Endometrial Stromal Decidualization Research Articles

Decreased thrombospondin-1 impairs endometrial stromal decidualization in unexplained recurrent spontaneous abortion†.

Inappropriate endometrial stromal decidualization has been implied as an important reason of many pregnancy-related complications, such as unexplained recurrent spontaneous abortion, preeclampsia, and intrauterine growth restriction. Here, we observed that thrombospondin-1, an adhesive glycoprotein, was significantly downregulated in endometrial decidual cells from patients with unexplained recurrent spontaneous abortion. The immortalized human endometrial stromal cell line was used to investigate the possible THBS1-mediated regulation of decidualization. In vitro experiments found that the expression level of THBS1 increased with the normal decidualization process. Knockdown of THBS1 could decrease the expression levels of prolactin and insulin-like growth factor binding protein-1, two acknowledged human decidualization markers, whereas THBS1 overexpression could reverse these effects. The RNA sequencing results demonstrated that the extracellular regulated protein kinases signaling pathway was potentially affected by the knockdown of THBS1. We further confirmed that the regulation of THBS1 on decidualization was achieved through the ERK signaling pathway by the treatment of inhibitors. Moreover, knockdown of THBS1 in pregnant mice could impair decidualization and result in an increased fetus resorption rate. Altogether, our study demonstrated a crucial role of THBS1 in the pathophysiological process of unexplained recurrent spontaneous abortion and provided some new insights into the research of pregnancy-related complications.

THU569 Steroid Receptor Coactivator 3 Is Required To Prevent Early Pregnancy Loss In The Mouse

Abstract Disclosure: V. Maurya: None. L. Hai: None. M.M. Szwarc: None. W. San-Pin: None. F.J. DeMayo: None. J.P. Lydon: None. Members of the Steroid Receptor Coactivator (SRC) family enact distinct transcriptional coregulator functions within a broad spectrum of physiologies and pathologies. We previously demonstrated that endometrial SRC-3 is critical for in vitro human endometrial stromal cell decidualization, a critical cellular process in the establishment of the maternofetal interface. To address SRC-3’s in vivo role in endometrial periimplantation biology, we recently generated a SRC-3 conditional knockout mouse model (SRC-3d/d) in which SRC-3 is selectively ablated in cells that express the progesterone receptor. A six-month breeding trial with wild type proven stud males demonstrated that the SRC-3d/d female fails to produce litters (n>10 per genotype). Unlike the SRC-2d/d female, however, both embryo attachment and implantation into the stromal compartment of the SRC-3d/d endometrium are not impaired at gestation day (GD) 5 or 6. While endometrial stromal decidualization initiates with embryo invasion, pregnant SRC-3d/d mice exhibit early pregnancy loss (EPL) by GD-7.5 (before placentation). At GD-6, the SRC-3d/d decidua is significantly smaller than those of sibling controls. Histological analyses reveal extensive decidual cell death in the SRC-3d/d decidua by GD-7, which results in the sudden demise of the embryo. These histological findings are supported by recent genome-wide RNA profiling studies that show a pro-survival role for SRC-3 in this tissue. Based on these results, we conclude that endometrial SRC-3 is critical for maintaining the functionality and survival of the decidual stromal cell and embryo developmental progression to placentation. Therefore, endometrial SRC-3 (and its downstream signaling networks) are predicted to represent new molecular determinants of non-aneuploid EPL, a poorly understood pregnancy complication for which diagnostic and treatment options are limited. Presentation Date: Thursday, June 15, 2023

Dysregulated Gln-Glu-α-ketoglutarate axis impairs maternal decidualization and increases the risk of recurrent spontaneous miscarriage

Recurrent spontaneous miscarriage (RSM) affects 1%-2% of fertile women worldwide and poses a risk of future pregnancy complications. Increasing evidence has indicated that defective endometrial stromal decidualization is a potential cause of RSM. Here, we perform liquid chromatography with mass spectrometry (LC-MS)-based metabolite profiling in human endometrial stromal cells (ESCs) and differentiated ESCs (DESCs) and find that accumulated α-ketoglutarate (αKG) derived from activated glutaminolysis contributes to maternal decidualization. Contrarily, ESCs obtained from patients with RSM show glutaminolysis blockade and aberrant decidualization. We further find that enhanced Gln-Glu-αKG flux decreases histone methylation and supports ATP production during decidualization. Invivo, feeding mice a Glu-free diet leads to a reduction of αKG, impaired decidualization, and an increase of fetal loss rate. Isotopic tracing approaches demonstrate Gln-dependent oxidative metabolism as a prevalent direction during decidualization. Our results demonstrate an essential prerequisite of Gln-Glu-αKG flux to regulate maternal decidualization, suggesting αKG supplementation as a putative strategy to rectify deficient decidualization in patients with RSM.

MAX deficiency impairs human endometrial decidualization through down-regulating OSR2 in women with recurrent spontaneous abortion

Human uterine stromal cell undergoes decidualization for pregnancy establishment and maintenance, which involved extensive proliferation and differentiation. Increasing studies have suggested that recurrent spontaneous abortion (RSA) may result from defective endometrial stromal decidualization. However, the critical molecular mechanisms underlying impaired decidualization during RSA are still elusive. By using our recently published single-cell RNA sequencing (scRNA-seq) atlas, we found that MYC-associated factor X (MAX) was significantly downregulated in the stromal cells derived from decidual tissues of women with RSA, followed by verification with immunohistochemistry (IHC) and quantitative real-time polymerase chain reaction (qRT-PCR). MAX knockdown significantly impairs human endometrial stromal cells (HESCs) proliferation as determined by MTS assay and Ki67 immunostaining, and decidualization determined by F-actin, and decidualization markers. RNA-seq together with chromatin immunoprecipitation sequencing (ChIP-seq) and cleavage under targets and release using nuclease sequencing (CUT&RUN-seq) analysis were applied to explore the molecular mechanisms of MAX in regulation of decidualization, followed by dual-luciferase reporter assay to verify that MAX targets to (odd-skipped related transcription factor 2) OSR2 directly. Reduced expression of OSR2 was also confirmed in decidual tissues in women with RSA by IHC and qRT-PCR. OSR2 knockdown also significantly impairs HESCs decidualization. OSR2-overexpression could at least partly rescue the downregulated insulin-like growth factor binding protein 1 (IGFBP1) expression level in response to MAX knockdown. Collectively, MAX deficiency observed in RSA stromal cells not only attenuates HESCs proliferation but also impairs HESCs decidualization by downregulating OSR2 expression at transcriptional level directly.

Scribble downregulation in adenomyosis compromises endometrial stromal decidualization by decreasing FOXO1 expression.

Does Scribble (SCRIB) contribute to aberrant decidualization of endometrial stromal cells (ESC) in adenomyosis? SCRIB knockdown impairs decidualization of ESC by decreasing Fork-head box O1A (FOXO1) expression through the protein kinase B (AKT) and atypical protein kinase C (aPKC) activated pathways. Stromal SCRIB is required for primary decidual zone formation and pregnancy success in mice. In our previous studies, decidualization was dampened in ESC isolated from adenomyosis patients, yet the underlying molecular mechanisms remain elusive. Eutopic endometrium tissue samples from diffuse adenomyosis and non-adenomyosis patients in proliferative, early-secretory and mid-secretory phase (n = 10 per phase for each group) were explored. In parallel, in vitro decidualization studies were carried out in ESC isolated from non-adenomyosis women (n = 8). The endometrial SCRIB expression was analyzed using immunohistochemistry staining and western blot. Quantitative RT-PCR (qRT-PCR), western blot and immunofluorescence staining were used to explore the expression of SCRIB in ESC during in vitro decidualization. siRNA-mediated SCRIB knockdown followed by decidual markers expression analysis, flow cytometry for cell cycle analysis and phalloidin staining for morphological analysis were performed to examine the function of SCRIB in ESC decidualization. RNA-sequencing was performed to examine the SCRIB-mediated transcriptional changes in decidualized ESC (DSC). Rescue experiments using an AKT inhibitor MK2206 and aPKC inhibitor NSC37044 were used to investigate the signaling pathways through which could mediate SCRIB-regulated FOXO1 protein expression and ESC decidualization. We found that the expression of SCRIB in the mid-secretory phase eutopic endometrial stroma of adenomyosis patients was significantly lower than that of non-adenomyosis. SCRIB knockdown reduced the expression of decidual markers, abrogated the epithelioid-like morphological changes, inhibited the mesenchymal-to-epithelial transitions process and promoted the cell cycle progression of ESC during in vitro decidualization. SCRIB knockdown-induced decidualization defects were attributed to a decrease in expression of transcription factor FOXO1, known to regulate decidualization. Furthermore, we found that SCRIB knockdown induced the aberrant activation of AKT and aPKC, which led to FOXO1 phosphorylation and degradation. Rescue assay confirmed that restoring the expression of FOXO1 effectively reversed the decidualization defects and cell cycle progression caused by SCRIB knockdown. N/A. In this study, it was demonstrated that SCRIB knockdown mediated the activation of AKT and aPKC, contributing to FOXO1 degradation and aberrant decidualization, however, the molecular link between AKT and aPKC signaling was not determined, and still requires further exploration. Our findings support the hypothesis that adenomyosis interferes with embryo implantation due to insufficient endometrial receptivity. Abnormal decidualization of the endometrial stroma may clarify the possible association between adenomyosis and infertility. Our findings may be clinically useful for counseling and treatment of infertile adenomyosis patients. This study was supported by the National Natural Science Foundation of China (82001523 and 82171639). The authors have no conflicts of interest to disclose. N/A.

Osteoprotegerin interacts with syndecan-1 to promote human endometrial stromal decidualization by decreasing Akt phosphorylation.

Does osteoprotegerin (OPG) promote human endometrial stromal decidualization? OPG is essential for human endometrial stromal decidualization through its interaction with syndecan-1 to decrease Akt phosphorylation. OPG (a cytokine receptor) levels are significantly increased in the circulation of pregnant women. However, the role and mechanism of OPG in human endometrial stromal cell (ESC) decidualization remain elusive. We analyzed the endometrial expression of OPG in endometrial tissue samples collected from women with regular menstrual cycles (ranging from 25 to 35 days), and decidual tissue samples collected from woman with normal early pregnancy or recurrent pregnancy loss (RPL) who visited the Department of Gynecology and Obstetrics at a tertiary care center from January to October 2018. None of the subjects had hormonal treatment for at least 3 months prior to the procedure. In total, 16 women with normal early pregnancy and 15 with RPL were selected as subjects for this study. The function of OPG in decidualization was explored in a human endometrial stromal cell (HESC) line and primary cultures of HESCs. We collected endometrial tissues (by biopsy) from the subjects during their menstrual cycle and decidual tissues from subjects with a normal early pregnancy and those with RPL at the time of dilation and curettage. The control group comprised randomly selected women who underwent termination of an apparently normal early pregnancy. The endometrial OPG expression was analyzed using immunohistochemical staining and quantitative RT-PCR (qRT-PCR). Immunofluorescence staining and western blot, and qRT-PCR were used to explore the mRNA and protein expression, respectively, of OPG in an immortalized HESC line and in primary cultures of HESC during proliferation and decidualization. siRNA-mediated knockdown experiments were performed to examine the function of OPG in HESC proliferation and decidualization. Flow cytometry and the cell proliferation MTS assay were performed to further examine the role of OPG in HESC proliferation. We also analyzed decidual marker gene expression by qRT-PCR to assess the consequences of OPG loss for HESC decidualization. A co-immunoprecipitation (IP) assay was used to determine the potential interaction between the OPG and Syndecan-1. Western blot analysis of the rescue experiments performed using the phosphatidylinositol 3-kinase (PI3K) signaling-specific inhibitor LY294002 was used to investigate the downstream signaling pathways through which OPG could mediate HESC decidualization. OPG was expressed in both the human endometrium and in vitro decidualized ESCs. Knockdown experiments revealed that OPG loss impaired the expression of IGF-binding protein-1 (IGFBP-1) (P < 0.05) and prolactin (PRL) (P < 0.05), two specific markers of decidualization, in HESC undergoing decidualization. We also uncovered that OPG knockdown induced the aberrant activation of Akt (protein kinase B) during HESC decidualization (P < 0.05). The inhibition of Akt activation could rescue the impaired expression of the decidual markers PRL (P < 0.05) and IGFBP-1 (P < 0.05) in response to OPG knockdown. Syndecan-1 was considered a potential receptor candidate, as it was expressed in both the endometrium and in vitro cultured stromal cells. Subsequent co-IP experiments demonstrated the interaction between OPG and Syndecan-1 during decidualization. In addition, Syndecan-1 knockdown not only clearly attenuated the decidualization markers PRL (P < 0.05) and IGFBP-1 (P < 0.05) but also induced the aberrant enhancement of Akt phosphorylation in decidualized cells, consistent with the phenotype of OPG knockdown cells. Finally, we revealed that the transcript and protein expression of both OPG and Syndecan-1 was significantly lower in the decidual samples of women with RPL than in those of women with normal pregnancy (P < 0.05). N/A. In this study, based on a number of approaches, it was demonstrated that OPG mediated the repression of Akt that occurs during human stromal cell decidualization, however, the molecular link between OPG and Akt signaling was not determined, and still requires further exploration. OPG is required for decidualization, and a decrease in OPG levels is associated with RPL. These findings provide a new candidate molecule for the diagnosis and potential treatment of RPL. This work was supported in part by the National Natural Science Foundation of China U1605223 (to G.S.), 81701457 (to Y.J.) and 81601349 (to Y.J.). The authors have no conflicts of interest to disclose.

Osteoprotegerin (OPG) Interacts with Syndecan-1 to Mediate Human Endometrial Stromal Decidualization &lt;i&gt;via&lt;/i&gt; Decreasing Akt Phosphorylation

Methods: Human endometrial tissues from proliferative stage to secretory stage of normal menstrual cycle, and decidua tissues from normal pregnancy and recurrent pregnancy loss patients were collected to analyze the expression of OPG. Human endometrial stromal cells (HESCs) and primary human endometrial stromal cells (ESCs) were performed to explore the function and mechanism of OPG during decidualization. Findings: OPG expressed in human endometrium across menstrual cycle and during the human endometrial stromal cell decidualization. Knockdown experiments revealed that decrease of OPG impaired the expression of decidual markers prolactin (PRL) and insulin-like growth factor binding protein-1 (IGFBP-1) without interference with the cell proliferation. Knockdown of OPG increased the phosphorylation of Akt and ERK1/2 during HESCs decidualization. Inhibition of PI3K signaling can rescue the impaired expression of decidual markers PRL and IGFBP-1 in response to OPG knockdown, but the ERK1/2 inhibition cannot. Further experiments revealed that OPG mediates human endometrial stromal decidualization by interacting with Syndecan-1 via decreasing Akt phosphorylation. Finally, endometrial expression of OPG was significantly lower in recurrent pregnancy loss (RPL) endometrial samples than that in normal pregnancy samples during the first trimester. Interpretation: OPG is required for decidualization, and the decrease of OPG is associated with recurrent pregnancy loss. These findings would provide a new candidate for diagnosis and treatment of infertility in women. Funding: This work was supported by the National Natural Science Foundation of China. Declaration of Interest: The authors declare that there is no conflict of interests. Ethical Approval: This study was approved by the Medical Ethics Committee of The First Affiliated Hospital of Xiamen University and written informed consent was obtained from all participants in accordance with the guidelines in The Declaration of Helsinki 2000.

Loss of high-mobility group N5 contributes to the promotion of human endometrial stromal cell decidualization.

PurposeHigh‐mobility group N (HMGN) proteins are the only non‐histone proteins that specifically bind within the nucleosome between core histones and DNA. Among them, HMGN5 is one of the candidates that could participate in mouse endometrial decidualization; however, the specific role of HMGN5 remains to be clarified in human endometrial stromal cells (HESCs).MethodsPrimary HESCs were isolated from hysterectomy specimens and incubated with or without 8‐bromo‐cyclic adenosine monophosphate (8‐br‐cAMP) and medroxyprogesterone acetate (MPA).ResultsWe demonstrated that HMGN5 expression in decidualized HESCs stimulated by 8‐br‐cAMP and MPA decreased significantly. The inhibition of HMGN5 expression by small interfering RNA (siRNA) induced the major decidual marker genes expression, including IGFBP1 (insulin‐like growth factor binding protein 1) and PRL (prolactin). In addition, microRNA‐542‐3p (miR‐542‐3p), which was identified as a regulatory miRNA of IGFBP1 during decidualization, was significantly suppressed by HMGN5 siRNA. However, the expression of HMGN5 was not alternated by miR‐542‐3p overexpression.ConclusionsThese findings suggest that the down‐regulation of HMGN5 plays a role in the promotion of human endometrial stromal decidualization and acts upstream of miR‐542‐3p.

Growth regulation by estrogen in breast cancer 1 (GREB1) is a novel progesterone-responsive gene required for human endometrial stromal decidualization.

Is Growth Regulation by Estrogen in Breast Cancer 1 (GREB1) required for progesterone-driven endometrial stromal cell decidualization? GREB1 is a novel progesterone-responsive gene required for progesterone-driven human endometrial stromal cell (HESC) decidualization. Successful establishment of pregnancy requires HESCs to transform from fibroblastic to epithelioid cells in a process called decidualization. This process depends on the hormone progesterone, but the molecular mechanisms by which it occurs have not been determined. Primary and transformed HESCs in which GREB1 expression was knocked down were decidualized in culture for up to 6 days. Wild-type and progesterone receptor (PR) knockout mice were treated with progesterone, and their uteri were assessed for levels of GREB1 expression. Analysis of previous data included data mining of expression profile data sets and in silico transcription factor-binding analysis. Endometrial biopsies obtained from healthy women of reproductive age during the proliferative phase (Days 8-12) of their menstrual cycle were used for isolating HESCs. Experiments were carried out with early passage (no more than four passages) HESCs isolated from at least three subjects. Transcript levels of decidualization markers prolactin (PRL) and insulin-like growth factor-binding protein-1 (IGFBP-1) were detected by quantitative RT-PCR as readouts for HESC decidualization. Cells were also imaged by phase-contrast microscopy. To assess the requirement for GREB1, PR and SRC-2, cells were transfected with specifically targeted small interfering RNAs. Results are shown as mean and SE from three replicates of one representative patient-derived primary endometrial cell line. Experiments were also conducted with transformed HESCs. Progesterone treatment of mice and transformed HESCs led to an ~5-fold (5.6 ± 0.81, P < 0.05, and 5.2 ± 0.26, P < 0.01, respectively) increase in GREB1 transcript levels. This increase was significantly reduced in the uteri of PR knock-out mice (P < 0.01), in HESCs treated with the PR antagonist RU486 (P < 0.01), or in HESCs in which PR expression was knocked down (P < 0.05). When GREB1 expression was knocked down, progesterone-driven decidualization markers in both immortalized and primary HESCs was significantly reduced (P < 0.05 and P < 0.01). Finally, GREB1 knock down signficantly reduced expression of the PR target genes WNT4 and FOXOA1 (P < 0.05 and P < 0.01, respectively). This study used the Nuclear Receptor Signaling Atlas. Although in vitro cell culture studies indicate that GREB1 is required for endoemtrial decidualization, the in vivo role of GREB1 in endometrial function and dysfunction should be assessed by using knock-out mouse models. Identification and functional analysis of GREB1 as a key molecular mediator of decidualization may lead to improved diagnosis and clinical management of women with peri-implantation loss due to inadequate endometrial decidualization. This research was funded in part by: a National Institutes of Health (NIH)/ National Institute of Child Health and Human Development (NICHD) grant (R00 HD080742) and Washington University School of Medicine start-up funds to R.K., an NIH/NICHD grant (RO1 HD-07857) to B.W.O.M., and a NIH/NICHD grant (R01 HD-042311) to J.P.L. The authors declare no conflicts of interests.

Endometrial Stromal Decidualization Responds Reversibly to Hormone Stimulation and Withdrawal.

Human endometrial stromal decidualization is required for embryo receptivity, angiogenesis, and placentation. Previous studies from our laboratories established that connexin (Cx)-43 critically regulates endometrial stromal cell (ESC) differentiation, whereas gap junction blockade prevents it. The current study evaluated the plasticity of ESC morphology and Cx43 expression, as well as other biochemical markers of cell differentiation, in response to decidualizing hormones. Primary human ESC cultures were exposed to 10 nM estradiol, 100 nM progesterone, and 0.5 mM cAMP for up to 14 days, followed by hormone withdrawal for 14 days, mimicking a biphasic ovulatory cycle. Reversible differentiation was documented by characteristic changes in cell shape. Cx43 was reversibly up- and down-regulated after the estradiol, progesterone, and cAMP treatment and withdrawal, respectively, paralleled by fluctuations in prolactin, vascular endothelial growth factor, IL-11, and glycodelin secretion. Markers of mesenchymal-epithelial transition (MET), and its counterpart epithelial-mesenchymal transition, followed reciprocal patterns corresponding to the morphological changes. Incubation in the presence of 18α-glycyrrhetinic acid, an inhibitor of gap junctions, partially reversed the expression of decidualization and MET markers. In the absence of hormones, Cx43 overexpression promoted increases in vascular endothelial growth factor and IL-11 secretion, up-regulated MET markers, and reduced N-cadherin, an epithelial-mesenchymal transition marker. The combined results support the hypothesis that Cx43-containing gap junctions and endocrine factors cooperate to regulate selected biomarkers of stromal decidualization and MET and suggest roles for both phenomena in endometrial preparation for embryonic receptivity.

FOXO1 is required for binding of PR on IRF4, novel transcriptional regulator of endometrial stromal decidualization.

The forkhead box O1A (FOXO1) is an early-induced target of the protein kinase A pathway during the decidualization of human endometrial stromal cells (HESCs). In this study we identified the cistrome and transcriptome of FOXO1 and its role as a transcriptional regulator of the progesterone receptor (PR). Direct targets of FOXO1 were identified by integrating RNA sequencing with chromatin immunoprecipitation followed by deep sequencing. Gene ontology analysis demonstrated that FOXO1 regulates a subset of genes in decidualization such as those involved in cancer, p53 signaling, focal adhesions, and Wnt signaling. An overlap of the FOXO1 and PR chromatin immunoprecipitation followed by deep sequencing intervals revealed the co-occupancy of FOXO1 in more than 75% of PR binding intervals. Among these intervals were highly enriched motifs for the interferon regulatory factor member 4 (IRF4). IRF4 was determined to be a genomic target of both FOXO1 and PR and also to be differentially regulated in HESCs treated with small interfering RNA targeting FOXO1 or PR prior to decidualization stimulus. Ablation of FOXO1 was found to abolish binding of PR to the shared binding interval downstream of the IRF4 gene. Finally, small interfering RNA-mediated ablation of IRF4 was shown to compromise morphological transformation of decidualized HESCs and to attenuate the expression of the decidual markers IGFBP1, PRL, and WNT4. These results provide the first evidence that FOXO1 is functionally required for the binding of PR to genomic targets. Most notably, FOXO1 and PR are required for the regulation of IRF4, a novel transcriptional regulator of decidualization in HESCs.

Marked stimulation of the lipoxin A4 synthesis pathway during endometrial stromal decidualization in women with and without endometriosis suggests a role in early pregnancy

Lipoxin A4 (LXA4) is an anti-inflammatory and pro-resolution eiconsanoid, produced by sequential actions of the enzymes ALOX15B and ALOX5. We have previously shown cycle-regulation of these enzymes in the epithelial cells as well as increased expression in endometriosis. We hypothesized that LXA4 could play an important anti-inflammatory role in early pregnancy and endometriosis might alter the expression of the biosynthetic enzymes. Therefore, the objective of these studies was to determine the effects of decidualization on ALOX15B and ALOX5 expression in stromal cells from women with and without endometriosis.

MiRNA Signature and Dicer Requirement during Human Endometrial Stromal Decidualization In Vitro

Decidualization is a morphological and biochemical transformation of endometrial stromal fibroblast into differentiated decidual cells, which is critical for embryo implantation and pregnancy establishment. The complex regulatory networks have been elucidated at both the transcriptome and the proteome levels, however very little is known about the post-transcriptional regulation of this process. miRNAs regulate multiple physiological pathways and their de-regulation is associated with human disorders including gynaecological conditions such as endometriosis and preeclampsia. In this study we profile the miRNAs expression throughout human endometrial stromal (hESCs) decidualization and analyze the requirement of the miRNA biogenesis enzyme Dicer during this process. A total of 26 miRNAs were upregulated and 17 miRNAs downregulated in decidualized hESCs compared to non-decidualized hESCs. Three miRNAs families, miR-181, miR-183 and miR-200, are down-regulated during the decidualization process. Using miRNAs target prediction algorithms we have identified the potential targets and pathways regulated by these miRNAs. The knockdown of Dicer has a minor effect on hESCs during in vitro decidualization. We have analyzed a battery of decidualization markers such as cell morphology, Prolactin, IGFBP-1, MPIF-1 and TIMP-3 secretion as well as HOXA10, COX2, SP1, C/EBPß and FOXO1 expression in decidualized hESCs with decreased Dicer function. We found decreased levels of HOXA10 and altered intracellular organization of actin filaments in Dicer knockdown decidualized hESCs compared to control. Our results provide the miRNA signature of hESC during the decidualization process in vitro. We also provide the first functional characterization of Dicer during human endometrial decidualization although surprisingly we found that Dicer plays a minor role regulating this process suggesting that alternative biogenesis miRNAs pathways must be involved in human endometrial decidualization.

The endometrial stromal decidualization miRNA signature

Endometrial stromal decidualization is required for the control of embryonic invasion during implantation, in fact defective decidualization may predispose to pregnancy complications such as preeclampsia, fetal growth restriction or miscarriage. Scarce information is available of how post-transcriprional regulators such us miRNAs control this process. Our objective is to analyze the expression and function of miRNAs during the human decidualization process. Human endometrial stromal cells (hESCs) were decidualized in vitro by estradiol (E2) and progesterone (P4) and the whole genome miRNA expression was analyzed during the decidualization process. hESCs (n = 3) were isolated from endometrial biopsies and then treated with E2 and P4 or vehicle for 9 days and the decidualization status was confirmed by the levels of secreted prolactin and IGFBP-1. Also natural decidualized endometria (n = 5) was obtained and the stromal compartment isolated. After total mRNA isolation and cDNA synthesis, we have investigated the expression of 704 miRNAs using the whole genome miRNA PCR array. T-Student test was used for statistical analysis. We have identified the miRNA decidualization signature using wide genome analysis in a standardized in vitro hESC decidualization model and validated in the in vivo human model. Several miRNAs included in this signature has been reported to be dysregulated in placentas from patients with preeclampsia. Specifically, miR-135b and miR-96 were selected because two of their top predicted gene targets are the transcriptional factors Hoxa10 and Foxo1, genes required for decidualization. miR-135b and miR-96 expression levels decrease significantly in in vitro and in vivo (late proliferative vs late secretory) decidualized stromal cells. hESCs have a specific miRNAs signature that is modified during the decidualization process. These results will be useful for the development of biomarkers of decidualization associated disorders such as preeclampsia.

Expression of tissue inhibitor of metalloproteinases-3 messenger RNA and protein in porcine endometrium during implantation

Recent evidence points to a stromal decidualization-like response in the pregnant porcine uterus. The objective of this study was to evaluate expression of tissue inhibitors of metalloproteinase-3 (TIMP-3), a sensitive indicator of endometrial stromal decidualization, in endometrium of pregnant sows and to further investigate this phenomenon. Real-time PCR, Western blot and immunostaining analysis were used to study TIMP-3 expression between/at attachment sites of endometrium of Days 13, 18 and 24 pregnant sows. The results indicate that TIMP-3 protein expression was lowest by Day 13 compared with Day 18 (P<0.01) and 24 (P<0.01), and the expression was higher at attachment sites than between attachment sites on Day 13 (P<0.01) and 18 (P<0.01). TIMP-3 intensive immunostaining was observed in stroma of endometrium on Days 13, 18 and 24, and the staining at attachment sites was stronger than between attachment sites. Collectively, these results suggest the crucial role of TIMP-3 in successful implantation and embryo survival and indicate the endometrial stromal decidualization-like in pigs.

Regulation of Expression of Cytokine Signal Transducer STAT3 by C/EBPbeta Contributes to Human Endometrial Stromal Decidualization.

The human endometrium is a dynamic tissue that undergoes proliferation, differentiation and shedding during each menstrual cycle. The sequential actions of ovarian steroids estrogen (E) and progesterone (P) induce a unique endometrial stromal differentiation and remodeling process, known as predecidualization, which is a prerequisite for successful implantation. The molecular pathways underlying this hormonally induced cellular transformation event remain poorly understood. We have recently identified C/EBPβ, a CCAAT box-binding transcription factor, as a unique regulator of decidualization in mice. In this study, using an in vitro decidualization system in which primary human endometrial stromal cells (HESC) undergo decidualization upon addition of E, P, and a cyclic AMP analog, we established that C/EBPβ plays an essential role in human stromal differentiation. Attenuation of endogenous C/EBPβ function by a dominant negative mutant of this transcription factor blocked the decidualization process. Similar results were obtained by siRNA-mediated down regulation of C/EBPβin HESCs. In order to identify the molecular pathways regulated by C/EBPβ during decidualization, we performed gene expression profiling experiments using normal and C/EBP β-deficient HESCs. Our studies revealed that several key regulators of decidualization, such as BMP2 and Wnt4, operate downstream of C/EBP beta. We also identified the interleukin 11 receptor alpha (IL-11Ralpha) and the signal transducer and activator of transcription 3 (STAT3) as prominent downstream targets of C/EBPβ. STAT3 mediates the signaling by several cytokines including IL11. Chromatin immunoprecipitation experiments indicated that C/EBPβ controls the expression of STAT3 by directly interacting with its promoter. siRNA-mediated down regulation of STAT3 expression in HESCs resulted in significantly reduced differentiation of these cells, indicating an important role for this cytokine signal transducer in the decidualization process. Collectively, our observations established that C/EBPβis a central regulator of human endometrial stromal differentiation. This study further revealed that STAT3 is a critical downstream mediator of the function of C/EBPβ, providing a novel link between C/EBPβ and cytokine signaling pathways that regulate endometrial functions during decidualization. (Supported by NIH grant U54 HD55787). (poster)

Role of activin A as a mediator of in vitro endometrial stromal cell decidualization via the cyclic adenosine monophosphate pathway

Objective To elucidate the regulation and role of activin A in endometrial stromal decidualization. Design In vitro model of human stromal cell decidualization with cyclic adenosine monophosphate (cAMP) used to evaluate expression of activin A and to evaluate the effect of the addition of follistatin, a known activin inhibitor, on expression of the decidualized phenotype (as measured by levels of insulin-like growth factor binding protein-1 [IGFBP-1]). Setting Academic research environment. Patient(s) Four premenopausal, normally cycling subjects (age range: 32–40 years). Intervention(s) Endometrial samples were obtained from the subjects after informed consent was obtained. Endometrial stromal cells were treated with cAMP (decidualizing stimulus) and 50 ng/mL, 100 ng/mL, and 200 ng/mL of follistatin for 48 hours. Main outcome measure(s) Levels of IGFBP-1 secreted from cells decidualized in the absence and presence of three different concentrations of follistatin. Result(s) Addition of follistatin, a known binding protein inhibitor of activin A, resulted in a dose-dependent inhibition of IGFBP-1 secreted into conditioned medium, with the greatest decrease observed at 4 days of decidualization. Cells treated with cAMP and 50 ng/mL, 100 ng/mL, and 200 ng/mL of follistatin demonstrated 67.3%, 58.6%, and 35.5%, respectively, of the IGFBP-1 levels observed with cAMP but without follistatin. Conclusion(s) These data suggest that activin A is a necessary component of the cAMP pathway leading to endometrial stromal decidualization. The role of activin A in regulating endometrial stromal decidualization and its known promotion of the invasive phenotype of the trophoblast suggest unique autocrine and paracrine interactions at the maternal/fetal interface during implantation, which might have important clinical implications for the understanding and treatment of fertility and pregnancy disorders.

Regulation of Human Endometrial Stromal Decidualization by Macrophage Colony-Stimulating Factor

Decidualized human endometrial stromal cells and human decidual CD56+ cells are reported to produce macrophage colony-stimulating factor (M-CSF), although its physiological roles remain unclear. In this study, the effects of M-CSF on cell viability and 8-Br-cAMP-stimulated decidualization of normal human endometrial stromal cells are examined by using an in vitro decidualization activity assay system. M-CSF alone affected neither the viable cell numbers nor PRL release of nonstimulated stromal cells, while high concentrations of M-CSF strongly suppressed the viable cell numbers and PRL secretion of stromal cells costimulated with 8-Br-cAMP and M-CSF. However, M-CSF did not suppress the viable cell numbers and PRL secretion of 8-Br-cAMP-induced decidualized cells. These results indicate that high concentrations of M-CSF can suppress the cell viability of stromal cells in the decidualization process, and inhibit decidualization of endometrial stromal cells. Thus, nonpregnant decidual M-CSF may autoregulate decidualization and the viable cell numbers of endometrial stromal cells in a paracrine manner.

Enhancement of human endometrial stromal decidualization by bestatin, an aminopeptidase-N inhibitor

We have re-evaluated the effects of bestatin, an aminopeptidase-N (CD13) inhibitor, on in vitro decidualization of normal human endometrial stromal cells by using an in vitro decidualization activity assay. Bestatin did not show any effects on the viability of both the decidualizing stromal cells co-stimulated with 8-Br-cAMP and bestatin and the 8-Br-cAMP-induced decidualized stromal cells, or on prolactin release from the decidualized stromal cells. However, bestatin dose-dependently enhanced prolactin release from the decidualizing stromal cells co-stimulated with 8-Br-cAMP and bestatin. These results indicate that bestatin enhances cAMP-mediated decidualization of human endometrial stromal cells and suggests that membrane aminopeptidase-N in the human endometrium is involved in the regulation of endometrial differentiation by inhibiting cAMP-mediated decidualization signals in endometrial stromal cells.

Common endometrial decidual reaction in postmenopausal breast cancer patients treated with tamoxifen and progestogens.

In order to assess endometrial reaction to the combined treatment of tamoxifen and progestogens in asymptomatic postmenopausal breast cancer patients, we evaluated all such patients by vaginal ultrasonography and by histological examination of endometrial samplings. All patients were initially treated with tamoxifen, and progestogens were then added when metastases became evident. Of 12 patients included in the study, eight (66.66%) showed evidence of strong endometrial stromal decidualization, while three (25%) had decidual reactions in endometrial polyps. Overall, 11 (91.66%) of the patients had decidual reactions, and all were treated with progestogens for > or = 3 consecutive months. One patient, treated with progestogens for 1 months, had an inactive endometrium. All but three had thickened endometria (> 10 mm) on ultrasonographic evaluation. These data show that postmenopausal breast cancer patients who received progestogens for < or = 3 months, and who concomitantly took tamoxifen, had a uniform decidual reaction in all uteri.