Upregulation of GPR133 expression impaired the phagocytosis of macrophages in recurrent spontaneous miscarriage.

Decidual macrophages are the second-largest immune cell group at the maternal-foetal interface. They participate in apoptotic cell removal, and protect the foetus from microorganisms or pathogens. Dysfunction of decidual macrophages gives rise to pregnancy complications such as preeclampsia and recurrent spontaneous miscarriage (RSM). However, the mechanisms by which decidual macrophages are involved in the occurrence of adverse pregnancy outcomes have not been elucidated. Here we integrated DNA methylation and gene expression data from decidua macrophages to identify potential risk factors related to RSM. GPR133 was significantly hypomethylated and upregulated in decidual macrophages from RSM patients. Further demethylation analysis demonstrated that GPR133 expression in decidual macrophages was significantly increased by 5-Aza-dC treatment. In addition, the influence of GPR133 on the phagocytic ability of macrophages was explored. Phagocytosis was impaired in the decidual macrophages of RSM patients with increased GPR133 expression. Increased GPR133 expression induced by demethylation treatment in the decidual macrophages of healthy control patients led to a significant decrease in phagocytic function. Importantly, knockdown of GPR133 resulted in a significant improvement in the phagocytic function of THP-1 macrophages. In conclusion, the existing studies have shown the influence of GPR133 on the phagocytic function of decidual macrophages and pregnancy outcomes, providing new data and ideas for future research on the role of decidual macrophages in RSM.

Differential gene expression in decidualized human endometrial stromal cells induced by different stimuli.

Decidualization can be induced by culturing human endometrial stromal cells (ESCs) with several decidualization stimuli, such as cAMP, medroxyprogesterone acetate (MPA) or Estradiol (E2). However, it has been unclear how decidualized cells induced by different stimuli are different. We compared transcriptomes and cellular functions of decidualized ESCs induced by different stimuli (MPA, E2 + MPA, cAMP, and cAMP + MPA). We also investigated which decidualization stimulus induces a closer in vivo decidualization. Differentially expressed genes (DEGs) and altered cellular functions by each decidualization stimuli were identified by RNA-sequence and gene-ontology analysis. DEGs was about two times higher for stimuli that use cAMP (cAMP and cAMP + MPA) than for stimuli that did not use cAMP (MPA and E2 + MPA). cAMP-using stimuli altered the cellular functions including angiogenesis, inflammation, immune system, and embryo implantation whereas MPA-using stimuli (MPA, E2 + MPA, and cAMP + MPA) altered the cellular functions associated with insulin signaling. A public single-cell RNA-sequence data of the human endometrium was utilized to analyze in vivo decidualization. The altered cellular functions by in vivo decidualization were close to those observed by cAMP + MPA-induced decidualization. In conclusion, decidualized cells induced by different stimuli have different transcriptome and cellular functions. cAMP + MPA may induce a decidualization most closely to in vivo decidualization.

Uterine macrophages and NK cells exhibit population and gene-level changes after implantation but maintain pro-invasive properties.

Introduction: Prior to pregnancy, hormonal changes lead to cellular adaptations in the endometrium allowing for embryo implantation. Critical for successful pregnancy establishment, innate immune cells constitute a significant proportion of uterine cells prior to arrival of the embryo and throughout the first trimester in humans and animal models. Abnormal uterine immune cell function during implantation is believed to play a role in multiple adverse pregnancy outcomes. Current work in humans has focused on uterine immune cells present after pregnancy establishment, and limited in vitro models exist to explore unique functions of these cells. Methods: With single-cell RNA-sequencing (scRNAseq), we comprehensively compared the human uterine immune landscape of the endometrium during the window of implantation and the decidua during the first trimester of pregnancy. Results: We uncovered global and cell-type-specific gene signatures for each timepoint. Immune cells in the endometrium prior to implantation expressed genes associated with immune metabolism, division, and activation. In contrast, we observed widespread interferon signaling during the first trimester of pregnancy. We also provide evidence of specific inflammatory pathways enriched in pre- and post-implantation macrophages and natural killer (NK) cells in the uterine lining. Using our novel implantation-on-a-chip (IOC) to model human implantation ex vivo, we demonstrate for the first time that uterine macrophages strongly promote invasion of extravillous trophoblasts (EVTs), a process essential for pregnancy establishment. Pre- and post-implantation uterine macrophages promoted EVT invasion to a similar degree as pre- and post-implantation NK cells on the IOC. Conclusions: This work provides a foundation for further investigation of the individual roles of uterine immune cell subtypes present prior to embryo implantation and during early pregnancy, which will be critical for our understanding of pregnancy complications associated with abnormal trophoblast invasion and placentation.

Increased levels of villus-derived exosomal miR-29a-3p in normal pregnancy than uRPL patients suppresses decidual NK cell production of interferon-γ and exerts a therapeutic effect in abortion-prone mice.

OBJECTIVE: Recurrent pregnancy loss (RPL) patients have higher absolute numbers of decidual natural killer (dNK) cells with elevated intracellular IFN-γ levels leading to a pro-inflammatory cytokine milieu, which contributes to RPL pathogenesis. The main objective of this study was twofold: first to explore the regulatory effects and mechanisms of villus-derived exosomes (vEXOs) from induced abortion patients or RPL patients at the level of intracellular IFN-γ in dNK cells; second to determine the validity of application of vEXOs in the treatment of unexplained RPL (uRPL) through in vitro experiments and mouse models. METHODS: Exosomes were isolated from villus explants by ultracentrifugation, co-cultured with dNK cells, and purified by enzymatic digestion and magnetically activated cell sorting. Flow cytometry, enzyme-linked immunosorbent assays, and RT-qPCR were used to determine IFN-γ levels. Comparative miRNA analysis of vEXOs from induced abortion (IA) and uRPL patients was used to screen potential candidates involved in dNK regulation, which was further confirmed by luciferase reporter assays. IA-vEXOs were electroporated with therapeutic miRNAs and encapsulated in a China Food and Drug Administration (CFDA)-approved hyaluronate gel (HA-Gel), which has been used as a clinical biomaterial in cell therapy for > 30 years. In vivo tracking was performed using 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyaine iodide (DiR) labelling. Tail-vein and uterine horn injections were used to evaluate therapeutic effects of the engineered exosomes in an abortion-prone mouse model (CBA/J × DBA/2 J). Placental growth was evaluated based on placental weight. IFN-γ mRNA levels in mouse placentas were measured by RT-qPCR. RESULTS: IFN-γ levels were significantly higher in dNK cells of uRPL patients than in IA patients. Both uRPL-vEXOs and IA-vEXOs could be efficiently internalized by dNK cells, whereas uRPL-vEXOs could not reduce the expression of IFN-γ by dNK cells as much as IA-vEXOs. Mechanistically, miR-29a-3p was delivered by vEXOs to inhibit IFN-γ production by binding to the 3' UTR of IFN-γ mRNA in dNK cells. For in vivo treatment, application of the HA-Gel effectively prolonged the residence time of vEXOs in the uterine cavity via sustained release. Engineered vEXOs loaded with miR-29a-3p reduced the embryo resorption rate in RPL mice with no signs of systemic toxicity. CONCLUSION: Our study provides the first evidence that villi can regulate dNK cell production of IFN-γ via exosome-mediated transfer of miR-29a-3p, which deepens our understanding of maternal-fetal immune tolerance for pregnancy maintenance. Based on this, we developed a new strategy to mix engineered vEXOs with HA-Gel, which exhibited good therapeutic effects in mice with uRPL and could be used for potential clinical applications in uRPL treatment.

Establishment of Murine Pregnancy Requires the Promyelocytic Leukemia Zinc Finger Transcription Factor.

Using an established human primary cell culture model, we previously demonstrated that the promyelocytic leukemia zinc finger (PLZF) transcription factor is a direct target of the progesterone receptor (PGR) and is essential for progestin-dependent decidualization of human endometrial stromal cells (HESCs). These in vitro findings were supported by immunohistochemical analysis of human endometrial tissue biopsies, which showed that the strongest immunoreactivity for endometrial PLZF is detected during the progesterone (P4)-dominant secretory phase of the menstrual cycle. While these human studies provided critical clinical support for the important role of PLZF in P4-dependent HESC decidualization, functional validation in vivo was not possible due to the absence of suitable animal models. To address this deficiency, we recently generated a conditional knockout mouse model in which PLZF is ablated in PGR-positive cells of the mouse (Plzf d/d). The Plzf d/d female was phenotypically analyzed using immunoblotting, real-time PCR, and immunohistochemistry. Reproductive function was tested using the timed natural pregnancy model as well as the artificial decidual response assay. Even though ovarian activity is not affected, female Plzf d/d mice exhibit an infertility phenotype due to an inability of the embryo to implant into the Plzf d/d endometrium. Initial cellular and molecular phenotyping investigations reveal that the Plzf d/d endometrium is unable to develop a transient receptive state, which is reflected at the molecular level by a blunted response to P4 exposure with a concomitant unopposed response to 17-ß estradiol. In addition to a defect in P4-dependent receptivity, the Plzf d/d endometrium fails to undergo decidualization in response to an artificial decidual stimulus, providing the in vivo validation for our earlier HESC culture findings. Collectively, our new Plzf d/d mouse model underscores the physiological importance of the PLZF transcription factor not only in endometrial stromal cell decidualization but also uterine receptivity, two uterine cellular processes that are indispensable for the establishment of pregnancy.

HLA-G isoforms, HLA-C allotype and their expressions differ between early abortus and placenta in relation to spontaneous abortions.

INTRODUCTION: Spontaneous abortion (SAB) affects approximately 10% of clinically recognized pregnancies. Fetal trophobalst invasion and remodeling of maternal spiral arteries is reported to be dependent on crosstalk between HLA-C/HLA-G expressed on extra villous trophoblast (EVTs)and Killer cell Immunoglobin like receptors (KIRs) of decidual NK (dNK). Immune dysfunction in decidua contributes to early miscarriage. METHODOLOGY: The study used mother neonate paired cord blood and term placenta samples (n = 46), elective abortus (n = 17,gestational age = 10-12 weeks of pregnancy) and SAB abortus (n = 24, gestational age = 12-15 weeks of pregnancy) for HLA-G, KIR2D and HLA-C. In addition, term placenta was collected from women with history of spontaneous pregnancy loss (n = 24) and women with history of live birth (n = 32). SSP-PCR was used for genotyping, RT-PCR for gene expression, copy number variation (CNVs) and HLA-C allotyping and ELISA for protein expression studies. RESULTS: Membrane bound HLA-G4 isoform proportion was higher 39.28%, p = 0.02) in term placenta. SAB abortus had higher proportion of HLA-G3 (50%),while elective abortus exhibited higher proportion of soluble isoforms (HLA-G5, = 5.9, HLA-G6 = 5.9%, HLA-G7 = 11.8%). Higher inhibitory KIR2DL1 content and copy numbers with lower HLA-C2 in SAB contrasted with higher copy numbers of KIR2DS1(p = 0.001), KIR2DS1+/2DL1+- HLA-C2 combined genotype in healthy placenta. Elevated KIR2D protein levels (p = 0.001), and concurrently, HLA-C levels were upregulated in healthy placenta. CONCLUSION: Our data supports lower cognate receptor ligand KIR2DS1+/2DL1+ HLA-C2 together with predominance of HLA-G3 isoform in SAB as confounding factors in spontaneous pregnancy loss. HLA-G isoforms and expression differed between first trimester abortus and term placenta suggesting temporal modulation and marks novelty of the study.

Single-cell transcriptome analyses reveal disturbed decidual homoeostasis in obstetric antiphospholipid syndrome.

OBJECTIVES: Obstetric antiphospholipid syndrome (OAPS) is an autoimmune disease characterised by the presence of antiphospholipid antibodies in circulation and pathological pregnancy. However, the pathogenesis of OAPS remains unknown. We aimed to reveal cellular compositions and molecular features of decidual cells involved in the development of OAPS using single-cell RNA sequencing (scRNA-seq). METHODS: We performed unbiased scRNA-seq analysis on the first-trimester decidua from five OAPS patients and five healthy controls (HCs), followed by validations with flow cytometry, immunohistochemical staining and immunofluorescence in a larger cohort. Serum chemokines and cytokines were measured by using ELISA. RESULTS: A higher ratio of macrophages but a lower ratio of decidual natural killer (dNK) cells was found in decidua from OAPS compared with HCs. Vascular endothelial cells shrinked in OAPS decidua while having upregulated chemokine expression and conspicuous responses to IFN-γ and TNF-α. Macrophages in OAPS had stronger phagocytosis function, complement activation signals and relied more on glycolysis. dNK cells were more activated in OAPS and had enhanced cytotoxicity and IFN-γ production. Downregulation of granules in OAPS dNK cells could be associated with suppressed glycolysis. Moreover, stromal cells had a prosenescent state with weakened immune surveillance for senescent cells in OAPS. In addition, the cellular interactions between decidual immune cells and those of immune cells with non-immune cells under disease state were altered, especially through chemokines, IFN-γ and TNF-α. CONCLUSION: This study provided a comprehensive decidual cell landscape and identified aberrant decidual microenvironment in OAPS, providing some potential therapeutic targets for this disease.

Decidual macrophage: a reversible role in immunotolerance between mother and fetus during pregnancy.

The tolerance of the semi-allogeneic fetus by the maternal immune system is an eternal topic of reproductive immunology for ensuring a satisfactory outcome. The maternal-fetal interface serves as a direct portal for communication between the fetus and the mother. It is composed of placental villi trophoblast cells, decidual immune cells, and stromal cells. Decidual immune cells engage in maintaining the homeostasis of the maternal-fetal interface microenvironment. Furthermore, growing evidence has shown that decidual macrophages play a crucial role in maternal-fetal tolerance during pregnancy. As the second largest cell population among decidual immune cells, decidual macrophages are divided into two subtypes: classically activated macrophages (M1) and alternatively activated macrophages (M2). M2 polarization is critical for placentation and embryonic development. Cytokines, exosomes, and metabolites regulate the polarization of decidual macrophages, and thereby modulate maternal-fetal immunotolerance. Explore the initial relationship between decidual macrophages polarization and maternal-fetal immunotolerance will help diagnose and treat the relevant pregnancy diseases, reverse the undesirable outcomes of mothers and infants.

Impaired bone morphogenetic protein (BMP) signaling pathways disrupt decidualization in endometriosis.

Endometriosis is linked to increased infertility and pregnancy complications due to defective endometrial decidualization. We hypothesized that identification of altered signaling pathways during decidualization could identify the underlying cause of infertility and pregnancy complications. Our study reveals that transforming growth factor ß (TGFß) pathways are impaired in the endometrium of individuals with endometriosis, leading to defective decidualization. Through detailed transcriptomic analyses, we discovered abnormalities in TGFß signaling pathways and key regulators, such as SMAD4, in the endometrium of affected individuals. We also observed compromised activity of bone morphogenetic proteins (BMP), a subset of the TGFß family, that control endometrial receptivity. Using 3-dimensional models of endometrial stromal and epithelial assembloids, we showed that exogenous BMP2 improved decidual marker expression in individuals with endometriosis. Our findings reveal dysfunction of BMP/SMAD signaling in the endometrium of individuals with endometriosis, explaining decidualization defects and subsequent pregnancy complications in these individuals.

Progesterone-induced progesterone receptor membrane component 1 rise-to-decline changes are essential for decidualization.

BACKGROUND: Decidualization of endometrial cells is the prerequisite for embryo implantation and subsequent placenta formation and is induced by rising progesterone levels following ovulation. One of the hormone receptors contributing to endometrial homeostasis is Progesterone Receptor Membrane Component 1 (PGRMC1), a non-classical membrane-bound progesterone receptor with yet unclear function. In this study, we aimed to investigate how PGRMC1 contributes to human decidualization. METHODS: We first analyzed PGRMC1 expression profile during a regular menstrual cycle in RNA-sequencing datasets. To further explore the function of PGRMC1 in human decidualization, we implemented an inducible decidualization system, which is achieved by culturing two human endometrial stromal cell lines in decidualization-inducing medium containing medroxyprogesterone acetate and 8-Br-cAMP. In our system, we measured PGRMC1 expression during hormone induction as well as decidualization status upon PGRMC1 knockdown at different time points. We further conferred proximity ligation assay to identify PGRMC1 interaction partners. RESULTS: In a regular menstrual cycle, PGRMC1 mRNA expression is gradually decreased from the proliferative phase to the secretory phase. In in vitro experiments, we observed that PGRMC1 expression follows a rise-to-decline pattern, in which its expression level initially increased during the first 6 days after induction (PGRMC1 increasing phase) and decreased in the following days (PGRMC1 decreasing phase). Knockdown of PGRMC1 expression before the induction led to a failed decidualization, while its knockdown after induction did not inhibit decidualization, suggesting that the progestin-induced 'PGRMC1 increasing phase' is essential for normal decidualization. Furthermore, we found that the interactions of prohibitin 1 and prohibitin 2 with PGRMC1 were induced upon progestin treatment. Knocking down each of the prohibitins slowed down the decidualization process compared to the control, suggesting that PGRMC1 cooperates with prohibitins to regulate decidualization. CONCLUSIONS: According to our findings, PGRMC1 expression followed a progestin-induced rise-to-decline expression pattern during human endometrial decidualization process; and the correct execution of this expression program was crucial for successful decidualization. Thereby, the results of our in vitro model explained how PGRMC1 dysregulation during decidualization may present a new perspective on infertility-related diseases.

GRIM-19 deficiency promotes macrophage polarization to the M1 phenotype partly through glycolysis in unexplained recurrent spontaneous abortion†.

The occurrence of unexplained recurrent spontaneous abortion (URSA) is closely related to immune system disorders, however, the underlying mechanisms remain unclear. The purpose of this study was to investigate the expression of GRIM-19 in URSA and the possible pathogenesis of URSA according to macrophage polarization. Here, we showed that GRIM-19 was downregulated in the uterine decidual macrophages of patients with URSA and that GRIM-19 downregulation was accompanied by increased M1 macrophage polarization. Furthermore, the expression levels of glycolytic enzymes were substantially enhanced in the uterine decidual macrophages of URSA patients, and glycolysis in THP-1-derived macrophages was further enhanced by the downregulation of GRIM-19. Additionally, the increase of M1 macrophages resulting from the loss of GRIM-19 was significantly reversed in cells treated with 2-deoxy-D-glucose (2-DG, an inhibitor of glycolysis). To provide more direct evidence, GRIM-19 deficiency was shown to promote macrophage polarization to the M1 phenotype in GRIM-19+/- mouse uteri. Overall, our study provides evidence that GRIM-19 deficiency may play a role in regulating macrophage polarization in URSA, and that glycolysis may participate in this process.

cAMP regulates the progesterone receptor gene expression through the protein kinase A pathway during decidualization in human immortalized endometrial stromal cells.

Decidualization, a crucial process for successful pregnancy establishment and maintenance, involves endometrial stromal cell differentiation. This process is orchestrated by estradiol (E2), progesterone, and other stimuli that increase intracellular cyclic adenosine monophosphate (cAMP) levels. The intracellular progesterone receptor (PR), encoded by the PGR gene, has a key role in decidualization. This study aimed to understand the role of sex steroids and cAMP in regulating PGR expression during the in vitro decidualization of the human immortalized endometrial stromal cell line, T-HESC. We subjected the cells to individual and combined treatments of E2, medroxyprogesterone (MPA), and cAMP. Additionally, we treated cells with PR and estrogen receptor antagonists and a protein kinase A (PKA) inhibitor. We evaluated the expression of PGR isoforms and decidualization-associated genes by RT-qPCR. Our findings revealed that cAMP induced PGR-B and PGR-AB expression by activating the PKA signaling pathway, while MPA downregulated their expression through the PR. Furthermore, downstream genes involved in decidualization, such as those coding for prolactin (PRL), insulin-like growth factor-binding protein-1 (IGFBP1), and Dickkopf-1 (DKK1), exhibited positive regulation via the cAMP-PKA pathway. Remarkably, MPA-activated PR signaling induced the expression of IGFBP1 and DKK1 but inhibited that of PRL. In conclusion, we have demonstrated that the PKA signaling pathway induces PGR gene expression during in vitro decidualization of the T-HESC human endometrial stromal cell line. This study has unraveled some of the intricate regulatory mechanisms governing PGR expression during this fundamental process for implantation and pregnancy maintenance.

Pregnancy trained decidual NK cells protect pregnancies from harmful Fusobacterium nucleatum infection.

Natural killer cells (NKs) found during pregnancy at the maternal-fetal interface named decidual (d)NKs, show signs of education following first pregnancy, resulting in better placentation and fetus-growth, hence termed pregnancy trained dNKs (PTdNKs). Here we show that PTdNKs provide increased protection of the fetus from Fusobacterium nucleatum (FN) infection. We demonstrate that PTdNKs secrete elevated amounts of the bacteriocidal protein granulysin (GNLY) upon incubation with FN compared to dNKs derived from first pregnancies, which leads to increased killing of FN. Furthermore, we showed mechanistically that the GNLY secretion is mediated through the interaction of the FN's Fap2 protein with Gal-GalNAc present on PTdNKs. Finally, we show in vivo, using GNLY-tg mice that enhanced protection of the fetuses from FN infection is observed, as compared to wild type and that this enhance protection is NK cell dependent. Altogether, we show a new function for PTdNKs as protectors of the fetus from bacterial infection.

USP7 promotes decidualization of ESCs by STAT3/PR axis during early pregnancy.

Decidualization is an essential process for embryo implantation during early pregnancy. Defective decidualization is a critical leading cause of early pregnancy loss (EPL). Ubiquitin-specific protease 7 (USP7) is a deubiquitinating enzyme that is involved in uterine function. This study aimed to explore the underlying mechanism by which USP7 regulates decidualization in EPL. We found that USP7 was downregulated in the decidual tissue of EPL patients. Upregulation of USP7 enhanced decidualization in endometrial stromal cells (ESCs), with increased decidualized biomarkers IGFBP1 and PRL and progesterone receptor A/B (PR-A/B) expression. Moreover, we found that signal transducer and activator of transcription 3 (STAT3) is a direct target of USP7 in ESCs. USP7 bound to STAT3 by deubiquitination and increased STAT3 levels in ESCs. Suppression of STAT3 impeded the USP7-promoted cell viability, decidualization, and PR-A/B expression of ESCs. USP7 promoted the decidualization of ESCs through the STAT3/PR signaling pathway during early pregnancy, which provides new insight into the pathological mechanism of EPL and may contribute to the clinical treatment of EPL.

Dysregulated expression of GATA2 and GATA6 transcription factors in adenomyosis: implications for impaired endometrial receptivity.

OBJECTIVE: To study the effect of adenomyosis on the localized expression of the GATA binding proteins 2 and 6 (GATA2 and GATA6) zinc-finger transcription factors that are involved in proliferation of hematopoietic and endocrine cell lineages, cell differentiation, and organogenesis, potentially leading to impaired endometrial implantation. DESIGN: Laboratory based experimental study. SETTING: Academic hospital and laboratory. PATIENTS: Human endometrial stromal cells (HESCs) of reproductive age patients, 18-45 years of age, with adenomyosis were compared with patients with no pathology and leiomyomatous uteri as controls (n = 4 in each group, respectively). Additionally, midsecretory phase endometrial sections were obtained from patients with adenomyosis and control patients with leiomyoma (n = 8 in each group, respectively). INTERVENTIONS: GATA2 and GATA6 immunohistochemistry and H-SCORE were performed on the midsecretory phase endometrial sections from adenomyosis and leiomyoma control patients (n = 8 each, respectively). Control and adenomyosis patient HESC cultures were treated with placebo or 10-8 M estradiol (E2), or decidualization media (EMC) containing 10-8 M E2, 10-7 M medroxyprogesterone acetate, and 5 × 10-5 M cAMP for 6 and 10 days. Additionally, control HESC cultures (n = 4) were transfected with scrambled small interfering RNA (siRNA) (control) or GATA2-specific siRNAs for 6 days while adenomyosis HESC cultures (n = 4) were transfected with human GATA2 expression vectors to silence or induce GATA2 overexpression. MAIN OUTCOME MEASURES: Immunohistochemistry was performed to obtain GATA2 and GATA6 H-SCORES in adenomyosis vs. control patient endometrial tissue. Expression of GATA2, GATA6, insulin-like growth factor-binding protein 1 (IGFBP1), prolactin (PRL), progesterone receptor (PGR), estrogen receptor 1 (ESR1), leukemia inhibitory factor (LIF), and Interleukin receptor 11 (IL11R) messenger RNA (mRNA) levels were analyzed using by qPCR with normalization to ACTB. Silencing and overexpression experiments also had the corresponding mRNA levels of the above factors analyzed. Western blot analysis was performed on isolated proteins from transfection experiments. RESULTS: Immunohistochemistry revealed an overall fourfold lower GATA2 and fourfold higher GATA6 H-SCORE level in the endometrial stromal cells of patients with adenomyosis vs. controls. Decidual induction with EMC resulted in significantly lower GATA2, PGR, PRL and IGFBP1 mRNA levels in HESC cultures from patients with adenomyosis patient vs. controls. Leukemia inhibitory factor and IL11R mRNA levels were also significantly dysregulated in adenomyosis HESCs compared with controls. . Silencing of GATA2 expression in control HESCs induced an adenomyosis-like state with significant reductions in GATA2, increases in GATA6 and accompanying aberrations in PGR, PRL, ESR1 and LIF levels. Conversely, GATA2 overexpression via vector in adenomyosis HESCs caused partial restoration of the defective decidual response with significant increases in GATA2, PGR, PRL and LIF expression. CONCLUSION: In-vivo and in-vitro experiment results demonstrate that there is an overall inverse relationship between endometrial GATA2 and GATA6 levels in patients with adenomyosis who have diminished GATA2 levels and concurrently elevated GATA6 levels. Additionally, lower GATA2 and higher GATA6 levels, together with aberrant levels of important receptors and implantation factors, such as ESR1, PGR, IGFBP1, PRL, LIF, and IL11R mRNA in HESCs from patients with adenomyosis or GATA2-silenced control HESCs, support impaired decidualization. These effects were partially restored with GATA2 overexpression in adenomyosis HESCs, demonstrating a potential therapeutic target.

Verification of the efficacy of deciduae for determining the genetic type of mouse embryos.

Mouse embryos in the early-implantation stage require manipulation under a microscope. While the extraction of DNA, RNA and proteins from a single sample allows for both determination of genetic type and analysis of gene expression, whole mount analysis is not possible. In this study, we explored the applicability of PCR using extraembryonic tissues, especially the decidual side tissue after isolating the embryos from implantation sites to establish a method for determining the genetic type of embryos. The implantation site was resected at each day from the date of vaginal plug confirmation, separated into embryos and deciduae. Genomic DNA were isolated separately from the embryos and the deciduae. PCR was performed using these genomic DNA, and the band patterns were compared after electrophoresis. As a result, we demonstrated that detecting embryo-derived cells in the decidua allows determination of the sex and presence of transgenes without harming the mouse embryos themselves, from 8.5 days of age. This method enables the determination of the genetic type of mouse embryos without damaging. This technique would expand the adaptations for analysis of mouse implanted embryos.

Progesterone and cAMP synergistically induce SHP2 expression via PGR and CREB1 during uterine stromal decidualization.

Decidualization of endometrial stroma is a key step in embryo implantation and its abnormality often leads to pregnancy failure. Stromal decidualization is a very complex process that is co-regulated by estrogen, progesterone and many local factors. The signaling protein SHP2 encoded by PTPN11 is dynamically expressed in decidualized endometrial stroma and mediates and integrates various signals to govern the decidualization. In the present study, we investigate the mechanism of PTPN11 gene transcription. Estrogen, progesterone and cAMP co-induced decidualization of human endometrial stromal cell in vitro, but only progesterone and cAMP induced SHP2 expression. Using the luciferase reporter, we refined a region from -229 bp to +1 bp in the PTPN11 gene promoter comprising the transcriptional core regions that respond to progesterone and cAMP. Progesterone receptor (PGR) and cAMP-responsive element-binding protein 1 (CREB1) were predicted to be transcription factors in this core region by bioinformatic methods. The direct binding of PGR and CREB1 on the PTPN11 promoter was confirmed by electrophoretic mobility and chromatin immunoprecipitation in vitro. Knockdown of PGR and CREB1 protein significantly inhibited the expression of SHP2 induced by medroxyprogesterone acetate and cAMP. These results demonstrate that transcription factors PGR and CREB1 bind to the PTPN11 promoter to regulate the expression of SHP2 in response to decidual signals. Our results explain the transcriptional expression mechanism of SHP2 during decidualization and promote the understanding of the mechanism of decidualization of stromal cells.

The Efficacy of Vitamin D Supplement in the Expression and Protein Levels of Endometrial Decidualization Factors in Women with Recurrent Implantation Failure.

Recurrent implantation failure (RIF) is a challenging situation for infertility specialists, and its treatment is introduced as a difficult case in the field of assisted reproductive technology (ART). Vitamin D (VD) is one of the supplements that have been suggested to improve the implantation process. In the present study, the effect of VD on the expression and protein levels of VD receptor (VDR), progesterone receptor (PR), prolactin (PRL), insulin-like growth factor binding protein-1 (IGFBP-1), and homeobox protein A10 (HOXA10) in the endometrial cells of unknown RIF women with and without VD deficiency were assessed by qRT-PCR and immunohistochemistry. Twelve women with unknown RIF and VD deficiency (≤ 20 ng/ml) and twelve women with unknown RIF without VD deficiency (≥ 30 ng/ml) from 2021 to 2022 were identified. Endometrial specimens were collected in the mid-luteal stage before treatment or pregnancy. In the group with VD deficiency, oral medication of VD 50,000 units was prescribed for 2 to 3 months and their serum levels of VD were re-measured, then an endometrial biopsy at the same stage of the menstrual cycle was performed. The expression and protein levels of VDR, PR, PRL, IGFBP1, and HOXA10 in RIF patients with VD deficiency were lower than the RIF patients without VD deficiency (P value < 0.05). Our findings suggest that VD can play a key role in the pregnancy process, especially during embryo implantation and decidualization of the endometrial cells.IRCT registration number: IRCT20220528055006N1, Registration date: 2022-10-15, Registration timing: retrospective.

Low- and high-level information analyses of transcriptome connecting endometrial-decidua-placental origin of preeclampsia subtypes: A preliminary study.

BACKGROUND: Existing proposed pathogenesis for preeclampsia (PE) was only applied for early onset subtype and did not consider pre-pregnancy and competing risks. We aimed to decipher PE subtypes by identifying related transcriptome that represents endometrial maturation and histologic chorioamnionitis. METHODS: We utilized eight arrays of mRNA expression for discovery (n=289), and other eight arrays for validation (n=352). Differentially expressed genes (DEGs) were overlapped between those of: (1) healthy samples from endometrium, decidua, and placenta, and placenta samples under histologic chorioamnionitis; and (2) placenta samples for each of the subtypes. They were all possible combinations based on four axes: (1) pregnancy-induced hypertension; (2) placental dysfunction-related diseases (e.g., fetal growth restriction [FGR]); (3) onset; and (4) severity. RESULTS: The DEGs of endometrium at late-secretory phase, but none of decidua, significantly overlapped with those of any subtypes with: (1) early onset (p-values ≤0.008); (2) severe hypertension and proteinuria (p-values ≤0.042); or (3) chronic hypertension and/or severe PE with FGR (p-values ≤0.042). Although sharing the same subtypes whose DEGs with which significantly overlap, the gene regulation was mostly counter-expressed in placenta under chorioamnionitis (n=13/18, 72.22%; odds ratio [OR] upper bounds ≤0.21) but co-expressed in late-secretory endometrium (n=3/9, 66.67%; OR lower bounds ≥1.17). Neither the placental DEGs at first-nor second-trimester under normotensive pregnancy significantly overlapped with those under late-onset, severe PE without FGR. CONCLUSIONS: We identified the transcriptome of endometrial maturation in placental dysfunction that distinguished early- and late-onset PE, and indicated chorioamnionitis as a PE competing risk. This study implied a feasibility to develop and validate the pathogenesis models that include pre-pregnancy and competing risks to decide if it is needed to collect prospective data for PE starting from pre-pregnancy including chorioamnionitis information.

ATOH8 Expression Is Regulated by BMP2 and Plays a Key Role in Human Endometrial Stromal Cell Decidualization.

During the secretory phase of the menstrual cycle, elongated fibroblast-like mesenchymal cells in the uterine endometrium begin to transdifferentiate into polygonal epithelioid-like (decidual) cells. This decidualization process continues more broadly during early pregnancy, and the resulting decidual tissue supports successful embryo implantation and placental development. This study was carried out to determine if atonal basic helix-loop-helix transcription factor 8 (ATOH8) plays a role in human endometrial stromal fibroblast (ESF) decidualization. ATOH8 messenger RNA and protein expression levels significantly increased in human ESF cells undergoing in vitro decidualization, with the protein primarily localized to the nucleus. When ATOH8 expression was silenced, the ability of the cells to undergo decidualization was significantly diminished. Overexpression of ATOH8 enhanced the expression of many decidualization markers. Silencing the expression of ATOH8 reduced the expression of FZD4, FOXO1, and several known FOXO1-downstream targets during human ESF cell decidualization. Therefore, ATOH8 may be a major upstream regulator of the WNT/FZD-FOXO1 pathway, previously shown to be critical for human endometrial decidualization. Finally, we explored possible regulators of ATOH8 expression during human ESF decidualization. BMP2 significantly enhanced ATOH8 expression when cells were stimulated to undergo decidualization, while an ALK2/3 inhibitor reduced ATOH8 expression. Finally, although the steroids progesterone plus estradiol did not affect ATOH8 expression, the addition of cyclic adenosine monophosphate (cAMP) analogue alone represented the major effect of ATOH8 expression when cells were stimulated to undergo decidualization. Our results suggest that ATOH8 plays a crucial role in human ESF decidualization and that BMP2 plus cAMP are major regulators of ATOH8 expression.