Uterine deficiency of Dnmt3b impairs decidualization and causes consequent embryo implantation defects.

Uterine deficiency of Dnmt3b impairs decidualization and consequent embryo implantation defects. Recent advances in molecular technologies have allowed the unprecedented mapping of epigenetic modifications during embryo implantation. DNA methyltransferase 3a (DNMT3A) and DNMT3B are responsible for establishing DNA methylation patterns produced through their de novo-type DNA methylation activity in implantation stage embryos and during germ cell differentiation. It was reported that conditional knockout of Dnmt3a in the uterus does not markedly affect endometrial function during embryo implantation, but the tissue-specific functions of Dnmt3b in the endometrium during embryo implantation remain poorly understood to investigate the role of Dnmt3b during peri-implantation period. Here, we generated Dnmt3b conditional knockout (Dnmt3bd/d) female mice using progesterone receptor-Cre mice and examined the role of Dnmt3b during embryo implantation. Dnmt3bd/d female mice exhibited compromised fertility, which was associated with defective decidualization, but not endometrial receptivity. Furthermore, results showed loss of Dnmt3b did not lead to altered genomic methylation patterns of the decidual endometrium during early pregnancy. Transcriptome sequencing analysis of uteri from day 6 pregnant mice identified phosphoglycerate kinase 1 (Pgk1) as one of the most variable genes in Dnmt3bd/d decidual endometrium. Potential roles of PGK1 in the decidualization process during early pregnancy were confirmed. Lastly, the compromised decidualization upon the downregulation of Dnmt3b could be reversed by overexpression of Pgk1. Collectively, our findings indicate that uterine deficiency of Dnmt3b impairs decidualization and consequent embryo implantation defects.

Clinical aspects of decidualization / A decidualizáció klinikai vonatkozásai

An essential component of successful conception and pregnancy is decidualization, which involves structural and functional transformation of the endometrium. The process involves structural changes in the uterine mucosa, transformation of spiral arterioles, numerical and functional adaptation of leukocytes in the endometrium and their subsequent migration, and functional and morphological changes in decidual stromal cells. As part of decidualization, trophoblast cells of embryonic origin perform a physiological invasion of maternal tissue to create the placenta. The success of the process is due to the special antigenicity of the trophoblast cells and the immune communication between the graft (fetus) and the host (mother) through hormones, cytokines and multiple receptorligand connections. Disorders of these processes are the basis of several diseases that threaten conception, implantation, and successful pregnancy, such as recurrent miscarriage, preeclampsia, intrauterine retardation, or preterm birth. In this article, we review the anatomical, immunological, and molecular basis of physiological decidualization to address common disorders in the clinical practice of obstetrics that are related to a dysfunctional decidualization.

Stromal cells of the endometrium and decidua: in search of a name and an identity†.

Human endometrial and decidual stromal cells are the same cells in different environments (nonpregnancy and pregnancy, respectively). Although some authors consider decidual stromal cells to arise solely from the differentiation of endometrial stromal cells, this is a debatable issue given that decidualization processes do not end with the formation of the decidua, as shown by the presence of stromal cells from both the endometrium and decidua in both undifferentiated (nondecidualized) and decidualized states. Furthermore, recent functional and transcriptomic results have shown that there are differences in the decidualization process of endometrial and decidual stromal cells, with the latter having a greater decidualization capacity than the former. These differences suggest that in the terminology and study of their characteristics, endometrial and decidual stromal cells should be clearly distinguished, as should their undifferentiated or decidualized status. There is, however, considerable confusion in the designation and identification of uterine stromal cells. This confusion may impede a judicious understanding of the functional processes in normal and pathological situations. In this article, we analyze the different terms used in the literature for different types of uterine stromal cells, and propose that a combination of differentiation status (undifferentiated, decidualized) and localization (endometrium, decidua) criteria should be used to arrive at a set of accurate, unambiguous terms. The cell identity of uterine stromal cells is also a debatable issue: phenotypic, functional, and transcriptomic studies in recent decades have related these cells to different established cells. We discuss the relevance of these associations in normal and pathological situations.

CD55 is upregulated by cAMP/PKA/AKT and modulates human decidualization via Src and ERK pathway and decidualization-related genes.

Decidualization is an essential process for embryo implantation and maintenance of pregnancy, and abnormal decidualization contributed to several pregnancy disorders like a miscarriage. The objective of this study was to explore the regulation and function of CD55 in human decidualization. By immunohistochemical staining, it was found that CD55 expression was higher in first-trimester decidua than in the endometrium. In both primary endometrial stromal cells and immortalized cell line T-hESCs, CD55 was upregulated by induction of in vitro decidualization with medroxyprogesterone acetate (MPA) and 8-Br-cAMP. During decidualization in vitro, CD55 was stimulated by 8-Br-cAMP in a time- and concentration-dependent manner, which was reversed by a PKA inhibitor H89 and partially by an AKT activator SC79. Knocking down CD55 expression diminished the expression of decidualization markers prolactin (PRL) and insulin-like growth factor-binding protein 1 (IGFBP1), accompanied by inhibition of Src, aberrant activation of ERK and decreased expression of several decidualization-related genes, including FOXO1, EGFR, and STAT3. Furthermore, the decidua of unexplained miscarriage women and the endometrium of unexplained infertile women both exhibited decreased CD55 expression. Collectively, these findings revealed that 8-Br-cAMP promotes CD55 expression via PKA activation and AKT dephosphorylation, and decreased CD55 impairs decidualization by inactivation of Src, aberrant activation of ERK pathway, and compromised expression of decidualization-related genes, indicating that CD55 deficiency may contribute to the pathogenesis of spontaneous miscarriage and infertility.

Single-cell analysis of prostaglandin E2-induced human decidual cell in vitro differentiation: a minimal ancestral deciduogenic signal†.

The decidua is a hallmark of reproduction in many placental mammals. Differentiation of decidual stromal cells is known to be induced by progesterone and the cyclic AMP/protein kinase A (cAMP/PKA) pathway. Several candidates have been identified as the physiological stimulus for adenylyl cyclase activation, but their relative importance remains unclear. To bypass this uncertainty, the standard approach for in vitro experiments uses membrane-permeable cAMP and progestin. We phylogenetically infer that prostaglandin E2 (PGE2) likely was the signal that ancestrally induced decidualization in conjunction with progesterone. This suggests that PGE2 and progestin should be able to activate the core gene regulatory network of decidual cells. To test this prediction, we performed a genome-wide study of gene expression in human endometrial fibroblasts decidualized with PGE2 and progestin. Comparison to a cAMP-based protocol revealed shared activation of core decidual genes and decreased induction of senescence-associated genes. Single-cell transcriptomics of PGE2-mediated decidualization revealed a distinct, early-activated state transitioning to a differentiated decidual state. PGE2-mediated decidualization was found to depend upon progestin-dependent induction of PGE2 receptor 2 (PTGER2) which in turn leads to PKA activation upon PGE2 stimulation. Progesterone-dependent induction of PTGER2 is absent in opossum, an outgroup taxon of placental mammals which is incapable of decidualization. Together, these findings suggest that the origin of decidualization involved the evolution of progesterone-dependent activation of the PGE2/PTGER2/PKA axis, facilitating entry into a PKA-dominant rather than AKT-dominant cellular state. We propose the use of PGE2 for in vitro decidualization as an alternative to 8-Br-cAMP.

Atrial natriuretic peptide promotes uterine decidualization and a TRAIL-dependent mechanism in spiral artery remodeling.

Atrial natriuretic peptide (ANP) is an important hormone in cardiovascular biology. It is activated by the protease corin. In pregnancy, ANP and corin promote uterine spiral artery remodeling, but the underlying mechanism remains unknown. Here we report an ANP function in uterine decidualization and TNF-related apoptosis-inducing ligand-dependent (TRAIL-dependent) death in spiral arterial smooth muscle cells (SMCs) and endothelial cells (ECs). In ANP- or corin-deficient mice, uterine decidualization markers and TRAIL expression were decreased, whereas in cultured human endometrial stromal cells (HESCs), ANP increased decidualization and TRAIL expression. In uterine spiral arteries from pregnant wild-type mice, SMC and EC loss occurred sequentially before trophoblast invasion. In culture, TRAIL from decidualized HESCs induced apoptosis in uterine SMCs, but not in ECs with low TRAIL receptor expression. Subsequently, cyclophilin B was identified from apoptotic SMCs that upregulated endothelial TRAIL receptor and caused apoptosis in ECs. These results indicate that ANP promotes decidualization and TRAIL expression in endometrial stromal cells, contributing to sequential events in remodeling of spiral arteries, including SMC death and cyclophilin B release, which in turn induces TRAIL receptor expression and apoptosis in ECs.

Modelling the impact of decidual senescence on embryo implantation in human endometrial assembloids.

Decidual remodelling of midluteal endometrium leads to a short implantation window after which the uterine mucosa either breaks down or is transformed into a robust matrix that accommodates the placenta throughout pregnancy. To gain insights into the underlying mechanisms, we established and characterized endometrial assembloids, consisting of gland-like organoids and primary stromal cells. Single-cell transcriptomics revealed that decidualized assembloids closely resemble midluteal endometrium, harbouring differentiated and senescent subpopulations in both glands and stroma. We show that acute senescence in glandular epithelium drives secretion of multiple canonical implantation factors, whereas in the stroma it calibrates the emergence of anti-inflammatory decidual cells and pro-inflammatory senescent decidual cells. Pharmacological inhibition of stress responses in pre-decidual cells accelerated decidualization by eliminating the emergence of senescent decidual cells. In co-culture experiments, accelerated decidualization resulted in entrapment of collapsed human blastocysts in a robust, static decidual matrix. By contrast, the presence of senescent decidual cells created a dynamic implantation environment, enabling embryo expansion and attachment, although their persistence led to gradual disintegration of assembloids. Our findings suggest that decidual senescence controls endometrial fate decisions at implantation and highlight how endometrial assembloids may accelerate the discovery of new treatments to prevent reproductive failure.

At the beginning of a human pregnancy, the embryo implants into the uterus lining, known as the endometrium. At this point, the endometrium transforms into a new tissue that helps the placenta to form. Problems in this transformation process are linked to pregnancy disorders, many of which can lead to implantation failure (the embryo fails to invade the endometrium altogether) or recurrent miscarriages (the embryo implants successfully, but the interface between the placenta and the endometrium subsequently breaks down). Studying the implantation of human embryos directly is difficult due to ethical and technical barriers, and animals do not perfectly mimic the human process, making it challenging to determine the causes of pregnancy disorders. However, it is likely that a form of cellular arrest called senescence, in which cells stop dividing but remain metabolically active, plays a role. Indeed, excessive senescence in the cells that make up the endometrium is associated with recurrent miscarriage, while a lack of senescence is associated with implantation failure. To study this process, Rawlings et al. developed a new laboratory model of the human endometrium by assembling two of the main cell types found in the tissue into a three-dimensional structure. When treated with hormones, these 'assembloids' successfully mimic the activity of genes in the cells of the endometrium during implantation. Rawlings et al. then exposed the assembloids to the drug dasatinib, which targets and eliminates senescent cells. This experiment showed that assembloids become very robust and static when devoid of senescent cells. Rawlings et al. then studied the interaction between embryos and assembloids using time-lapse imaging. In the absence of dasatinib treatment, cells in the assembloid migrated towards the embryo as it expanded, a process required for implantation. However, when senescent cells were eliminated using dasatinib, this movement of cells towards the embryo stopped, and the embryo failed to expand, in a situation that mimicks implantation failure. The assembloid model of the endometrium may help scientists to study endometrial defects in the lab and test potential treatments. Further work will include other endometrial cell types in the assembloids, and could help increase the reliability of the model. However, any drug treatments identified using this model will need further research into their safety and effectiveness before they can be offered to patients.

TAZ as a novel regulator of oxidative damage in decidualization via Nrf2/ARE/Foxo1 pathway.

TAZ, as a crucial effector of Hippo pathway, is required for spermatogenesis and fertilization, but little is known regarding its physiological function in uterine decidualization. In this study, we showed that TAZ was localized in the decidua, where it promoted stromal cell proliferation followed by accelerated G1/S phase transition via Ccnd3 and Cdk4 and induced the expression or activity of stromal differentiation markers Prl8a2, Prl3c1 and ALP, indicating the importance of TAZ in decidualization. Knockdown of TAZ impeded HB-EGF induction of stromal cell proliferation and differentiation. Under oxidative stress, TAZ protected stromal differentiation against oxidative damage by reducing intracellular ROS and enhancing cellular antioxidant capacity dependent on the Nrf2/ARE/Foxo1 pathway. TAZ strengthened the transcriptional activity of Nrf2 which directly bound to the antioxidant response element (ARE) of Foxo1 promoter region. Additionally, silencing TAZ caused accumulation of intracellular ROS through heightening NOX activity whose blockade by APO reversed the disruption in stromal differentiation. Further analysis revealed that TAZ might restore mitochondrial function, as indicated by the increase in ATP level, mtDNA copy number and mitochondrial membrane potential with the reduction in mitochondrial superoxide. Additionally, TAZ modulated the activities of mitochondrial respiratory chain complexes I and III whose suppression by ROT and AA resulted in the inability of TAZ to defend against oxidative damage to stromal differentiation. Moreover, TAZ prevented stromal cell apoptosis by upregulating Bcl2 expression and inhibiting Casp3 activity and Bax expression. In summary, TAZ might mediate HB-EGF function in uterine decidualization through Ccnd3 and ameliorate oxidative damage to stromal cell differentiation via Nrf2/ARE/Foxo1 pathway.

Uterine Epithelial LIF Receptors Contribute to Implantation Chamber Formation in Blastocyst Attachment.

Recent studies have demonstrated that the formation of an implantation chamber composed of a uterine crypt, an implantation-competent blastocyst, and uterine glands is a critical step in blastocyst implantation in mice. Leukemia inhibitory factor (LIF) activates signal transducer and activator of transcription 3 (STAT3) precursors via uterine LIF receptors (LIFRs), allowing successful blastocyst implantation. Our recent study revealed that the role of epithelial STAT3 is different from that of stromal STAT3. However, both are essential for blastocyst attachment, suggesting the different roles of epithelial and stromal LIFR in blastocyst implantation. However, how epithelial and stromal LIFR regulate the blastocyst implantation process remains unclear. To investigate the roles of LIFR in the uterine epithelium and stroma, we generated Lifr-floxed/lactoferrin (Ltf)-iCre (Lifr eKO) and Lifr-floxed/antimüllerian hormone receptor type 2 (Amhr2)-Cre (Lifr sKO) mice with deleted epithelial and stromal LIFR, respectively. Surprisingly, fertility and blastocyst implantation in the Lifr sKO mice were normal despite stromal STAT3 inactivation. In contrast, blastocyst attachment failed, and no implantation chambers were formed in the Lifr eKO mice with epithelial inactivation of STAT3. In addition, normal responsiveness to ovarian hormones was observed in the peri-implantation uteri of the Lifr eKO mice. These results indicate that the epithelial LIFR-STAT3 pathway initiates the formation of implantation chambers, leading to complete blastocyst attachment, and that stromal STAT3 regulates blastocyst attachment without stromal LIFR control. Thus, uterine epithelial LIFR is critical to implantation chamber formation and blastocyst attachment.

Preimplantation factor modulates trophoblastic invasion throughout the decidualization of human endometrial stromal cells.

BACKGROUND: Successful human embryo implantation requires the differentiation of endometrial stromal cells (ESCs) into decidual cells during a process called decidualization. ESCs express specific markers of decidualization, including prolactin, insulin-like growth factor-binding protein-1 (IGFBP-1), and connexin-43. Decidual cells also control of trophoblast invasion by secreting various factors, such as matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases. Preimplantation factor (PIF) is a recently identified, embryo-derived peptide with activities at the fetal-maternal interface. It creates a favorable pro-inflammatory environment in human endometrium and directly controls placental development by increasing the human trophoblastic cells' ability to invade the endometrium. We hypothesized that PIF's effects on the endometrium counteract its pro-invasive effects. METHODS: We tested sPIF effect on the expression of three decidualization markers by RT-qPCR and/or immunochemiluminescence assay. We examined sPIF effect on human ESC migration by performing an in vitro wound healing assay. We analyzed sPIF effect on endometrial control of human trophoblast invasion by performing a zymography and an invasion assay. RESULTS: Firstly, we found that a synthetic analog of PIF (sPIF) significantly upregulates the mRNA expression of IGFBP-1 and connexin-43, and prolactin secretion in ESCs - suggesting a pro-differentiation effect. Secondly, we showed that the HTR-8/SVneo trophoblastic cell line's invasive ability was low in the presence of conditioned media from ESCs cultured with sPIF. Thirdly, this PIF's anti-invasive action was associated with a specifically decrease in MMP-9 activity. CONCLUSION: Taken as a whole, our results suggest that PIF accentuates the decidualization process and the production of endometrial factors that limit trophoblast invasion. By controlling both trophoblast and endometrial cells, PIF therefore appears to be a pivotal player in the human embryo implantation process.

Mesenchymal stem cells induce expansion of regulatory T cells in abortion-prone mice.

Recurrent pregnancy loss (RPL) is one of the most common complications of early pregnancy associated in most cases with local or systemic immune abnormalities such as the diminished proportion of regulatory T cells (Tregs). Mesenchymal stem cells (MSCs) have been shown to modulate the immune responses by de novo induction and expansion of Tregs. In this study, we analyzed the molecular and cellular mechanisms involved in Treg-associated pregnancy protection following MSCs administration in an abortion-prone mouse mating. In a case-control study, syngeneic abdominal fat-derived MSCs were administered intraperitoneally (i.p) to the DBA/2-mated CBA/J female mice on day 4.5 of pregnancy. Abortion rate, Tregs proportion in spleen and inguinal lymph nodes, Ho1, Foxp3, Pd1 and Ctla4 genes expression at the feto-maternal interface were then measured on day 13.5 of pregnancy using flow cytometry and quantitative RT-PCR, respectively. The abortion rate in MSCs-treated mice reduced significantly and normalized to the level observed in normal pregnant animals. We demonstrated a significant induction of Tregs in inguinal lymph nodes but not in the spleen following MSCs administration. Administration of MSCs remarkably upregulated the expression of Ho1, Foxp3, Pd1 and Ctla4 genes in both placenta and decidua. Here, we show that MSCs therapy could protect the fetus in the abortion-prone mice through Tregs expansion and upregulation of Treg-related genes. These events could establish an immune-privileged microenvironment, which participates in the regulation of detrimental maternal immune responses against the semi-allogeneic fetus.

Insulin regulation of solute carrier family 2 member 1 (glucose transporter 1) expression and glucose uptake in decidualizing human endometrial stromal cells: an in vitro study.

BACKGROUND: Solute carrier family 2 member 1 (SLC2A1; previously known as glucose transporter 1), is the most abundant glucose transporter in human endometrium and is up-regulated during decidualization, whereas high insulin may have a negative impact on this process. The present study aimed to investigate the effect of insulin on the expression of SLC2A1 and glucose uptake in decidualizing human endometrial stromal cells. METHODS: We induced in vitro decidualization of endometrial stromal cells obtained from regularly menstruating healthy non-obese women. The cells were treated with increasing concentrations of insulin, and the involvement of the transcription factor forkhead box O1 (FOXO1) was evaluated using a FOXO1 inhibitor. SLC2A1 mRNA levels were measured by Real-Time PCR and protein levels were evaluated by immunocytochemistry. Glucose uptake was estimated by an assay quantifying the cellular uptake of radioactive glucose. One-way ANOVA, Dunnett's multiple comparisons test and paired t-test were used to determine the statistical significance of the results. RESULTS: We found that insulin dose-dependently decreased SLC2A1 mRNA levels and decreased protein levels of SLC2A1 in decidualizing human endometrial stromal cells. Transcriptional inactivation of FOXO1 seems to explain at least partly the down-regulation of SLC2A1 by insulin. Glucose uptake increased upon decidualization, whereas insulin treatment resulted in a slight inhibition of the glucose uptake, although not significant for all insulin concentrations. CONCLUSIONS: These results indicate an impairment of decidualization by high concentrations of insulin. Future studies will determine the clinical significance of our results for endometrial function and decidualization in women with insulin resistance and hyperinsulinemia.

Decidualization Process Induces Maternal Monocytes to Tolerogenic IL-10-Producing Dendritic Cells (DC-10).

Decidualization is a process that involves phenotypic and functional changes of endometrial stromal cells to sustain endometrial receptivity and the participation of immunoregulatory factors to maintain immune homeostasis. In this context, tolerogenic dendritic cells (DCs) can induce regulatory T cells, which are essential to manage the pro- to anti-inflammatory transition during embryo implantation. Recently, Myeloid Regulatory Cells (MRCs) were proposed as immunosuppressants and tolerance-inducer cells, including the DC-10 subset. This novel and distinctive subset has the ability to produce IL-10 and to induce type 1 regulatory T cells (Tr1) through an HLA-G pathway. Here we focus on the impact of the decidualization process in conditioning peripheral monocytes to MRCs and the DC-10 subset, and their ability to induce regulatory T cells. An in vitro model of decidualization with the human endometrial stromal cell line (HESC), decidualized by medroxyprogesterone and dibutyryl-cAMP was used. Monocytes isolated from peripheral blood mononuclear cells from healthy women were cultured with rhGM-CSF + rhIL-4 and then, the effect of conditioned media from decidualized (Dec-CM) and non-decidualized cells (Non-dec-CM) was tested on monocyte cultures. We found that Dec-CM inhibited the differentiation to the CD1a+CD14- immature DC profile in a concentration-dependent manner. Dec-CM also significantly increased the frequency of CD83+CD86low and HLA-DR+ cells in the monocyte-derived culture. These markers, associated with the increased production of IL-10, are consistent with a MRCs tolerogenic profile. Interestingly, Dec-CM treatment displayed a higher expression of the characteristic markers of the tolerogenic DC-10 subset, HLA-G and ILT2/CD85j; while this modulation was not observed in cultures treated with Non-dec-CM. Moreover, when monocyte cultures with Dec-CM were challenged with LPS, they sustained a higher IL-10 production and prevented the increase of CD83, CD86, IL-12p70, and TNF-α expression. Finally, the DC-10 subset was able to induce a CD4+HLA-G+ regulatory T cells subset. These results suggest that the decidualization process might induce different subsets of MRCs, like DC-10, able to induce regulatory T cells as a novel CD4+HLA-G+ subset which might play an immunoregulatory role in embryo implantation.

Bisphenol A-induced mechanistic impairment of decidualization.

Decidualization is a crucial precedent to embryo implantation, as its impairment is a major contributor to female infertility and pregnancy complications. Unraveling the molecular mechanisms involved in the impairment of decidualization has been a subject of interest in the field of reproductive medicine. Evidence from several experimental settings show that exposure to bisphenol A (BPA), an endocrine-disrupting chemical, affects the expression of several molecules that are involved in decidualization. Both low and high doses of BPA impair decidualization through the dysregulation of estrogen (ER) and progesterone (PR) receptors. Exposure to low doses of BPA leads to decreased levels and activities of several antioxidant enzymes, increased activity of endothelial nitric oxide synthase (eNOS), and increased production of nitric oxide (NO) via the upregulation of ER and PR. Consequently, oxidative stress is induced and decidualization becomes impaired. On the other hand, exposure to high doses of BPA downregulates ER and PR and impairs decidualization through two distinct pathways. One is through the upregulation of early growth response-1 (EGR1) via increased phosphorylation of extracellular signal-regulated protein kinases 1 and 2; and the other is through a reduced serum glucocorticoid-induced kinase-1 (SGK1)-mediated downregulation of epithelial sodium channel-α and the induction of oxidative stress. Thus, regardless of the dose, BPA can impair decidualization to trigger infertility and pregnancy complications. This warrants the need to adopt lifestyles that will decrease the tendency of getting exposed to BPA.

Endometrial Decidualization: The Primary Driver of Pregnancy Health.

Interventions to prevent pregnancy complications have been largely unsuccessful. We suggest this is because the foundation for a healthy pregnancy is laid prior to the establishment of the pregnancy at the time of endometrial decidualization. Humans are one of only a few mammalian viviparous species in which decidualization begins during the latter half of each menstrual cycle and is therefore independent of the conceptus. Failure to adequately prepare (decidualize) the endometrium hormonally, biochemically, and immunologically in anticipation of the approaching blastocyst-including the downregulation of genes involved in the pro- inflammatory response and resisting tissue invasion along with the increased expression of genes that promote angiogenesis, foster immune tolerance, and facilitate tissue invasion-leads to abnormal implantation/placentation and ultimately to adverse pregnancy outcome. We hypothesize, therefore, that the primary driver of pregnancy health is the quality of the soil, not the seed.

Human Endometrial Stromal Cell Differentiation is Stimulated by PPARß/δ Activation: New Targets for Infertility?

CONTEXT: Implantation is a reproductive bottleneck in women, regulated by fluctuations in ovarian steroid hormone concentrations. However, other nuclear receptor ligands are modifiers of endometrial differentiation leading to successful pregnancy. In the present study we analyzed the effects of peroxisome-proliferator-activated receptor ß/δ (PPARß/δ) activation on established cellular biomarkers of human endometrial differentiation (decidualization). OBJECTIVE: The objective of this work is to test the effects of PPARß/δ ligation on human endometrial cell differentiation. DESIGN: Isolated primary human endometrial stromal cells (ESCs) were treated with synthetic (GW0742) or natural (all trans-retinoic acid, RA) ligands of PPARß/δ, and also with receptor antagonists (GSK0660, PT-S58, and ST247) in the absence or presence of decidualizing hormones (10 nM estradiol, 100 nM progesterone, and 0.5 mM dibutyryl cAMP [3',5'-cyclic adenosine 5'-monophosphate]). In some cases interleukin (IL)-1ß was used as an inflammatory stimulus. Time course and dose-response relationships were evaluated to determine effects on panels of well characterized in vitro biomarkers of decidualization. RESULTS: PPARß/δ, along with estrogen receptor α (ERα) and PR-A and PR-B, were expressed in human endometrial tissue and isolated ESCs. GW0742 treatment enhanced hormone-mediated ESC decidualization in vitro as manifested by upregulation of prolactin, insulin-like growth factor-binding protein 1, IL-11, and vascular endothelial growth factor (VEGF) secretion and also increased expression of ERα, PR-A and PR-B, and connexin 43 (Cx43). RA treatment also increased VEGF, ERα, PR-A, and PR-B and an active, nonphosphorylated isoform of Cx43. IL-1ß and PPARß/δ antagonists inhibited biomarkers of endometrial differentiation. CONCLUSION: Ligands that activate PPARß/δ augment the in vitro expression of biomarkers of ESC decidualization. By contrast, PPARß/δ antagonists impaired decidualization markers. Drugs activating these receptors may have therapeutic benefits for embryonic implantation.

Canine conceptus-maternal communication during maintenance and termination of pregnancy, including the role of species-specific decidualization.

Among domestic animal species, the reproductive biology of the dog belongs to the most peculiar. This includes the conceptus-maternal communication and endocrine mechanisms involved in maintenance of pregnancy. Dogs fully depend on luteal progesterone (P4) throughout pregnancy, with similar steroid secretion patterns in pregnant and non-pregnant bitches until prepartum luteolysis. Thus, dogs lack the classical recognition of pregnancy. The luteal P4 is the most important hormone regulating the onset and maintenance of pregnancy in previously estrogenized bitches. Although the canine uterus is exposed to high P4 levels, decidualization is not spontaneous but induced by the presence of embryos. Following implantation, decidualization continues, associated with development of the invasive endotheliochorial placenta, leading to establishment of maternal decidual cells expressing the nuclear P4 receptor (PGR). Consequently, although not producing steroids, the canine placenta remains highly sensitive to circulating ovarian steroids. The placental conceptus-maternal communication is responsible for the maintenance of pregnancy, with functional withdrawal of PGR evoking a luteolytic cascade with prepartum PGF2α release. The fetal trophoblast is the major source of prepartum placental prostaglandins. This conceptus-maternal communication is unique to the dog and has clinical implications. Due to luteal steroids, there is no prepartum estradiol increase. Elevated cortisol levels are observed irregularly. This emphasizes the unique character of canine reproductive physiology and the challenges in transferring translational research to the dog. Further research is needed for better understanding of canine reproduction and improving clinical protocols, including the latest results obtained from applying modern laboratory technologies such as the transcriptomic approach.

Seminal plasma promotes decidualization of endometrial stromal fibroblasts in vitro from women with and without inflammatory disorders in a manner dependent on interleukin-11 signaling.

STUDY QUESTION: Do seminal plasma (SP) and its constituents affect the decidualization capacity and transcriptome of human primary endometrial stromal fibroblasts (eSFs)? SUMMARY ANSWER: SP promotes decidualization of eSFs from women with and without inflammatory disorders (polycystic ovary syndrome (PCOS), endometriosis) in a manner that is not mediated through semen amyloids and that is associated with a potent transcriptional response, including the induction of interleukin (IL)-11, a cytokine important for SP-induced decidualization. WHAT IS KNOWN ALREADY: Clinical studies have suggested that SP can promote implantation, and studies in vitro have demonstrated that SP can promote decidualization, a steroid hormone-driven program of eSF differentiation that is essential for embryo implantation and that is compromised in women with the inflammatory disorders PCOS and endometriosis. STUDY DESIGN, SIZE, DURATION: This is a cross-sectional study involving samples treated with vehicle alone versus treatment with SP or SP constituents. SP was tested for the ability to promote decidualization in vitro in eSFs from women with or without PCOS or endometriosis (n = 9). The role of semen amyloids and fractionated SP in mediating this effect and in eliciting transcriptional changes in eSFs was then studied. Finally, the role of IL-11, a cytokine with a key role in implantation and decidualization, was assessed as a mediator of the SP-facilitated decidualization. PARTICIPANTS/MATERIALS, SETTING, METHODS: eSFs and endometrial epithelial cells (eECs) were isolated from endometrial biopsies from women of reproductive age undergoing benign gynecologic procedures and maintained in vitro. Assays were conducted to assess whether the treatment of eSFs with SP or SP constituents affects the rate and extent of decidualization in women with and without inflammatory disorders. To characterize the response of the endometrium to SP and SP constituents, RNA was isolated from treated eSFs or eECs and analyzed by RNA sequencing (RNAseq). Secreted factors in conditioned media from treated cells were analyzed by Luminex and ELISA. The role of IL-11 in SP-induced decidualization was assessed through Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-9-mediated knockout experiments in primary eSFs. MAIN RESULTS AND THE ROLE OF CHANCE: SP promoted decidualization both in the absence and presence of steroid hormones (P < 0.05 versus vehicle) in a manner that required seminal proteins. Semen amyloids did not promote decidualization and induced weak transcriptomic and secretomic responses in eSFs. In contrast, fractionated SP enriched for seminal microvesicles (MVs) promoted decidualization. IL-11 was one of the most potently SP-induced genes in eSFs and was important for SP-facilitated decidualization. LARGE SCALE DATA: RNAseq data were deposited in the Gene Expression Omnibus repository under series accession number GSE135640. LIMITATIONS, REASONS FOR CAUTION: This study is limited to in vitro analyses. WIDER IMPLICATIONS OF THE FINDINGS: Our results support the notion that SP promotes decidualization, including within eSFs from women with inflammatory disorders. Despite the general ability of amyloids to induce cytokines known to be important for implantation, semen amyloids poorly signaled to eSFs and did not promote their decidualization. In contrast, fractionated SP enriched for MVs promoted decidualization and induced a transcriptional response in eSFs that overlapped with that of SP. Our results suggest that SP constituents, possibly those associated with MVs, can promote decidualization of eSFs in an IL-11-dependent manner in preparation for implantation. STUDY FUNDING/COMPETING INTEREST(S): This project was supported by NIH (R21AI116252, R21AI122821 and R01AI127219) to N.R.R. and (P50HD055764) to L.C.G. The authors declare no conflict of interest.

HOXA10 co-factor MEIS1 is required for the decidualization in human endometrial stromal cell.

Decidualization is a critical process for embryo implantation and pregnancy maintenance in humans. The homeobox gene HOXA10 has been widely studied in endometrial receptivity establishment and decidualization. MEIS1, a three-amino-acid loop extension (TALE) family homeobox gene, has been proven to be a co-factor for HOXA10 in mouse uterus. However, the interaction between MEIS1 and HOXA10 in the human decidual cells remains to be elucidated. siRNA and CRISPR-Cas9 were employed to knockdown and knockout MEIS1 in the cultured human endometrial stromal cells, and it was found that MEIS1 deficiency leads to impaired decidualization. The physical interaction between the MEIS1 and HOXA10 in human endometrial stromal cell was confirmed by immunoprecipitation. Moreover, KAT2B and ETA were proved to be downregulated in the absence of MEIS1, and luciferase reporter and ChIP assays demonstrated that MEIS1-HOXA10 complex binds to the promoters of KAT2B and ETA and regulates their activity. Overexpression of KAT2B and ETA can partially rescue the decidualization defects in MEIS1-knockout HESCs. Taken together, these data suggest that MEIS1 plays an indispensable role in decidualization in human endometrial stromal cells, and MEIS1 interacts with HOXA10 to regulate the downstream genes, such as KAT2B and ETA. These findings will contribute to our understanding about the regulatory network in the process of decidualization in humans.