IL11 activates the placental inflammasome to drive preeclampsia.

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.

Cyclic processes in the uterine tubes, endometrium, myometrium, and cervix: pathways and perturbations.

This review leads the 2023 Call for Papers in MHR: 'Cyclical function of the female reproductive tract' and will outline the complex and fascinating changes that take place in the reproductive tract during the menstrual cycle. We will also explore associated reproductive tract abnormalities that impact or are impacted by the menstrual cycle. Between menarche and menopause, women and people who menstruate living in high-income countries can expect to experience ∼450 menstrual cycles. The primary function of the menstrual cycle is to prepare the reproductive system for pregnancy in the event of fertilization. In the absence of pregnancy, ovarian hormone levels fall, triggering the end of the menstrual cycle and onset of menstruation. We have chosen to exclude the ovaries and focus on the other structures that make up the reproductive tract: uterine tubes, endometrium, myometrium, and cervix, which also functionally change in response to fluctuations in ovarian hormone production across the menstrual cycle. This inaugural paper for the 2023 MHR special collection will discuss our current understanding of the normal physiological processes involved in uterine cyclicity (limited specifically to the uterine tubes, endometrium, myometrium, and cervix) in humans, and other mammals where relevant. We will emphasize where knowledge gaps exist and highlight the impact that reproductive tract and uterine cycle perturbations have on health and fertility.

Endometrial stromal cell miR-19b-3p release is reduced during decidualization implying a role in decidual-trophoblast cross-talk.

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. Discussion: Our data demonstrates that decidualization represses miR release by hESFs and overexpression of miR-19b-3p was found in endometrial tissue from patients with a history of early pregnancy loss. miR-19b-3p impaired HTR8/Svneo proliferation implying a role in trophoblast function. Overall we speculate that miR release by hESF may regulate other cell types within the decidua and that appropriate release of miRs by decidualized hESF is essential for healthy implantation and placentation.

Radiotherapy exposure directly damages the uterus and causes pregnancy loss.

Female cancer survivors are significantly more likely to experience infertility than the general population. It is well established that chemotherapy and radiotherapy can damage the ovary and compromise fertility, yet the ability of cancer treatments to induce uterine damage, and the underlying mechanisms, have been understudied. Here, we show that in mice total-body γ-irradiation (TBI) induced extensive DNA damage and apoptosis in uterine cells. We then transferred healthy donor embryos into ovariectomized adolescent female mice that were previously exposed to TBI to study the impacts of radiotherapy on the uterus independent from effects to ovarian endocrine function. Following TBI, embryo attachment and implantation were unaffected, but fetal resorption was evident at midgestation in 100% of dams, suggesting failed placental development. Consistent with this hypothesis, TBI impaired the decidual response in mice and primary human endometrial stromal cells. TBI also caused uterine artery endothelial dysfunction, likely preventing adequate blood vessel remodeling in early pregnancy. Notably, when pro-apoptotic protein Puma-deficient (Puma-/-) mice were exposed to TBI, apoptosis within the uterus was prevented, and decidualization, vascular function, and pregnancy were restored, identifying PUMA-mediated apoptosis as a key mechanism. Collectively, these data show that TBI damages the uterus and compromises pregnancy success, suggesting that optimal fertility preservation during radiotherapy may require protection of both the ovaries and uterus. In this regard, inhibition of PUMA may represent a potential fertility preservation strategy.

Pre-eclampsia.

Pre-eclampsia is a life-threatening disease of pregnancy unique to humans and a leading cause of maternal and neonatal morbidity and mortality. Women who survive pre-eclampsia have reduced life expectancy, with increased risks of stroke, cardiovascular disease and diabetes, while babies from a pre-eclamptic pregnancy have increased risks of preterm birth, perinatal death and neurodevelopmental disability and cardiovascular and metabolic disease later in life. Pre-eclampsia is a complex multisystem disease, diagnosed by sudden-onset hypertension (>20 weeks of gestation) and at least one other associated complication, including proteinuria, maternal organ dysfunction or uteroplacental dysfunction. Pre-eclampsia is found only when a placenta is or was recently present and is classified as preterm (delivery <37 weeks of gestation), term (delivery ≥37 weeks of gestation) and postpartum pre-eclampsia. The maternal syndrome of pre-eclampsia is driven by a dysfunctional placenta, which releases factors into maternal blood causing systemic inflammation and widespread maternal endothelial dysfunction. Available treatments target maternal hypertension and seizures, but the only 'cure' for pre-eclampsia is delivery of the dysfunctional placenta and baby, often prematurely. Despite decades of research, the aetiology of pre-eclampsia, particularly of term and postpartum pre-eclampsia, remains poorly defined. Significant advances have been made in the prediction and prevention of preterm pre-eclampsia, which is predicted in early pregnancy through combined screening and is prevented with daily low-dose aspirin, starting before 16 weeks of gestation. By contrast, the prediction of term and postpartum pre-eclampsia is limited and there are no preventive treatments. Future research must investigate the pathogenesis of pre-eclampsia, in particular of term and postpartum pre-eclampsia, and evaluate new prognostic tests and treatments in adequately powered clinical trials.

miR-23b-3p regulates human endometrial epithelial cell adhesion implying a role in implantation.

In brief: miR-23b-3p expression is increased in fertile endometrium during receptivity. This study investigates the function of miR-23b-3p on endometrial adhesion and its downstream targets. Abstract: The human endometrium undergoes dramatic remodeling throughout the menstrual cycle that is essential for successful blastocyst attachment and implantation in the mid-secretory (receptive) phase. microRNA (miR) plays a role in the preparation of endometrial receptivity. miR-23b-3p expression is increased in fertile endometrium during receptivity. Here, we aimed to investigate miR-23b-3p function during receptivity. qPCR and in situ hybridization were used to investigate the expression and localization of miR-23b-3p in human endometrium, respectively. Ishikawa cells (endometrial epithelial cell line) and endometrial organoid-derived epithelial cells were transfected with miR-23b-3p mimic, and trophoblast progenitor spheroid (blastocyst surrogate) adhesion assay was used to determine effects on blastocyst adhesion to endometrial cells. We demonstrated that miR-23b-3p was significantly upregulated in the fertile endometrium of the receptive phase compared to the non-receptive, proliferative phase. No difference was identified for the expression of miR-23b-3p between fertile and infertile mid-secretory phase endometrium. miR-23b-3p localized to the epithelium and stroma in the mid-secretory phase but was undetectable in the proliferative phase of fertile endometrium. Functionally, miR-23-3p overexpression in Ishikawa cells and fertile endometrial organoid-derived epithelial cells significantly improved their adhesive capacity to trophoblast progenitor spheroids. miR-23b-3p overexpression in infertile endometrial organoid-derived epithelial cells did not improve adhesion. Among 10 miR-predicted gene targets examined, miR-23b-3p overexpression in Ishikawa cells significantly reduced the expression of MET, secreted frizzled-related protein 4 (SFRP4) and acyl-CoA dehydrogenase short/branched chain (ACADSB) compared to control. The reduction of SFRP4 after miR23b-3p overexpression was confirmed by immunoblotting in fertile organoid-derived epithelial cells. SFRP4 expression in fertile endometrium exhibited an inverse expression pattern compared to miR-23b-3p and was higher in the proliferative phase compared to the mid-secretory phase. Overall, miR-23b-3p is likely a critical regulator of endometrial epithelial adhesion and receptivity.

Galectin-7 dysregulates renin-angiotensin-aldosterone and NADPH oxide synthase pathways in preeclampsia.

OBJECTIVES: Preeclampsia is a life-threatening disorder of pregnancy unique to humans. Poor placentation in the first trimester of pregnancy is widely accepted to be an underlying cause of preeclampsia. Galectin-7 is abnormally elevated in chorionic villous samples and serum from women that subsequently develop pre-term preeclampsia. Administration of exogenous galectin-7 to pregnant mice causes preeclampsia-like features (hypertension, proteinuria), associated with dysregulation of the renin-angiotensin system (RAS). In this study investigated the mechanism by which galectin-7 induces alterations to tissue RAS homeostasis and ROS production. We hypothesized that galectin-7 induces alterations in the production of either placental RAS or NADPH oxidases (or both) to drive the dysregulated RAS and ROS production seen in preeclampsia. STUDY DESIGN: Mated female mice (n = 5-6/group) received single (embryonic day [E]12/13) or multiple (E8-12) subcutaneous injections of 400 µg/kg/day galectin-7 or vehicle control and killed on E13 or E18. Human first trimester placental villous and decidual tissue (n = 11) was cultured under 8 % oxygen with 1 µg/mL galectin-7 or vehicle control for 16 h. RESULTS: Galectin-7 administration to pregnant mice impaired placental labyrinth formation, suppressed circulating aldosterone and altered placental RAS (Agt, Renin) and NADPH oxidase (Cyba, Cybb and Icam1) mRNA expression. In vitro, galectin-7 regulated human placental villous RAS (AGT) and NADPH oxidase (CYBA, ICAM1 and VCAM1) mRNA expression. CONCLUSIONS: Overall, galectin-7 likely drives hypertension in preeclampsia via its direct regulation of multiple pathways associated with preeclampsia in the placenta. Galectin-7 may therefore be a therapeutic target to improve placental function and prevent preeclampsia.

Characterization of chloride intracellular channel 4 in the regulation of human trophoblast function.

INTRODUCTION: Proper placentation requires well controlled extravillous trophoblast cell (EVT) migration and invasion. Transforming growth factor ß (TGFß) signaling has been well characterized as negatively regulating EVT migration and invasion. CLIC4 is an enhancer of TGFß signaling, however CLIC4's function in placentation and its association to placental TGFß signaling is unknown. Here we aimed to investigate the role of CLIC4 on trophoblast cell function and its relationship to TGFß signaling. METHODS: CLIC4 was immunolocalized in human placenta throughout gestation and the first trimester decidua. siRNA was used to knockdown CLIC4 in a human trophoblast cell line (HTR8/SVneo) to reveal functional consequences of CLIC4 loss on cell adhesion, proliferation, migration and invasion via xCELLigence. qPCR was used to identify downstream targets of CLIC4 in HTR8/SVNeo cells. RESULTS: CLIC4 was widely expressed in the syncytiotrophoblast, cytotrophoblast and decidual cells across all trimesters of pregnancy with no significant difference in staining intensity in the different cellular compartments both across gestation and between compartments. Using immunofluorescent co-localization of CLIC4 and EVT marker HLA-G, we confirmed that CLIC4 localized to the cytoplasm of cell column EVTs in the first trimester decidua and nuclei of some EVTs that invaded in the decidua. Knockdown of CLIC4 in HTR8/SVneo cells significantly elevated cell adhesion, migration and invasion. Analysis of TGFß signaling downstream targets identified that CDH2 and BAMBI expression were significantly increased after CLIC4 knockdown in HTR8/SVneo cells. DISCUSSION: Our data support an inhibitory role for CLIC4 in regulating trophoblast migration and invasion, likely acting in part via BAMBI and CDH2.

Jagged1 regulates endometrial receptivity in both humans and mice.

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.

Prednisolone Alters Endometrial Decidual Cells and Affects Decidual-Trophoblast Interactions.

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.

MAML1: a coregulator that alters endometrial epithelial cell adhesive capacity.

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 control. MAML1 knockdown significantly affected the expression of classical receptivity markers (SPP1, DPP4) and this response was not directly via hormone receptors. The expression level of Hippo pathway target Ankyrin repeat domain-containing protein 1 (ANKRD1) was also affected after MAML1 knockdown in Ishikawa cells. CONCLUSION: Our data strongly suggest that MAML1 is involved in regulating the endometrial adhesive capacity and may facilitate embryo attachment, either directly or indirectly through the Notch signaling pathway.

Medawar's PostEra: Galectins Emerged as Key Players During Fetal-Maternal Glycoimmune Adaptation.

Lectin-glycan interactions, in particular those mediated by the galectin family, regulate many processes required for a successful pregnancy. Over the past decades, increasing evidence gathered from in vitro and in vivo experiments indicate that members of the galectin family specifically bind to both intracellular and membrane bound carbohydrate ligands regulating angiogenesis, immune-cell adaptations required to tolerate the fetal semi-allograft and mammalian embryogenesis. Therefore, galectins play important roles in fetal development and placentation contributing to maternal and fetal health. This review discusses the expression and role of galectins during the course of pregnancy, with an emphasis on maternal immune adaptions and galectin-glycan interactions uncovered in the recent years. In addition, we summarize the galectin fingerprints associated with pathological gestation with particular focus on preeclampsia.

Recurrent pregnancy loss.

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.

Galectin-7 Impairs Placentation and Causes Preeclampsia Features in Mice.

Preeclampsia is a serious pregnancy-induced disorder unique to humans. The etiology of preeclampsia is poorly understood; however, poor placental formation is thought causal. Galectin-7 is produced by trophoblast and is elevated in first-trimester serum of women who subsequently develop preeclampsia. We hypothesized that elevated placental galectin-7 may be causative of preeclampsia. Here, we demonstrated increased galectin-7 production in chorionic villous samples from women who subsequently develop preterm preeclampsia compared with uncomplicated pregnancies. In vitro, galectin-7 impaired human first-trimester trophoblast outgrowth, increased placental production of the antiangiogenic sFlt-1 splice variant, sFlt-1-e15a, and reduced placental production and secretion of ADAM12 (a disintegrin and metalloproteinase12) and angiotensinogen. In vivo, galectin-7 administration (E8-E12) to pregnant mice caused elevated systolic blood pressure, albuminuria, impaired placentation (reduced labyrinth vascular branching, impaired decidual spiral artery remodeling, and a proinflammatory placental state demonstrated by elevated IL1ß, IL6 and reduced IL10), and dysregulated expression of renin-angiotensin system components in the placenta, decidua, and kidney, including angiotensinogen, prorenin, and the angiotensin II type 1 receptor. Collectively, this study demonstrates that elevated galectin-7 during placental formation contributes to abnormal placentation and suggests that it leads to the development of preeclampsia via altering placental production of sFlt-1 and renin-angiotensin system components. Targeting galectin-7 may be a new treatment option for preeclampsia.

Profilin-1 is dysregulated in endometroid (type I) endometrial cancer promoting cell proliferation and inhibiting pro-inflammatory cytokine production.

Endometrial cancer (EC) is the most common gynaecological malignancy. Alarmingly its incidence and mortality rate is increasing particularly in younger women of reproductive age. Despite this, there are limited treatment options for EC. Profilin-1 (PFN1) regulates tumorigenesis in numerous cancers, but the role of PFN1 in EC has not been investigated. We hypothesized that PFN1 would have altered expression in EC and contribute to the development of EC. We quantified PFN1 in type 1 EC and benign/normal endometrium by RT-qPCR and IHC. The effect of silencing PFN1 on cell adhesion and proliferation was investigated using 2 EC cell lines (HEC1A and AN3CA). The effect of recombinant PFN1 (100 µM) on pro-inflammatory cytokine gene expression was investigated using THP1 monocyte cell line. PFN1 immunolocalized to glandular epithelial cells, vascular endothelial cells and leukocytes in the stromal compartment of normal endometrium and EC. PFN1 immunostaining intensity was significantly elevated in grade (G)I EC compared to normal endometrium, GI-II and GIII EC. In endometrial epithelial cancer cells alone, PFN1 immunostaining intensity was significantly reduced in GII and III EC compared to normal endometrium and GI EC. The stromal compartment of EC had strong PFN1 expression compared to benign and normal endometrium. Silencing PFN1 in the AN3CA endometrial epithelial cancer cell line significantly enhanced cell adhesion and proliferation. PFN1 treatment significantly down-regulated TNFα and IL1ß mRNA expression by THP1 cells. This study demonstrated that whilst PFN1 production is retained in the stromal compartment of EC, PFN1 production is lost in endometrial epithelial cancer cells with increasing cancer grade. PFN1 may play a role in the tumorigenesis of EC. Loss of PFN1 in GII and GIII endometrial epithelial cancer cells associated with sustained PFN1 by infiltrating immune cells may promote EC tumorigenesis due to increased endometrial epithelial cancer cell proliferation coupled with a pro-tolerance tumor microenvironment.

Restoration of microRNA-29c in type I endometrioid cancer reduced endometrial cancer cell growth.

Endometrial cancer is the most common gynaecological cancer worldwide, and the prognosis of patients with advanced disease remains poor. MicroRNAs (miRs) are dysregulated in endometrial cancer. miRs-29-a, -b and -c expression levels are downregulated in endometrial cancer; however, a specific role for miR-29c and its target genes remain to be elucidated. The aim of the present study was to determine the functional effect of restoring miR-29c expression in endometrial cancer cell lines and to identify miR-29c targets involved in cancer progression. miR-29c expression in human endometrial tumour grades 1-3 and benign tissue as well as in the endometrial cancer cell lines Ishikawa, HEC1A and AN3CA were analysed using reverse transcriptase-quantitative PCR (RT-qPCR). The cell lines were transfected with miR-29c mimic, miR-29c inhibitor or scrambled control. xCELLigence real-time cell monitoring analysed proliferation and migration, and flow cytometry was used to analyse apoptosis and cell cycle. The expression of miR-29c target genes in transfected cell lines was analysed using RT-qPCR. miR-29c was downregulated in grade 1-3 endometrial cancer samples compared with benign endometrium. miR-29c was reduced in Ishikawa and AN3CA cells, but not in HEC1A cell lines compared with non-cancerous primary human endometrial epithelial cells. Overexpression of miR-29c variably reduced proliferation, increased apoptosis and reduced the expression levels of miR-29c target genes, including cell division cycle 42, HMG-box transcription factor 1, integrin subunit ß 1, MCL1 apoptosis regulator BCL2 family member, MDM2 proto-oncogene, serum/glucocorticoid regulated kinase 1, sirtuin 1 and vascular endothelial growth factor A, across the three cell lines investigated. Inhibition of miR-29c in HEC1A cells increased proliferation and collagen type IV α 1 chain expression. The re-introduction of miR-29c to endometrial cancer cell lines reduced proliferation, increased apoptosis and reduced miR-29c target gene expression in vitro. The present results suggested that miR-29c may be a potential therapeutic target for endometrial cancer.

Galectin-7 is elevated in endometrioid (type I) endometrial cancer and promotes cell migration.

Endometrial cancer (EC) is the most commonly diagnosed gynecological malignancy in Australian women. Notably, its incidence and mortality rate is increasing. Despite this, there are limited treatment options for EC. Galectin-7 regulates tumorigenesis in numerous epithelial cancer types, but the role of galectin-7 has not been investigated in EC. It was hypothesized that galectin-7 expression would be altered in EC and contribute to the development of EC. Galectin-7 levels in EC and benign endometrium were quantified by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and ELISA. The effect of recombinant galectin-7 (1 µg/ml) on cell adhesion, proliferation, apoptosis (xCELLigence and flow cytometry), migration (wound healing assay) and gene expression (RT-qPCR) was investigated using three human EC cell lines (Ishikawa, HEC1A and AN3CA). Galectin-7 gene and protein expression was significantly elevated in Grade 3 EC, compared with benign tissues. Galectin-7 was almost undetectable in Ishikawa and AN3CA cells, but highly expressed by HEC1A cells. Recombinant galectin-7 had no significant effect on cell proliferation or apoptosis in any cell line, but significantly reduced cell adhesion in Ishikawa (at 4 and 6 h) and AN3CA (at 2, 3, 4 and 6 h). Galectin-7 significantly promoted Ishikawa migration and significantly elevated collagen type IV α 1 chain and intercellular adhesion molecule 1 (ICAM1) gene expression during wound healing. The present study demonstrated that galectin-7 production increased in EC with increasing cancer grade; therefore, galectin-7 may promote the metastasis of EC by reducing cell-cell adhesion and enhancing cell migration.

Interleukin 11 blockade during mid to late gestation does not affect maternal blood pressure, pregnancy viability or subsequent fertility in mice.

Interleukin (IL)11 is a crucial regulator during the initiation of pregnancy in humans and mice. Elevated levels are detected in serum, placenta and decidua of women with pre-eclampsia. Elevated IL11 during placentation recapitulates pre-eclampsia in mice, although withdrawal rescues pre-eclampsia features, suggesting that IL11 could provide a novel therapeutic target. The aim of this study was to determine the safety profile of an IL11 antagonist ligated to polyethylene glycol (PEGIL11A) during pregnancy in mice. Blocking IL11 signalling during mid to late gestation pregnancy in mice did not affect pregnancy viability, or alter placental or fetal weight, or morphology. Importantly, decidual area remained unchanged. PEGIL11A did not affect maternal blood pressure, urinary protein or term pup weight. PEGIL11A administration to non-pregnant mice did not affect subsequent fertility; there was no difference in number of implantation sites, or placental or fetal weight between PEGIL11A and PEG-treated mice. These data show that blocking IL11Rα during placentation does not alter the placenta, decidua, fetus, maternal blood pressure or kidneys. These findings highlight the potential of IL11 signalling inhibition as a safe therapy to alleviate pre-eclampsia symptoms and demonstrate the potential for IL11 inhibition as a novel fertility-preserving therapy for women with cancer.

Mouse double minute homologue 2 (MDM2) downregulation by miR-661 impairs human endometrial epithelial cell adhesive capacity.

Human blastocysts that fail to implant following IVF secrete elevated levels of miR-661, which is taken up by primary human endometrial epithelial cells (HEECs) and impairs their adhesive capability. MicroRNA miR-661 downregulates mouse double minute homologue 2 (MDM2) and MDM4 in other epithelial cell types to activate p53; however, this has not been examined in the endometrium. In this study MDM2 protein was detected in the luminal epithelium of the endometrium, the site of blastocyst attachment, during the mid secretory receptive phase of the menstrual cycle. The effects of miR-661 on gene expression in and adhesion of endometrial cells was also examined. MiR-661 overexpression consistently downregulated MDM2 but not MDM4 or p53 gene expression in the Ishikawa endometrial epithelial cell line and primary HEEC. Adhesion assays were performed on the real-time monitoring xCELLigence system and by co-culture using Ishikawa cells and HEECs with HTR8/SVneo trophoblast spheroids. Targeted siRNA-mediated knockdown of MDM2 in endometrial epithelial cells reduced Ishikawa cell adhesion (P<0.001) and also reduced HTR8/SVneo trophoblast spheroid adhesion to Ishikawa cells (P<0.05) and HEECs (P<0.05). MDM2 overexpression using recombinant protein treatment resulted in enhanced HTR8/SVneo trophoblast spheroid adhesion to Ishikawa cells (P<0.01) and HEECs (P<0.05). This study highlights a potential new mechanism by which human blastocyst-secreted miR-661 reduces endometrial epithelial cell adhesion; via downregulation of MDM2. These findings suggest that MDM2 contributes to endometrial-blastocyst adhesion, implantation and infertility in women.