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INTRODUCTION: Preeclampsia is a life-threatening disorder of pregnancy unique to humans. Interleukin (IL)11 is elevated in serum from pregnancies that subsequently develop early-onset preeclampsia and pharmacological elevation of IL11 in pregnant mice causes the development of early-onset preeclampsia-like features (hypertension, proteinuria, and fetal growth restriction). However, the mechanism by which IL11 drives preeclampsia is unknown. METHOD: Pregnant mice were administered PEGylated (PEG)IL11 or control (PEG) from embryonic day (E)10-16 and the effect on inflammasome activation, systolic blood pressure (during gestation and at 50/90 days post-natal), placental development, and fetal/post-natal pup growth measured. RNAseq analysis was performed on E13 placenta. Human 1st trimester placental villi were treated with IL11 and the effect on inflammasome activation and pyroptosis identified by immunohistochemistry and ELISA. RESULT: PEGIL11 activated the placental inflammasome causing inflammation, fibrosis, and acute and chronic hypertension in wild-type mice. Global and placental-specific loss of the inflammasome adaptor protein Asc and global loss of the Nlrp3 sensor protein prevented PEGIL11-induced fibrosis and hypertension in mice but did not prevent PEGIL11-induced fetal growth restriction or stillbirths. RNA-sequencing and histology identified that PEGIL11 inhibited trophoblast differentiation towards spongiotrophoblast and syncytiotrophoblast lineages in mice and extravillous trophoblast lineages in human placental villi. DISCUSSION: Inhibition of ASC/NLRP3 inflammasome activity could prevent IL11-induced inflammation and fibrosis in various disease states including preeclampsia.

INTRODUCTION: A healthy pregnancy requires successful blastocyst implantation into an adequately prepared or 'receptive' endometrium. Decidualization of uterine endometrial stromal fibroblast cells (hESF) is critical for the establishment of a healthy pregnancy. microRNAs (miRs) are critical regulators of cellular function that can be released by a donor cell to influence the physiological state of recipient cells. We aimed to determine how decidualization affects hESF miR release and investigated the function of one decidualization regulated miR, miR-19b-3p, previously shown to be associated with recurrent pregnancy loss. METHOD: miR release by hESF was determined by miR microarray on culture media from hESF decidualized in vitro for 3 and 14 days by treatment with oestradiol and medroxyprogesterone acetate. Cellular and whole endometrial/decidual tissue miR expression was quantified by qPCR and localized by in situ hybridization. The function of miR-19b-3p in HTR8/Svneo trophoblast cells was investigated using real time cell analysis (xCELLigence) and gene expression qPCR. RESULTS: From our miR screen we found that essentially all hESF miR release was reduced following in vitro decidualization, significantly so for miR-17-5p, miR-21-3p, miR-34c-3p, miR-106b-5p, miR-138-5p, miR-296-5p, miR-323a-3p, miR-342-3p, miR-491-5p, miR-503-5p and miR-542-5p. qPCR demonstrated that miR-19b-3p, 181a-2-3p and miR-409-5p likewise showed a significant reduction in culture media following decidualization but no change was found in cellular miR expression following decidualization. In situ hybridization localized miR-19b-3p to epithelial and stromal cells in the endometrium and qPCR identified that miR-19b-3p was significantly elevated in the cycling endometrium of patients with a history of early pregnancy loss compared to normally fertile controls. Functionally, overexpression of miR-19b-3p significantly reduced HTR8/Svneo trophoblast proliferation and increased HOXA9 expression.

Endometrial cancer is the most diagnosed gynecological malignancy. Despite numerous scientific advances, the incidence and mortality rate of endometrial cancer continues to rise. Emerging evidence suggests a putative role of the (pro)renin receptor ((P)RR), in the ontogenesis of endometrial cancer. The (P)RR is implicated in breast cancer and pancreatic carcinoma pathophysiology by virtue of its role in proliferation, angiogenesis, fibrosis, migration and invasion. Thus, we aimed to investigate the functional role of the (P)RR in human endometrial cancer. We employed an siRNA-mediated knockdown approach to abrogate (P)RR expression in the endometrial epithelial cell lines; Ishikawa, AN3CA and HEC-1-A and examined cellular proliferation and viability. We also carried out a sophisticated proteomic screen to explore potential pathways via which the (P)RR is acting in endometrial cancer physiology. These data confirmed that the (P)RR is critical for endometrial cancer development, contributing to both its proliferative capacity and in the maintenance of cell viability. This is likely mediated through proteins such as MGA, SLC4A7, SLC7A11 or DHRS2, which were reduced following (P)RR knockdown. These putative protein interactions/pathways, which rely on the presence of the (P)RR, are likely to contribute to endometrial cancer progression and could therefore, represent several novel therapeutic targets for endometrial cancer.

Fetal growth restriction (FGR) is commonly associated with placental insufficiency and inflammation. Nonetheless, the role played by inflammasomes in the pathogenesis of FGR is poorly understood. We hypothesised that placental inflammasomes are differentially expressed and contribute to the aberrant trophoblast function. Inflammasome gene expression profiles were characterised by real-time PCR on human placental tissues collected from third trimester FGR and gestation-matched control pregnancies (n = 25/group). The functional significance of a candidate inflammasome was then investigated using lipopolysaccharide (LPS)-induced models of inflammation in human trophoblast organoids, BeWo cells in vitro, and a murine model of FGR in vivo. Placental mRNA expression of NLRP3, caspases 1, 3, and 8, and interleukin 6 increased (>2-fold), while that of the anti-inflammatory cytokine, IL-10, decreased (<2-fold) in FGR compared with control pregnancies. LPS treatment increased NLRP3 and caspase-1 expression (>2-fold) in trophoblast organoids and BeWo cell cultures in vitro, and in the spongiotrophoblast and labyrinth in the murine model of FGR. However, the LPS-induced rise in NLRP3 was attenuated by its siRNA-induced down-regulation in BeWo cell cultures, which correlated with reduced activity of the apoptotic markers, caspase-3 and 8, compared to the control siRNA-treated cells. Our findings support the role of the NLRP3 inflammasome in the inflammation-induced aberrant trophoblast function, which may contribute to FGR.

Proper placentation in the first trimester is essential for a healthy pregnancy in humans. A recent proteomics study of human placental tissue has identified that tripeptidyl peptidase 1 (TPP1) production is reduced in the placenta in early-onset preeclampsia compared to uncomplicated pregnancy. However, it remains to be investigated if TPP1 plays a role in regulating trophoblast cell function during early pregnancy. In this study, immunohistochemistry was used to determine the production and localization of TPP1 in human placenta throughout gestation and the first-trimester decidua/implantation sites. TPP1 siRNA (20 nM) was transfected into a human trophoblast cell line (HTR8/SVneo) to knock down TPP1, and functional consequences on cell adhesion, proliferation, migration, and invasion were analyzed via xCELLigence real-time monitoring. The expression of TPP1 downstream targets was examined by qPCR. Our data show that TPP1 localized to the discrete foci in the cytoplasm in syncytiotrophoblast, cytotrophoblast, and decidual cells across all trimesters of pregnancy. In the first-trimester human decidua, TPP1 exhibited similar staining patterns in the cytotrophoblast cells based at the cell columns. However, minimal/no staining was identified in the HLA-G positive extravillous trophoblast cells (EVTs), especially in the EVTs that invaded in the decidua. Knockdown of TPP1 in HTR8/SVneo cells by 95% significantly impaired cell adhesion and proliferation without affecting cell migration and invasion. qPCR revealed that the expression of cell proliferation markers P21 and MKI67 and TPP1-related genes MRE11, CLN3, and CLN8 was significantly changed after TPP1 knockdown in HTR8/SVneo cells compared to control. Overall, our data demonstrate that TPP1 alters trophoblast cell line function suggesting that it may be involved in regulating human placentation in the first trimester via controlling trophoblast cell adhesion and proliferation.

Background: A couple’s infertility can originate from the male and/or the female. In women, the uterus provides the site where the maternal-fetal interface is established and maintained. Final blastocyst development occurs within the uterine cavity, then the blastocyst must attach to and implant into the endometrium (the inner uterine surface), via its outermost trophectodermal cells. Beneath the epithelium, these differentiate into syncytial trophoblast and invasive trophoblast — the latter progress through the endometrium to invade the spiral arteries converting them to the flaccid blood sacs of the placenta. Therefore, the endometrium plays a critical role in establishment of pregnancy. Objectives: To critically examine current knowledge of endometrial preparation for blastocyst implantation and placental development at the cellular and molecular level and to evaluate measures to improve implantation success. Mechanism: Literature searching by leading experts in the field. Findings: A wealth of new knowledge resulting from ‘omics’ technologies and new functional models has greatly enhanced our knowledge, but this information is yet to be translated into enhanced outcomes. Conclusions: The endometrium remains the ‘black box’ of infertility. Extensive trials do not support current adjuvant therapies as being better than placebo while effectively timed testing for endometrial preparedness for implantation is still urgently needed.

INTRODUCTION: Embryo implantation failure leads to infertility. As an important approach to regulate implantation, endometrial epithelial cells produce and secrete factors apically into the uterine cavity in the receptive phase to prepare the initial blastocyst adhesion and implantation. Organoids were recently developed from human endometrial epithelium with similar apical-basal polarity compared to endometrial gland making it an ideal model to study endometrial epithelial secretions. METHODS: Endometrial organoids were established using endometrial biopsies from women with primary infertility and normal fertility. Fertile and infertile organoids were treated with hormones to model receptive phase of the endometrial epithelium and intra-organoid fluid (IOF) was collected to compare the apical protein secretion profile and function on trophoblast cell adhesion. RESULTS: Our data show that infertile organoids were dysregulated in their response to estrogen and progesterone treatment. Proteomic analysis of organoid apical secretions identified 150 dysregulated proteins between fertile and infertile groups (>1.5-fold change). Trophoblast progenitor spheroids (blastocyst surrogates) treated with infertile organoid apical secretions significantly compromised their adhesion to organoid epithelial cell monolayers compared to fertile group (P < 0.0001). DISCUSSION: This study revealed that endometrial organoid apical secretions alter trophoblast cell adhesiveness relative to fertility status of women. It paves the way to determine the molecular mechanisms by which endometrial epithelial apical released factors regulate blastocyst initial attachment and implantation.

The human endometrium undergoes cycle-dependent changes and is only receptive to an implanting blastocyst within a narrow window of 2-4 days in the mid-secretory phase. Such functional changes require delicate interplay between a diversity of factors including cytokines and signaling pathways. The Notch signaling pathway members are expressed in human endometrium. We have previously demonstrated that Notch ligand Jagged1 (JAG1) localizes in the endometrial luminal epithelium (LE) and is abnormally reduced in infertile women during receptivity. However, the functional consequences of reduced JAG1 production on endometrial receptivity to implantation of the blastocyst are unknown. This study aimed to determine the role of JAG1 in regulating endometrial receptivity in humans and mice. Knockdown of JAG1 in both primary human endometrial epithelial cells and Ishikawa cells significantly reduced their adhesive capacity to HTR8/SVneo (trophoblast cell line) spheroids. We confirmed that in human endometrial epithelial cells, JAG1 interacted with Notch Receptor 3 (NOTCH3) and knockdown of JAG1 significantly reduced the expression of Notch signaling downstream target HEY1 and classical receptivity markers. Knockdown of Jag1 in mouse LE significantly impaired blastocyst implantation. We identified ten genes (related to tight junction, infertility, and cell adhesion) that were differentially expressed by Jag1 knockdown in LE in mice. Further analysis of the tight junction family members in both species revealed that JAG1 altered the expression of tight junction components only in mice. Together, our data demonstrated that JAG1 altered endometrial epithelial cell adhesive capacity and regulated endometrial receptivity in both humans and mice likely via different mechanisms.

BACKGROUND: Abnormalities in endometrial receptivity has been identified as a major barrier to successful embryo implantation. Endometrial receptivity refers to the conformational and biochemical changes occurring in the endometrial epithelial layer which make it adhesive and receptive to blastocyst attachment. This takes place during the mid-secretory phase of woman's menstrual cycle and is a result of a delicate interplay between numerous hormones, cytokines and other factors. Outside of this window, the endometrium is refractory to an implanting blastocyst. It has been shown that Notch ligands and receptors are dysregulated in the endometrium of infertile women. Mastermind Like Transcriptional Coactivator 1 (MAML1) is a known coactivator of the Notch signaling pathway. This study aimed to determine the role of MAML1 in regulating endometrial receptivity. METHODS: The expression and localization of MAML1 in the fertile human endometrium (non-receptive proliferative phase versus receptive mid-secretory phase) were determined by immunohistochemistry. Ishikawa cells were used as an endometrial epithelial model to investigate the functional consequences of MAML1 knockdown on endometrial adhesive capacity to HTR8/SVneo (trophoblast cell line) spheroids. After MAML1 knockdown in Ishikawa cells, the expression of endometrial receptivity markers and Notch dependent and independent pathway members were assessed by qPCR. Two-tailed unpaired or paired student's t-test were used for statistical analysis with a significance threshold of P < 0.05. RESULTS: MAML1 was localized in the luminal epithelium, glandular epithelium and stroma of human endometrium and the increased expression identified in the mid-secretory phase was restricted only to the luminal epithelium (P < 0.05). Functional analysis using Ishikawa cells demonstrated that knockdown of MAML1 significantly reduced epithelial adhesive capacity (P < 0.01) to HTR8/SVneo (trophoblast cell line) spheroids compared to cont

An amendment to this paper has been published and can be accessed via the original article.

Egyptian spiny mice are the only known species to have human-like menstruation and a postpartum ovulation. Unfortunately, no endocrine or morphological evidence has been provided for a postpartum ovulation in spiny mice, and while later stages of pregnancy have been well studied, early events including embryo implantation and spiral artery remodelling have not been reported. This study compared the sex steroid endocrinology and reproductive tract morphology of dams at eight timepoints (n = 40) postpartum to determine the timing of ovulation and the timing and invasiveness of embryo implantation in A. cahirinus. Reproductive tracts were fixed and stained for histology and immunohistochemistry, and plasma was prepared for enzyme-linked immunosorbent assay. Ovarian histology and estradiol-17B concentrations indicate ovulation within 48 h of parturition and then immediate resumption of follicular growth. Uterine histology and immunohistochemistry revealed progressive epithelial repair, endometrial growth and spiral artery assembly and remodelling in dams postpartum. Blastocysts were seen in the uterine lumen at day 4-5 postpartum and embryos had implanted superficially with minimal stromal invasion by day 5-6. This study provides further evidence for the unique, humanesque reproductive biology of spiny mice and for a postpartum ovulation using endocrine and morphological changes observed during early pregnancy. Taken together, our data suggest that spiny mice may act as appropriate models of human pregnancy disorders such as implantation failure or pre-eclampsia.

Poor pregnancy outcomes such as recurrent pregnancy loss (RPL) and preeclampsia are associated with impaired decidualization and abnormal trophoblast invasion. Emerging evidence suggests that use of corticosteroids, including prednisolone affects fertility by altering uterine function and may be associated with preeclampsia incidence. In this study, using primary and gestational-age appropriate tissue, we aimed to define the effect of prednisolone on human endometrial stromal fibroblast (hESF) decidualization and determine whether hESF decidualization in the presence of prednisolone would alter hESF regulation of trophoblast function. We found that prednisolone treatment reduced hESF cytokine expression (IL6, IL11, IL18, LIF, and LIFR) but had no effect on hESF expression or secretion of the classic markers of decidualization [prolactin (PRL) and IGFBP1]. Using proteomics we determined that prednisolone altered decidualized hESF protein production, enriching hESF proteins associated with acetylation and mitrochondria. Conditioned media from hESF decidualized in the presence of prednisolone significantly enhanced trophoblast outgrowth and trophoblast mRNA expression of cell motility gene PLCG1 and reduced trophoblast production of PGF. Prednisolone treatment during the menstrual cycle and 1st trimester of pregnancy might alter decidual interactions with other cells, including invasive trophoblast.

[This corrects the article DOI: 10.1371/journal.pone.0030916.].

Recurrent pregnancy loss is a distressing pregnancy disorder experienced by ~2.5% of women trying to conceive. Recurrent pregnancy loss is defined as the failure of two or more clinically recognized pregnancies before 20-24 weeks of gestation and includes embryonic and fetal losses. The diagnosis of an early pregnancy loss is relatively straightforward, although progress in predicting and preventing recurrent pregnancy loss has been hampered by a lack of standardized definitions, the uncertainties surrounding the pathogenesis and the highly variable clinical presentation. The prognosis for couples with recurrent pregnancy loss is generally good, although the likelihood of a successful pregnancy depends on maternal age and the number of previous losses. Recurrent pregnancy loss can be caused by chromosomal errors, anatomical uterine defects, autoimmune disorders and endometrial dysfunction. Available treatments target the putative risk factors of pregnancy loss, although the effectiveness of many medical interventions is controversial. Regardless of the underlying aetiology, couples require accurate information on their chances of having a baby and appropriate support should be offered to reduce the psychological burden associated with multiple miscarriages. Future research must investigate the pathogenesis of recurrent pregnancy loss and evaluate novel diagnostic tests and treatments in adequately powered clinical trials.

Embryo implantation failure is considered a leading cause of infertility and a significant bottleneck for in vitro fertilization (IVF) treatment. Confirmed factors that lead to implantation failure involve unhealthy embryos, unreceptive endometrium, and asynchronous development and communication between the two. The quality of embryos is further dependent on sperm parameters, oocyte quality, and early embryo development after fertilization. The extensive involvement of such different factors contributes to the variability of implantation potential across different menstrual cycles. An ideal approach to predict the implantation outcome should not compromise embryo implantation. The use of clinical material, including follicular fluid, cumulus cells, sperm, seminal exosomes, spent blastocyst culture medium, blood, and uterine fluid, that can be collected relatively non-invasively without compromising embryo implantation in a transfer cycle opens new perspectives for the diagnosis of embryo implantation potential. Compositional comparison of these samples between fertile women and women or couples with implantation failure has identified both quantitative and qualitative differences in the expression of microRNAs (miRs) that hold diagnostic potential for implantation failure. Here, we review current findings of secreted miRs that have been identified to potentially be useful in predicting implantation outcome using material that can be collected relatively non-invasively. Developing non-invasive biomarkers of implantation potential would have a major impact on implantation failure and infertility.

BACKGROUND: Abnormal endometrial receptivity is one of the major causes of embryo implantation failure and infertility. The plasma membrane transformation (PMT) describes the collective morphological and molecular alterations occurring to the endometrial luminal epithelium across the mid-secretory phase of the menstrual cycle to facilitate implantation. Dysregulation of this process directly affects endometrial receptivity and implantation. Multiple parallels between these alterations to confer endometrial receptivity in women have been drawn to those seen during the epithelial-mesenchymal transition (EMT) in tumorigenesis. Understanding these similarities and differences will improve our knowledge of implantation biology, and may provide novel therapeutic targets to manage implantation failure. METHODS: A systematic review was performed using the Medline (Ovid), Embase, and Web of Science databases without additional limits. The search terms used were "(plasma membrane* or cell membrane*) and transformation*" and "endometrium or endometrial." Research studies on the PMT or its regulation in women, discussing either the endometrial epithelium, decidualized stroma, or both, were eligible for inclusion. RESULTS: A total of 198 articles were identified. Data were extracted from 15 studies that matched the inclusion criteria. Collectively, these included studies confirmed the alterations occurring to the endometrial luminal epithelium during the PMT are similar to those seen during the EMT. Such similarities included alterations to the actin cytoskeleton remodeling of adherens junctions, integrin expression and epithelial-stromal communication. These were also some differences between these processes, such as the regulation of tight junctions and mucins, which need to be further researched. CONCLUSIONS: This review raised the prospect of shared and distinct mechanisms existing in PMT and EMT. Further investigation into similarities between the PMT in the endometrium and

Preeclampsia (PE) is a pregnancy-specific multisystem disorder and is associated with maladaptation of the maternal cardiovascular system and abnormal placentation. One of the important characteristics in the pathophysiology of PE is a dysfunction of the placenta. Placental insufficiency is associated with poor trophoblast uterine invasion and impaired transformation of the uterine spiral arterioles to high capacity and low impedance vessels and/or abnormalities in the development of chorionic villi. Significant progress in identifying potential molecular targets in the pathophysiology of PE is underway. The human placenta is immunologically functional with the trophoblast able to generate specific and diverse innate immune-like responses through their expression of multimeric self-assembling protein complexes, termed inflammasomes. However, the type of response is highly dependent upon the stimuli, the receptor(s) expressed and activated, the downstream signaling pathways involved, and the timing of gestation. Recent findings highlight that inflammasomes can act as a molecular link for several components at the syncytiotrophoblast surface and also in maternal blood thereby directly influencing each other. Thus, the inflammasome molecular platform can promote adverse inflammatory effects when chronically activated. This review highlights current knowledge in placental inflammasome expression and activity in PE-affected pregnancies, and consequently, vascular dysfunction in PE that must be addressed as an interdependent interactive process.

BACKGROUND: The endometrial luminal epithelium is the first point of attachment of embryos during implantation. Failure of embryos to firmly adhere results in implantation failure and infertility. A receptive endometrial luminal epithelium is achieved through the expression of adhesion molecules in the mid-secretory phase and is a requirement for implantation. Cadherin 6 (CDH6) is an adhesion molecule localizing to the endometrial luminal epithelial cell surface in the mid-secretory/receptive phase and knockdown of CDH6 in the Ishikawa cells (receptive endometrial epithelial cell line) compromises cell integrity. However, there are no studies investigating the role of CDH6 on receptivity and infertility. This study aimed to investigate whether CDH6 is dysregulated in the endometrium of women with infertility during the receptive window and the effect of CDH6 on endometrial adhesion and receptivity. METHODS: The expression and the localization of CDH6 in the human endometrium were determined by immunohistochemistry. Ishikawa cells were used to investigate the functional consequences of CDH6 knockdown on endometrial adhesive capacity to HTR8/SVneo (trophoblast cell line) spheroids in vitro. CDH6 knockdown was assessed by qPCR and immunoblotting. After CDH6 knockdown, the expression of type II cadherin family members and CDH6 functional partners were assessed by qPCR. Two-tailed unpaired student's t-test or one-way ANOVA as appropriate were used for statistical analysis with a significance threshold of P < 0.05. RESULTS: A significant reduction of CDH6 immunolocalization was recorded in the luminal and glandular epithelium of endometrium from women with infertility (P < 0.05) compared to fertile group respective cellular compartments in the mid-secretory phase. Functional analysis using Ishikawa cells demonstrated that knockdown of CDH6 (treated with 50 nM CDH6 siRNA) significantly reduced epithelial adhesive capacity (P < 0.05) to HTR8/SVneo spheroids compared to cont

We reported earlier that an anti-inflammatory small peptide receptor-formyl peptide receptor-2 (FPR2) was significantly decreased in placentas from third trimester pregnancies complicated with fetal growth restriction (FGR), compared to placentas from uncomplicated control pregnancies, suggesting FPR2 may play a role in the development of FGR. The aim of this study is to investigate whether the actions of FPR2 alters placental growth process in humans. Accordingly, using small-for-gestation age (SGA) as a proxy for FGR, we hypothesize that FPR2 expression is decreased in first-trimester placentas of women who later manifest FGR, and contributes to aberrant trophoblast function and the development of FGR. Chorionic villus sampling (CVS) tissues were collected at 10-12 weeks gestation in 70 patients with singleton fetuses; surplus tissue was used. Real-time PCR and immunoassays were performed to quantitate FPR2 gene and protein expression. Silencing of FPR2 was performed in two independent, trophoblast-derived cell lines, HTR-8/SVneo and JEG-3 to investigate the functional consequences of FPR2 gene downregulation. FPR2 mRNA relative to 18S rRNA was significantly decreased in placentae from SGA-pregnancies (n = 28) compared with controls (n = 52) (p < 0.0001). Placental FPR2 protein was significantly decreased in SGA compared with control (n = 10 in each group, p < 0.05). Proliferative, migratory and invasive potential of the human placental-derived cell lines, HTR-8/SVneo and JEG-3 were significantly reduced in siFPR2 treated cells compared with siCONT control groups. Down-stream signaling molecules, STAT5B and SOCS3 were identified as target genes of FPR2 action in the trophoblast-derived cell lines and in SGA and control chorionic villous tissues. FPR2 is a novel regulator of key molecular pathways and functions in placental development, and its decreased expression in women destined to develop FGR reinforces a placental origin of SGA/FGR, and that it contributes to