Decidualization Process Research Articles

Exploring the role of Luman/CREB3 in regulating decidualization of mice endometrial stromal cells by comparative transcriptomics

BackgroundLuman is a member of CREB3 (cAMP responsive element-binding) subfamily of the basic leucine-zipper (bZIP) transcription factors. It may play an important regulatory role during the decidualization process since Luman was highly expressed in the decidual cells. However, the exact molecular mechanisms of how Luman regulating decidualization is unknown.ResultsUsing an in vitro model, we prove that Luman knockdown significantly affects the decidualization process of mice endometrial stromal cells (ESCs) as the expression of two decidual markers PRL8a2 and PRL3c1 were repressed. We employed massively parallel RNA sequencing (RNA-Seq) to understand the changes in the transcriptional landscape associated with knockdown of Luman in ESCs during in vitro decidualization. We found significant dysregulation of genes related to protein processing in the endoplasmic reticulum (ER). Several genes involved in decidualization including bone morphogenetic proteins (e.g. BMP1, BMP4, BMP8A, BMP2, and BMP8B), growth factor-related genes (e.g. VEGFB, FGF10, and FGFR2), and transcription factors (IF4E, IF4A2, WNT4, WNT9A, ETS1, NOTCH1, IRX1, IDB1, IDB2, and IDB3), show altered expression. We also found that the knockdown of Luman is associated with increased expression of cell cycle-related genes including cycA1, cycB1, cycB2, CDK1, CDK2, and PLPK1, which resulted in an increased proportion of ESCs in the G1 phase. Differentially expressed genes (DEGs) were highly enriched on ECM-receptor interaction signaling, endoplasmic reticulum protein processing, focal adhesion, and PI3K-Akt signaling pathways.ConclusionsLuman knockdown results in widespread gene dysregulation during decidualization of ESCs. Genes involved in protein processing in ER, bone morphogenetic protein, growth factor, and cell cycle progression were identified as particularly important for explaining the decidual deficiency observed in this in vitro model. Therefore, this study provides clues as to the underlying mechanisms that may expand our understanding of gene regulation during decidualization.

Copper and lead exposures disturb reproductive features of primary endometrial stromal and epithelial cells

This study investigates if Cu and Pb act as endocrine disruptors affecting endometrial cells. Primary EnSCs and EnECs were exposed to Cu (0, 50, 100 and 200 μM) or Pb (0, 30, 100 and 500 μM) and assessed for viability, decidualization, apoptosis and proliferation on EnSCs, and wound healing and adhesion capabilities on EnECs. Cu exposure decreased significantly cell viability in a dose-dependent manner. Cu and Pb negatively affected in vitro decidualization, showing a significant decrease in PRL secretion. HOXA10 and ERα mRNA levels significantly decreased in decidualized cells (dEnSCs) exposed to Cu. Cu and Pb decreased adhesion and regeneration capability in EnEC. This study reveals that Cu and Pb could negatively affect endometrial functionality, compromising the decidualization process and disrupting endometrial regeneration and embryo adhesion. Therefore, special care should be taken considering heavy metals exposure if pregnancy is being pursued.

Epigenetic modifications working in the decidualization and endometrial receptivity.

Decidualization is a critical event for the blastocyst implantation, placental development and fetal growth and the normal term. In mice, the embryo implantation to the uterine epithelial would trigger the endometrial stromal cells to differentiate into decidual stromal cells. However, decidualization in women takes place from the secretory phase of each menstrual cycle and continues to early pregnancy if there is conceptus. Deficient decidualization is often associated with pregnancy specific complications and reproductive disorders. Dramatic changes occur in the gene expression profiles during decidualization, which is coordinately regulated by steroid hormones, growth factors, and molecular and epigenetic mechanisms. Recently, emerging evidences showed that epigenetic modifications, mainly including DNA methylation, histone modification, and non-coding RNAs, play an important role in the decidualization process via affecting the target genes' expression. In this review, we will focus on the epigenetic modifications in decidualization and open novel avenues to predict and treat the pregnancy complications caused by abnormal decidualization.

Insulin Signaling Via Progesterone-Regulated Insulin Receptor Substrate 2 is Critical for Human Uterine Decidualization.

Decidualization, the process by which fibroblastic human endometrial stromal cells (HESC) differentiate into secretory decidual cells, is a critical event during the establishment of pregnancy. It is dependent on the steroid hormone progesterone acting through the nuclear progesterone receptor (PR). Previously, we identified insulin receptor substrate 2 (IRS2) as a factor that is directly regulated by PR during decidualization. IRS2 is an adaptor protein that functionally links receptor tyrosine kinases, such as insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF1R), and their downstream effectors. IRS2 expression was induced in HESC during in vitro decidualization and siRNA-mediated downregulation of IRS2 transcripts resulted in attenuation of this process. Further use of siRNAs targeted to IR or IGF1R transcripts showed that downregulation of IR, but not IGF1R, led to impaired decidualization. Loss of IRS2 transcripts in HESC suppressed phosphorylation of both ERK1/2 and AKT, downstream effectors of insulin signaling, which mediate gene expression associated with decidualization and regulate glucose uptake. Indeed, downregulation of IRS2 resulted in reduced expression and membrane localization of the glucose transporters GLUT1 and GLUT4, resulting in lowered glucose uptake during stromal decidualization. Collectively, these data suggest that the PR-regulated expression of IRS2 is necessary for proper insulin signaling for controlling gene expression and glucose utilization, which critically support the decidualization process to facilitate pregnancy. This study provides new insight into the mechanisms by which steroid hormone signaling intersects with insulin signaling in the uterus during decidualization, which has important implications for pregnancy complications associated with insulin resistance and infertility.

Bisphenol compounds regulate decidualized stromal cells in modulating trophoblastic spheroid outgrowth and invasion in vitro†.

Bisphenol A (BPA) is commonly found in epoxy resins used in the manufacture of plastic coatings in food packaging and beverage cans. There is a growing concern about BPA as a weak estrogenic compound that can affect human endocrine function. Chemicals structurally similar to BPA, such as bisphenol F (BPF) and bisphenol S (BPS), have been developed as substitutes in the manufacturing industry. Whether these bisphenol substitutes have adverse effects on human endocrine and reproductive systems remains largely unknown. This study investigated the effects of BPA, BPF, and BPS on regulating the function of decidualized human primary endometrial stromal cells on trophoblast outgrowth and invasion by indirect and direct co-culture models. All three bisphenols did not affect the stromal cell decidualization process. However, BPA- and BPF-treated decidualized stromal cells stimulated trophoblastic spheroid invasion in the indirect coculture model. The BPA-treated decidualized stromal cells had upregulated expressions of several invasion-related molecules including leukemia inhibitory factor (LIF), whereas both BPA- and BPF-treated decidualized stromal cells had downregulated expressions of anti-invasion molecules including plasminogen activator inhibitor type 1 (PAI-1) and tumor necrosis factor (TNFα) . Taken together, BPA and BPF altered the expression of invasive and anti-invasive molecules in decidualized stromal cells modulating its function on trophoblast outgrowth and invasion, which could affect the implantation process and subsequent pregnancy outcome.

CGMP-dependent protein kinase II determines β-catenin accumulation that is essential for uterine decidualization in mice.

In the early phase of pregnancy, decidualization is an indispensable event after mammal embryo implantation, accompanied by proliferation and differentiation of uterine stromal cells. Type II cGMP-dependent protein kinase (Prkg2) belongs to the family of serine/threonine kinase, which plays multiple roles in cellular signaling pathways to control proliferation and differentiation. However, the regulatory function and molecular mechanism of Prkg2 in decidualization are still unknown. In this study, we show that Prkg2 has a gradually increased expression pattern during peri-implantation and artificial decidualization, and the expression of Prkg2 is induced by estrogen and progesterone in the ovariectomized mouse uteri and primary cultured uterine stromal cells, the process of which is blocked by treating with estrogen receptor (ER) antagonist (ICI-182,780) and progesterone receptor (PR) antagonist (RU-486). Inhibition of Prkg2 activity by HA-100 promotes uterine stromal cell proliferation but compromises decidualization with decreased expression of prolactin family 8, subfamily a, member 2. In addition, the functional regulation of decidualization by Prkg2 is accomplished by its induced phosphorylation of glycogen synthase kinase-3β (GSK-3β) at serine-9, which results in accumulation of β-catenin in the decidual cells. Taken together, our findings demonstrate that estrogen and progesterone upregulate the expression of Prkg2 in uterine stromal cells depending on ER and PR; Prkg2 promotes phosphorylation of GSK-3β at serine-9 and inactivates it, leading to the accumulation of β-catenin and promoting the process of decidualization. In addition to revealing the regulatory mechanism of Prkg2 that ensures the success of uterine decidualization, our findings will contribute to the understanding in the maintenance of early pregnancy.

Anandamide targets aromatase: A breakthrough on human decidualization

In each menstrual cycle endometrial stromal cells (hESC) proliferate and differentiate into specialized decidual cells, a process termed decidualization, which regulates endometrial receptivity. Decidualization is mainly controlled by sex ovarian hormones, estradiol (E2) and progesterone. E2 plays an important role in the expression of the progesterone receptor and promotes the endometrial stromal cells differentiation. Our group previously reported that anandamide (AEA) impairs decidualization through cannabinoid receptor 1 (CB1). In this study, we hypothesized whether AEA inhibitory effect on cell decidualization could be mediated through interaction with aromatase and consequent interference in estradiol production/signaling. We used an immortalized human endometrial stromal cell line (St-T1b) and human decidual fibroblasts (HdF) derived from human term placenta. In cells exposed to a differentiation stimulus, AEA-treatment prevents the increase of the expression of CYP19A1 gene encoding aromatase, E2 levels and of estradiol receptor expression, that are observed in differentiating cells. Regarding CYP19A1 mRNA levels, the effect was partially reverted by a CB1 receptor antagonist and by a COX2 inhibitor. In addition, we report that AEA presents anti-aromatase activity in placental microsomes, the nature of the inhibition being the uncommon mixed type as revealed by the kinetic studies. Structural analysis of the AEA-Aromatase complexes determined that AEA may bind to the active site pocket of the enzyme. In overall we report that AEA inhibits aromatase activity and may affect E2 signaling crucial for the decidualization process, indicating that a deregulation of the endocannabinoid system may be implicated in endometrial dysfunction and in fertility/infertility disorders.

Sex steroids influence organizational but not functional decidualization of feline endometrial cells in a 3D culture system†.

Successful implantation requires complex signaling between the uterine endometrium and the blastocyst. Prior to the blastocyst reaching the uterus, the endometrium is remodeled by sex steroids and other signals to render the endometrium receptive. In vitro models have facilitated major advances in our understanding of endometrium preparation and endometrial-blastocyst communication in mice and humans, but these systems have not been widely adapted for use in other models which might generate a deeper understanding of these processes. The objective of our study was to use a recently developed, three-dimensional culture system to identify specific roles of female sex steroids in remodeling the organization and function of feline endometrial cells. We treated endometrial cells with physiologically relevant concentrations of estradiol and progesterone, either in isolation or in combination, for 1 week. We then examined size and density of three-dimensional structures, and quantified expression of candidate genes known to vary in response to sex steroid treatments and that have functional relevance to the decidualization process. Combined sex steroid treatments recapitulated organizational patterns seen in vivo; however, sex steroid manipulations did not induce expected changes to expression of decidualization-related genes. Our results demonstrate that sex steroids may not be sufficient for complete decidualization and preparation of the feline endometrium, thereby highlighting key areas of opportunity for further study and suggesting some unique functions of felid uterine tissues.

Phytoestrogen exposure alters endometrial stromal cells and interferes with decidualization signaling

To investigate whether phytoestrogens (genistein and daidzein) alter invitro decidualization of human endometrial stromal cells (ESCs). Isolated primary ESCs were exposed to phytoestrogens and decidualized invitro. Academic fertility center. Twenty fertile oocyte donors attending the IVI Valencia clinic. Treatment of ESC with phytoestrogens at 0, 10, 20, 50, and 100μM. The ESC proliferation was analyzed by MTS assay. Invitro decidualization was induced in the presence of phytoestrogens by medroxyprogesterone acetate/cyclic adenosine 3':5' monophosphate and evaluated by prolactin (PRL) ELISA and F-actin immunostaining. The Ki67 proliferative marker was analyzed by immunofluorescence. The ESC apoptosis was assessed by annexin V/propidium iodide detection using flow cytometry. Estrogen (ERβ) and P receptor (PR) localization were evaluated by immunofluorescence. The ESC exposed to 0, 19, 20, 50, and 100μM of genistein, daidzein, and genistein + daidzein showed a dose-dependent proliferation decrease. After 48-96hours of culture, this reduction was significant in the presence of 50μM of phytoestrogens versus 10μM untreated ESC. The ESC decidualized in the presence of phytoestrogens did not rearrange their cytoskeletons and showed a significant decrease in PRL secretion compared with untreated decidualized ESCs (dESCs). However, phytoestrogens did not alter proliferative status or the percentage of viable/apoptotic cells in dESC compared with untreated dESC. During decidualization, phytoestrogens induced the same nuclear translocation of ERβ and PR as the control dESC. This study reveals that high doses of phytoestrogens could affect the invitro decidualization process.

Identifying the BMP Pathway Type II Receptors in Decidualization

Decidualization is a process driven by hormones and growth factors that results in the differentiation of endometrial stromal cells into decidual cells that are capable of supporting a developing embryo. The overall effect of decidualization is the differentiation of the endometrial stromal cells into decidual cells, which facilitate early blastocyst development, placental function and parturition. Despite the importance of decidualization in reproduction, the molecular mechanisms controlling decidualization are not fully understood. Identifying the signaling pathways responsible for decidualization will provide opportunity to enhance the process of decidualization, which can benefit women experiencing infertility, early pregnancy loss or other pregnancy‐related complications. Past research has shown that Bone Morphogenetic Protein (BMP) signaling is one of the mechanisms controlling decidualization, but the roles of each member of this pathway in decidualization are unknown. Identifying the precise genes in the BMP pathway responsible for decidualization will enable us to develop drugs with clinical applications. The BMP signaling pathway is initiated when a BMP dimer associates with a tetrameric complex of two type one (ALK2, ALK3, ALK6) and two type two receptor proteins (BMPR2, ACVR2A, ACVR2B), which then phosphorylate the SMAD1 and SMAD5 transcription factors. Human endometrial stromal cell (HESC) decidualization can be recapitulated in vitro by exposure to progesterone, estrogen and 8‐bromo‐cyclic‐AMP. Using this technique, we performed siRNA experiments in primary cultures of HESC's to determine the roles of various BMP signaling pathway members in decidualization. We found that SMAD1 and SMAD5 transcription factors and the BMPR2 receptor are essential for in vitro HESC cell decidualization. We also identified that ACVR2A and ACVR2B receptors may be playing a redundant role in decidualization as well. These results indicate that the BMP signaling receptor complex responsible for HESC decidualization is controlled by the concerted action of cell surface receptors that act redundantly but require the activity of both SMAD1 and SMAD5 transcription factors.Support or Funding InformationNCI P30 CURE Grant (CA125123)IRACDA Training GrantPDEP Burroughs Wellcome FundThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Resveratrol inhibits decidualization by accelerating downregulation of the CRABP2-RAR pathway in differentiating human endometrial stromal cells

Pregnancy critically depends on the transformation of the human endometrium into a decidual matrix that controls embryo implantation and placenta formation, a process driven foremost by differentiation and polarization of endometrial stromal cells into mature and senescent decidual cells. Perturbations in the decidual process underpin a spectrum of prevalent reproductive disorders, including implantation failure and early pregnancy loss, emphasizing the need for new therapeutic interventions. Resveratrol is a naturally occurring polyphenol, widely used for its antioxidant and anti-inflammatory properties. Using primary human endometrial stromal cell (HESC) cultures, we demonstrate that resveratrol has anti-deciduogenic properties, repressing not only the induction of the decidual marker genes PRL and IGFBP1 but also abrogating decidual senescence. Knockdown of Sirtuin 1, a histone deacetylase activated by resveratrol, restored the expression of IGFBP1 but not the induction of PRL or senescence markers in decidualizing HESCs, suggesting involvement of other pathways. We demonstrate that resveratrol interferes with the reprogramming of the retinoic acid signaling pathway in decidualizing HESCs by accelerating down-regulation of cellular retinoic acid-binding protein 2 (CRABP2) and retinoic acid receptor (RAR). Notably, knockdown of CRABP2 or RAR in HESCs was sufficient to recapitulate the anti-deciduogenic effects of resveratrol. Thus, while resveratrol has been advanced as a potential fertility drug, our results indicate it may have detrimental effects on embryo implantation by interfering with decidual remodeling of the endometrium.

Heparin prevents oxidative stress-induced apoptosis in human decidualized endometrial stromal cells.

Clinical trials have shown that administering heparin during the luteal phase has beneficial effects on implantation and live birth rates. Heparin exerts direct effects on decidual human endometrial stromal cells (HESCs), which are independent of its anticoagulant effect. However, the accurate effects of heparin on the decidualization process remain unidentified. Here, we demonstrate that HESCs become dramatically resistant to oxidative stress upon decidualization, and we hypothesize a possible direct action of heparin on the decidualization of HESCs, which would lead to improved implantation. To test this hypothesis, we established primary HESC cultures and propagated them, and then we decidualized confluent cultures with 8-bromo-cAMP, with medroxyprogesterone acetate, and with or without heparin. We treated the cells with hydrogen peroxide (H2O2) as a source of reactive oxygen species (ROS). Adding heparin to decidualized HESCs induced prolactin secretion. Decidualized HESCs treated with heparin were prevented from undergoing apoptosis induced by oxidative stress. Heparin induced nuclear accumulation of the forkhead transcription factor FOXO1 and expression of its downstream target, the ROS scavenger superoxide dismutase 2. These results demonstrate that heparin-treated decidualized HESCs acquired further resistance to oxidative stress, suggesting that heparin may improve the implantation environment.

Hemodynamic forces enhance decidualization via endothelial-derived prostaglandin E2 and prostacyclin in a microfluidic model of the human endometrium.

STUDY QUESTIONDoes the uterine vasculature play a localized role in promoting stromal cell decidualization in the human endometrium?SUMMARY ANSWEROur study demonstrated that hemodynamic forces induced secretion of specific endothelial cell-derived prostanoids that enhanced endometrial perivascular decidualization via a paracrine mechanism.WHAT IS KNOWN ALREADYDifferentiation of stromal cell fibroblasts into the specialized decidua of the placenta is a progesterone-dependent process; however, histologically, it has long been noted that the first morphological signs of decidualization appear in the perivascular stroma. These observations suggest that the human endometrial vasculature plays an active role in promoting stromal differentiation.STUDY DESIGN, SIZE, DURATIONPrimary human endometrial stromal cells were co-cultured for 14 days with primary uterine microvascular endothelial cells within a microfluidic Organ-on-Chip model of the endometrium.PARTICIPANTS/MATERIALS, SETTING, METHODSCultures were maintained with estradiol and a progestin, with or without continuous laminar perfusion to mimic hemodynamic forces derived from the blood flow. Some cultures additionally received exogenous agonist-mediated challenges. Decidualization in the microfluidic model was assessed morphologically and biochemically. ELISA was used to examine the culture effluent for expression of decidualization markers and prostaglandins. Immunofluorescence was used to monitor cyclooxygenase-2 expression in association with decidualization.MAIN RESULTS AND THE ROLE OF CHANCEA significantly enhanced stromal decidualization response was observed in the co-cultures when the endothelial cells were stimulated with hemodynamic forces (e.g. laminar shear stress) derived from controlled microfluidic perfusion (<0.001). Furthermore, the enhanced progestin-driven stromal differentiation was mediated via cyclooxygenase-2 and the paracrine action of prostaglandin E2 and prostacyclin. Altogether, these translational findings indicate that the vascular endothelium plays a key physiologic role during the early events of perivascular decidualization in the human endometrium.LARGE SCALE DATAN/A.LIMITATIONS, REASONS FOR CAUTIONThis report is largely an in vitro study. Although we were able to experimentally mimic hemodynamic forces in our microfluidic model, we have not yet determined the contribution of additional cell types to the decidualization process or determined the precise physiological rates of shear stress that the microvasculature of the endometrium undergoes in vivo.WIDER IMPLICATIONS OF THE FINDINGSIdentification of specific endothelial-derived prostaglandins and their role during endometrial reproductive processes may have clinical utility as therapeutic targets for reproductive disorders such as infertility, endometriosis, adenomyosis, pre-eclampsia and poor pregnancy outcomes.STUDY FUNDING/COMPETING INTEREST(S)This work was supported by the Veterans Affairs (I01 BX002853), the Bill and Melinda Gates Foundation Grand Challenges Exploration (OPP1159411), the Environmental Toxicology Training Grant (NIH T32 ES007028) and the Environmental Protection Agency STAR Center Grant (83573601).CONFLICT OF INTERESTThe authors report no conflicts of interest.TRIAL REGISTRATION NUMBERN/A.

Endometrial stromal cell proteome mapping in repeated implantation failure and recurrent pregnancy loss cases and fertile women.

Are there proteomic differences between endometrial stromal cells of repeated implantation failure (RIF), recurrent pregnancy loss (RPL) and normal fertile women, and is there differential protein expression upon decidualization? This exploratory study investigated the proteome of in-vitro cultured endometrial stromal cells of women with RIF (n = 4), women with RPL (n = 3) and normal fertile women (n = 4), comparing day 0 with 5 days of decidualization. Total proteins extracted from cell lysates were analysed by high-definition mass spectrometry. Data analysis was performed using significance analysis of microarray in R (P<0.05; false discovery rate [FDR] 10%). In the RIF group, ANXA6, PSMC5 and FSCN1 were up-regulated (1.9-fold, 2.5-fold and 1.9-fold, respectively), whereas PBXIP1 was down-regulated (7.7-fold) upon decidualization. In the RPL group, RPS25 and ACADVL were down-regulated (1.9-fold and 2.4-fold, respectively; FDR 10%) between the non-decidualized and the decidualized samples. In the normal fertile group VIM and RPL23A were down-regulated (1.9-fold and 2.4-fold, respectively). Comparing ratios of expression of decidualized over non-decidualized samples in the different groups revealed six differentially expressed proteins: DUX4L2, CNPY4, PDE7A, CTSK, PCBP2 and PSMD4. Comparison of RPL versus normal fertile in the decidualized condition revealed serotransferrin to be differentially expressed. The changes in expression levels for serotransferrin, ANX6, ACDVL and VIM were confirmed by western blot. Results show a varying response of endometrial stromal cells in distinct clinical groups (RIF, RPL and normal fertile) upon in-vitro decidualization. Serotransferrin could serve as a marker for the aberrant decidualization process in RPL.

53 Pre-implantation exposure to bisphenol A and 4-tert-octylphenol result in disruption of calcium channels

Miscarriage due to blastocyst implantation failure occurs in up to two-thirds of all miscarriage cases in humans. Calcium (Ca2+) has been shown to involve many cellular signal transduction pathways as well as regulation of cell adhesion, which is necessary for the physiology process of endometrial epithelial cell transformation and stromal cell decidualization during embryo implantation. Exposure to endocrine-disrupting chemicals (ED) can regulate the expression of genes associated with calcium transport in during pregnancy such as TRPV5, TRPV6, PMCA, and NCX1. Additionally, exposure to ED during early gestation results in disrupted intrauterine implantation, uterine receptive, leading to implantation failure. In this study, oestrogen (E2), bisphenol A (BPA), octylphenol (OP), and/or ICI 182,780 (oestrogen receptor antagonist, ICI) were injected subcutaneously from gestation Day 1 to gestation Day 3 post coitus. The number of implantation sites was significantly lower in the OP group, and no implantation site was observed in the E2 and ED+ICI groups. There were differences in the expression of calcium transient transport channel between maternal uterine and implantation. The level of TRPV6 and TRPV5 mRNA and protein was significantly increased by ED and/or ICI treatment in the uterus. The levels of TRPV5 and TRPV6 gene expression were significantly increased by ED with/without ICI treatment in the uterus. However, TRPV5 and TRPV6 gene expression was significantly lower in implantation site samples. The NCX1 and PMCA1 mRNA levels were significantly decreased by OP and BPA in the implantation site samples. Both mRNA and protein levels of MUC1 were markedly higher in all groups, except the BPA group when compared with the vehicle group in the uterus. The LIF and HOXA-10 mRNA were significantly low in E2; BPA+ICI; OP; and/or ICI in both the uterus and implantation site. Expression of the oestrogen receptor (ERa) and progesterone receptor (PR) was significantly lower in all groups except the BPA group when compared with the vehicle group. Taken together, E2, BPA, and OP disrupt the success of implantation through altered expression of calcium transport genes.

Metformin inhibits estradiol and progesterone-induced decidualization of endometrial stromal cells by regulating expression of progesterone receptor, cytokines and matrix metalloproteinases

BackgroundPolycystic ovary syndrome (PCOS) is a serious threat for reproductive-aged women. Metformin has been used for the treatment of PCOS. However, its molecular mechanism in decidualization process of PCOS has not been well featured. MethodsRT-qPCR analysis was used to detect expression patterns of progesterone receptor (PGR), estradiol receptor alpha (ERα), Cytokeratin 8 and Vimentin in endometrial tissues of PCOS and non-PCOS patients. RT-qPCR assay was also employed to determine mRNA expression of prolactin, Insulin-like growth factor-binding protein 1 (IGFBP-1), matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP9). Cytokine secretion were measured by matching ELISA kits. Protein expression of p-ERK1/2, ERK1/2, p-p38 MAPK, p38 MAPK, and PGR (PGRA and PGRB) was tested by western blot assay. ResultsPGR expression was upregulated in PCOS patients. Metformin alleviated estradiol (E2) and progesterone (P4) (EP)-induced decidualization of endometrial stromal cells. Abnormal cytokine secretion was observed in EP-stimulated endometrial stromal cells in the absence or presence of metfromin. Metformin suppressed EP-induced MMP-2 and MMP-9 upregulation. Metformin alleviated EP-triggered p38 MAPK inactivation and PGR (PGRA and PGRB) expression. Metfromin had no effect on ERK1/2 signaling in EP-stimulated endometrial stromal cells. ConclusionMetformin alleviated EP-induced decidualization of endometrial stromal cells by modulating secretion of multiple cytokines, inhibiting expression of MMP-2 and MMP-9, activating p38-MAPK signaling and reducing PGR expression, providing a deep insight into the molecular basis of metfromin therapy for PCOS patients.

FOXO3a is essential for murine endometrial decidualization through cell apoptosis during early pregnancy.

Embryo implantation is essential for normal pregnancy, and the process of decidualization is critical for embryo implantation. However, the mechanism of decidualization during early pregnancy is still unknown. Forkhead box O3a (FOXO3a) is the most important functional transcription factor of the forkhead box family and is a highly conserved transcription factor of apoptosis-related genes. In the mouse uterus, FOXO3a was found to be expressed regularly from Days 1-7 of early pregnancy. Upon further exploration, it was found that FOXO3a was expressed at significantly higher levels at the implantation site than at the interimplantation site on Days 5-7 of pregnancy. Under artificial decidualization, FOXO3a was highly expressed in the first and second decidual zones. After decidualization, the expression of FOXO3a was significantly increased both in vivo and vitro. In primary stromal cells, apoptosis was reduced by decreased expression of FOXO3a after inducing decidualization. Moreover, when FOXO3a-small interfering RNA was transfected into the uteri of mice, the expression of decidualization- and apoptosis-related factors was impaired. Thus, FOXO3a might play an important role in decidualization during early pregnancy, and cell apoptosis might be one of pathways for FOXO3a-regulated decidualization.

Preeclampsia as a parental epigenetic disease

Objectives: Preeclampsia still remains a major cause of maternal and perinatal mortality and morbidity. The aim of this study is to provide an overview of the evidence from epigenetic regulation of preeclampsia development, with a focus on candidate imprinted genes and their roles. Materials and Methods: A PubMed search of the relevant literature published between 2005 and 2016 was performed to identify the preeclampsia susceptibility genes and imprinted genes. Results: Several susceptibility genes that have highlighted the potential role of preeclampsia development have been identified. There appears to be at least two types of genes in the placenta: the paternity-related genes appear to be evolved as the trophoblast invasion and fetal growth, while the maternity-related genes act as the defence and protective mechanism for the mother. A number of imprinting genes are essential for the biological functions such as decidualization, pregnancy maintenance, tumor suppressor, and fetal developmental processes, which include paternally expressed/maternally imprinted genes and maternally expressed/paternally imprinted genes. Some biological aspects of preeclampsia are explained from an imbalanced expression of genomic imprinting (epigenetic conflict). Although the genotypic and phenotypic extent of multilocus imprinting epimutations is poorly understood, the preeclamptic placenta showed unique epigenetic features. Changes of parental life experiences with stress or early-life conditions during pregnancy may display impaired reproductive outcome through the dynamic alterations in the patterns of the specific imprinted genes. Conclusion: In conclusion, preeclampsia may be recognized as a parental imprinting disease.

Human chorionic gonadotropin induces decidualization of ectopic human endometrium more effectively than forskolin in an in-vivo endometriosis model.

Endometriosis, characterized by the presence of endometrial tissue at ectopic sites, is a leading cause of pelvic pain and subfertility in women. The stromal compartment of the endometrium is considered to play a pivotal role in the establishment and persistence of endometriotic lesions, thus impaired decidualization of these cells may result in enhanced invasion capacity at ectopic sites. Consequently, stimulation of decidualization may alleviate this disease. To analyze the effect of systemically applied compounds on decidualization of ectopic endometrial tissue, endometriosis was induced by suturing human eutopic endometrium to the peritoneum of 22 NOD/SCID mice. Each mouse received four tissue fragments from the same patient. Mice were randomly allocated either to one control and three experimental groups ( n = 4/group) which were treated with progesterone alone or in combination with forskolin or human chorionic gonadotropin for seven days or to one control and one experimental group ( n = 3/group) which was treated with progesterone and human chorionic gonadotropin for 10 days followed by 7 days without treatment. At the end of the experiments, lesions were measured and analyzed for markers of decidualization (FOXO-1, prolactin) and proliferation (Ki-67). Decidualization was induced in the ectopic lesions by systemic treatment in vivo. This induction was significantly stronger after treatment with progesterone in combination with human chorionic gonadotropin than with forskolin or with progesterone alone. Only the combination with human chorionic gonadotropin led to induction of FOXO1 protein expression and a significant physiologic transformation of the ectopic endometrial stromal cells after seven days of treatment. After termination of human chorionic gonadotropin treatment, the decidualization process continued, leading to a significant inhibition of proliferation. Thus, decidualization of human ectopic endometrial tissue can be induced in a humanized endometriosis mouse model in vivo. This model may help to decipher the signal pathways involved in this decidualization process and to develop novel therapeutical approaches to alleviate this painful disease. Impact statement Impaired decidualization of endometrial stromal cells may contribute to the development of endometriosis, and an increased decidualization reaction may prevent or alleviate this prevalent gynecological disease. Human chorionic gonadotropin (hCG) has been shown to promote decidualization in eutopic endometrium. Up to now in vitro studies mainly used cAMP for successful induction of decidualization of isolated endometrial stromal cells. Here, for the first time, decidualization of ectopic endometrial lesions is induced in an experimental endometriosis mouse model, comparing the effectiveness of hCG with that of the direct adenylyl cyclase activator Forskolin. In this 3D-organ structure in vivo, hCG proved to be more effective in the induction of decidualization than forskolin. Particularly in case of progesterone resistance, alternative pathways inducing decidualization could alleviate endometriosis, and the sophisticated hCG action could constitute a therapeutical tool to induce terminal differentiation in ectopic endometrial lesions.

Dysfunction of pseudogene PGK1P2 is involved in preeclampsia by acting as a competing endogenous RNA of PGK1.

Normal decidualization is essential for normal pregnancy and abnormal decidualization is thought to cause preeclampsia (PE). Phosphoglycerate kinase 1 (PGK1) is an enzyme involved in the glycolytic pathway which is the main metabolism process decidual cells exhibit. Phosphoglycerate kinase 1, pseudogene 2 (PGK1P2), which is also a long non-coding RNA (lncRNA), has a high sequence similarity to PGK1 and therefore acquires an ability for sequence-specific regulation. The expression of PGK1 and PGK1P2 in human decidua, as well as their relationship and functions during decidualization was investigated using in vitro cultured human endometrial stromal cell lines (hESCs) and primary ESCs by real-time PCR, immunohistochemistry, western blotting, siRNA techniques and miRNA inhibitor or mimic transfection. The levels of PGK1 and PGK1P2 mRNA and PGK1 protein in severe preeclamptic decidua were lower than those in normal pregnant controls. PGK1 and PGK1P2 mRNAs were both induced after in vitro decidualization and their deficiency caused impaired decidualization in turn. We also found PGK1P2 acted as a competing endogenous RNA (ceRNA) to regulate PGK1 expression through miR-330-5p. We proved that PGK1 and PGK1P2 are a pair of ceRNAs against miR-330-5p and they play a vital role in human decidualization by regulating angiogenesis and glycolysis metabolism. The deficiency of PGK1 and PGK1P2 in the decidua jeopardizes the decidualization process and subsequently might lead to the occurrence of PE. These findings may help in promoting novel predictive, diagnostic and prognostic biomarkers of PE in future.