid-secretory-infertile whole endometrial tissue (has all cells of the tissue), and primary epithelial and stromal cells while no differences were found in DICER, AGO1, and AGO2 mRNA. In the luminal epithelium, DROSHA staining intensity was reduced in early and mid-secretory-infertile while DICER staining was reduced in the early secretory-infertile compared with their respective fertile groups. DICER and DROSHA were dynamically regulated across the menstrual cycle and reduced levels during receptivity phase could underlie implantation failure/infertility.

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WOMEN IN REPRODUCTIVE SCIENCE: My WOMBan’s life: understanding human endometrial function

Reproduction ◽

10.1530/rep-18-0518 ◽

2019 ◽

Vol 158 (6) ◽

pp. F55-F67 ◽

Cited By ~ 3

Author(s):

Lois A Salamonsen

Keyword(s):

Uterine Cavity ◽

Abnormal Uterine Bleeding ◽

Endometrial Receptivity ◽

Uterine Bleeding ◽

Menstrual Disorders ◽

Secretory Phase ◽

Infertile Women ◽

Proliferative Phase ◽

Cellular Changes ◽

Reproductive Science

The focus of my life in science has been to improve reproductive health for women, with an emphasis on the endometrium, the most dynamic tissue in the human body: its remarkable cyclical remodelling is essential for the establishment of pregnancy. The most notable events in a woman’s endometrial cycle are menstruation and endometrial repair, regeneration of the endometrium during the proliferative phase, attainment of receptivity by the mid-secretory phase of the cycle and the embryo–maternal interactions that initiate peri-implantation events within the microenvironment of the uterine cavity. I have contributed to understanding the molecular and cellular changes underpinning these events, and how disturbance of them leads to menstrual disorders, infertility and endometrial diseases including abnormal uterine bleeding, endometriosis and endometrial cancer. My team has contributed to changes in clinical IVF practice, to a new diagnostic for endometrial receptivity in infertile women and to enhancing endometrial repair. I have shared my world with many amazing younger scientists: it has indeed been a privileged journey.

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Transcriptomics of receptive endometrium in women with sonographic features of adenomyosis

Reproductive Biology and Endocrinology ◽

10.1186/s12958-021-00871-5 ◽

2022 ◽

Vol 20 (1) ◽

Author(s):

Erika Prašnikar ◽

Tanja Kunej ◽

Mario Gorenjak ◽

Uroš Potočnik ◽

Borut Kovačič ◽

...

Keyword(s):

Embryo Implantation ◽

Endometrial Receptivity ◽

Molecular Organization ◽

Cycle Phase ◽

P Value ◽

Rna Seq ◽

Uterine Receptivity ◽

Tumour Necrosis Factor Production ◽

Transcriptomics Data ◽

Induced Genes

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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