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A successful pregnancy requires a synchronous dialogue between endometrium and embryo within the endometrial milieu. The aim of this study was to assess the role in the implantation of mediators in the endometrial milieu. Total secretome (TS), soluble secretome (SS) and small extracellular vesicles (containing exosomes) were generated from hormonally primed human endometrial epithelial cell culture medium. Human trophectoderm stem cell-derived spheroids were cultured with TS, SS or exosomes (30 µg/ml) on hormonally primed epithelial cells, with exosomes significantly increasing cell adhesion and outgrowth. Furthermore, F1 mouse 2-cell embryos were cultured in groups for 48 h followed by culture with each secretome fraction (30 µg/ml) for 48 h. Blastocyst cell number and hatching were quantified. In addition, blastocysts were further cultured on a fibronectin matrix for 72 h or transferred to recipient mice (with corresponding secretomes) with embryo implantation assessed after 6 days. Exosomes significantly increased total cell number in mouse embryos and complete hatching from zona pellucida, with both exosomes and SS significantly enhancing mouse embryo outgrowth. Importantly, exosomes increased the embryo implantation rate in comparison to other secretome fractions (normalized based on treatment amount) from the endometrial epithelia. These data indicate that endometrial epithelial exosomes support embryo growth, development and implantation while the SS has selective involvement specifically on mouse embryo outgrowth. This finding provides new insights into the molecular differences of endometrial secretome components in implantation and early embryo development and may implicate endometrial exosomes in the pathophysiology of implantation failure in infertility.

The endometrium lines a women's uterus becoming receptive, and allowing embryo implantation to occur, for just a few days during the post-ovulatory mid-secretory phase of each menstrual cycle. We investigated whether concentrations of proposed receptivity biomarkers (VEGF, IL8, FGF2, CSF3 sFlt-1, sGP130 and PlGF) secreted by the endometrium into the uterine cavity and forming the microenvironment for embryo implantation is altered among a population of age-matched women with unexplained (idiopathic) infertility compared to fertile women during the receptive mid-secretory phase (n = 16 fertile, 18 infertile) and the prior pre-receptive early secretory phase (n = 19 fertile, 18 infertile) of their cycle. In the mid-secretory cohort significantly elevated concentrations of five biomarkers; PlGF (p = 0.001), IL8 (p = 0.004), sGP130 (p = 0.009), sFlt-1 (p = 0.021), and CSF3 (p = 0.029) was present in uterine fluid of infertile women during the mid-secretory phase, but only CSF3 was significantly elevated in the pre-receptive early secretory phase (p = 0.006). In vitro studies of glycosylated and non-glycosylated forms of CSF3 at representative fertile (20 ng/mL) and infertile (70 ng/mL) effects on endometrium and embryo behaviour were performed. Non-glycosylated CSF3 at fertile concentrations significantly (p 0.001) elevated endometrial epithelial cell proliferation however chronic treatment or elevated (infertile) concentrations of CSF3 in glycosylated form abrogated the positive effects. Both forms of CSF3 increased trophoblast cell invasion (p 0.001) regardless of concentration. Mouse embryo outgrowth was significantly (p 0.01) increased at fertile but not at infertile concentrations. The study confirmed potential utility of five biomarkers of endometrial receptivity for future application in the mid-secretory phase while highlighting CSF3 is elevated in the earlier pre-receptive phase. Our data provides evidence that CSF3 acts on both human endometrium and embryo in a manner that is concentration and glycosylation dependent.

Idiopathic pulmonary fibrosis (IPF) is characterized by chronic inflammation, severe scarring, and stem cell senescence. Stem cell‐based therapies modulate inflammatory and fibrogenic pathways by release of soluble factors. Stem cell‐derived extracellular vesicles should be explored as a potential therapy for IPF. Human amnion epithelial cell‐derived exosomes (hAEC Exo) were isolated and compared against human lung fibroblasts exosomes. hAEC Exo were assessed as a potential therapy for lung fibrosis. Exosomes were isolated and evaluated for their protein and miRNA cargo. Direct effects of hAEC Exo on immune cell function, including macrophage polarization, phagocytosis, neutrophil myeloperoxidase activity and T cell proliferation and uptake, were measured. Their impact on immune response, histological outcomes, and bronchioalveolar stem cell (BASC) response was assessed in vivo following bleomycin challenge in young and aged mice. hAEC Exo carry protein cargo enriched for MAPK signaling pathways, apoptotic and developmental biology pathways and miRNA enriched for PI3K‐Akt, Ras, Hippo, TGFβ, and focal adhesion pathways. hAEC Exo polarized and increased macrophage phagocytosis, reduced neutrophil myeloperoxidases, and suppressed T cell proliferation directly. Intranasal instillation of 10 μg hAEC Exo 1 day following bleomycin challenge reduced lung inflammation, while treatment at day 7 improved tissue‐to‐airspace ratio and reduced fibrosis. Administration of hAEC Exo coincided with the proliferation of BASC. These effects were reproducible in bleomycin‐challenged aged mice. The paracrine effects of hAECs can be largely attributed to their exosomes and exploitation of hAEC Exo as a therapy for IPF should be explored further. Stem Cells Translational Medicine 2018;7:180–196

In vitro fertilization has overcome infertility issues for many couples. However, achieving implantation of a viable embryo into the maternal endometrium remains a limiting step in optimizing pregnancy success. The molecular mechanisms which characterize the transient state of endometrial receptivity, critical in enabling embryo-endometrial interactions, and proteins which underpin adhesion at the implantation interface, are limited in humans despite these temporally regulated processes fundamental to life. Hence, failure of implantation remains the "final frontier" in infertility. A human coculture model is utilized utilizing spheroids of a trophectoderm (trophoblast stem) cell line, derived from pre-implantation human embryos, and primary human endometrial epithelial cells, to functionally identify "fertile" versus "infertile" endometrial epithelium based on adhesion between these cell types. Quantitative proteomics identified proteins associated with human endometrial epithelial receptivity ("epithelial receptome") and trophectoderm adhesion ("adhesome"). As validation, key "epithelial receptome" proteins (MAGT-1/CDA/LGMN/KYNU/PC4) localized to the epithelium of receptive phase (mid-secretory) endometrium obtained from fertile, normally cycling women but is largely absent from non-receptive (proliferative) phase tissues. Factors involved in embryo-epithelium interaction in successive temporal stages of endometrial receptivity and implantation are demonstrated and potential targets for improving fertility are provided, enhancing potential to become pregnant either naturally or in a clinical setting.

STUDY QUESTION: Does vascular endothelial growth factor (VEGF) have important roles during early embryo development and implantation? SUMMARY ANSWER: VEGF plays key roles during mouse preimplantation embryo development, with beneficial effects on time to cavitation, blastocyst cell number and outgrowth, as well as implantation rate and fetal limb development. WHAT IS KNOWN ALREADY: Embryo implantation requires synchronized dialog between maternal cells and those of the conceptus. Following ovulation, secretions from endometrial glands increase and accumulate in the uterine lumen. These secretions contain important mediators that support the conceptus during the peri-implantation phase. Previously, we demonstrated a significant reduction of VEGFA in the uterine cavity of women with unexplained infertility. Functional studies demonstrated that VEGF significantly enhanced endometrial epithelial cell adhesive properties and embryo outgrowth. STUDY DESIGN, SIZE, DURATION: Human endometrial lavages (n = 6) were obtained from women of proven fertility. Four-week old Swiss mice were superovulated and mated with Swiss males to obtain embryos for treatment with VEGF in vitro. Preimplantation embryo development was assessed prior to embryo transfer (n = 19-30/treatment group/output). Recipient F1 female mice (8-12 weeks of age) were mated with vasectomized males to induce pseudopregnancy and embryos were transferred. On Day 14.5 of pregnancy, uterine horns were collected for analysis of implantation rates as well as placental and fetal development (n = 14-19/treatment). PARTICIPANTS/MATERIALS, SETTING, METHODS: Lavage fluid was assessed by western immunoblot analysis to determine the VEGF isoforms present. Mouse embryos were treated with either recombinant human (rh)VEGF, or VEGF isoforms 121 and 165. Preimplantation embryo development was quantified using time-lapse microscopy. Blastocysts were (i) stained for cell number, (ii) transferred to wells coated with fibronectin to examine trophoblast outgrowth or (iii) transferred to pseudo pregnant recipients to analyze implantation rates, placental and fetal development. MAIN RESULTS AND THE ROLE OF CHANCE: Western blot analysis revealed the presence of VEGF121 and 165 isoforms in human uterine fluid. Time-lapse microscopy analysis revealed that VEGF (n = 22) and VEGF121 (n = 23) treatment significantly reduced the preimplantation mouse embryo time to cavitation (P < 0.05). VEGF and VEGF165 increased both blastocyst cell number (VEGF n = 27; VEGF165 n = 24: P < 0.001) and outgrowth (n = 15/treatment: 66 h, P < 0.001; 74, 90, 98 and 114 h, P < 0.01) on fibronectin compared with control. Furthermore, rhVEGF improved implantation rates and enhanced fetal limb development (P < 0.05). LIMITATIONS, REASONS FOR CAUTION: Due to the nature of this work, embryo development and implantation was only examined in the mouse. WIDER IMPLICATIONS OF THE FINDINGS: The absence or reduction in levels of VEGF during the preimplantation period likely affects key events during embryo development, implantation and placentation. The potential for improvement of clinical IVF outcomes by the addition of VEGF to human embryo culture media needs further investigation. STUDY FUNDING/COMPETING INTERESTS: This study was supported by a University of Melbourne Early Career Researcher Grant #601040, the NHMRC (L.A.S., Program grant #494802; Fellowship #1002028; N.J.H., Fellowship # 628927; J.E.; project grant #1047756) and L.A.S., Monash IVF Research and Education Foundation. N.K.B. was supported by an Australian Postgraduate Award. Work at PHI-MIMR Institute was also supported by the Victorian Government's Operational Infrastructure Support Program. There are no conflicts of interest to declare.

Background Improvements in vitrification now make frozen embryo transfers (FETs) a viable alternative to fresh embryo transfer, with reports from observational studies and randomized controlled trials suggesting that: (i) the endometrium in stimulated cycles is not optimally prepared for implantation; (ii) pregnancy rates are increased following FET and (iii) perinatal outcomes are less affected after FET. Methods This review integrates and discusses the available clinical and scientific evidence supporting embryo transfer in a natural cycle. Results Laboratory-based studies demonstrate morphological and molecular changes to the endometrium and reduced responsiveness of the endometrium to hCG, resulting from controlled ovarian stimulation. The literature demonstrates reduced endometrial receptivity in controlled ovarian stimulation cycles and supports the clinical observations that FET reduces the risk of ovarian hyperstimulation syndrome and improves outcomes for both the mother and baby. Conclusions This review provides the basis for an evidence-based approach towards changes in routine IVF, which may ultimately result in higher delivery rates of healthier term babies.

Embryo implantation into maternal endometrium is critical for initiation and establishment of pregnancy, requiring developmental synchrony between endometrium and blastocyst. However, factors regulating human endometrial-embryo cross talk and facilitate implantation remain largely unknown. Extracellular vesicles (EVs) are emerging as important mediators of this process. Here, a trophectoderm spheroid-based in vitro model mimicking the pre-implantation human embryo is used to recapitulate important functional aspects of blastocyst implantation. Functionally, human endometrial EVs, derived from hormonally treated cells synchronous with implantation, are readily internalized by trophectoderm cells, regulating adhesive and invasive capacity of human trophectoderm spheroids. To gain molecular insights into mechanisms underpinning endometrial EV-mediated enhancement of implantation, quantitative proteomics reveal critical alterations in trophectoderm cellular adhesion networks (cell adhesion molecule binding, cell-cell adhesion mediator activity, and cell adherens junctions) and metabolic and gene expression networks, and the soluble secretome from human trophectodermal spheroids. Importantly, transfer of endometrial EV cargo proteins to trophectoderm to mediate changes in trophectoderm function is demonstrated. This is highlighted by correlation among endometrial EVs, the trophectodermal proteome following EV uptake, and EV-mediated trophectodermal cellular proteome, important for implantation. This work provides an understanding into molecular mechanisms of endometrial EV-mediated regulation of human trophectoderm functions-fundamental in understanding human endometrium-embryo signaling during implantation.

STUDY QUESTION Does prolonged exposure of the endometrium to hCG, as experienced after ovulation induction in an assisted reproduction technology (ART) cycle, affect functional measures of endometrial receptivity? SUMMARY ANSWER Prolonged endometrial hCG exposure detrimentally affects the manner in which the endometrium can respond to hCG secreted by the blastocyst. WHAT IS KNOWN ALREADY Prolonged hCG exposure down-regulates endometrial LH-CG receptor (LHCGR) expression in a baboon model. HCG exposure during the proliferative phase of oocyte-donation cycles and frozen embryo transfer cycles is associated with a lower pregnancy rate. STUDY DESIGN, SIZE, DURATION LHCGR was examined in endometria of women undergoing ART cycles (GnRH agonist/antagonist) and across the menstrual cycle in normally cycling fertile women. To determine whether prolonged hCG exposure affects the subsequent endometrial response to hCG, endometrial epithelial cells (HES cell line and primary cultures of human endometrial epithelial cells) were exposed to a low dose of hCG (0.5-5 IU) for up to 5 days, to mimic the chronic exposure during an ART cycle, and subsequently exposed to an acute 'blastocyst mimic' dose of hCG (20 IU). PARTICIPANTS/MATERIALS, SETTING, METHODS Endometrial tissues were collected at hCG + 2 (n = 37) from women undergoing ART between August 2006 and August 2008, and across the cycle from women with known fertility (n = 40). LHCGR localization and staining intensity were determined by immunohistochemistry and semi-quantitative scoring. HES cells were treated with hCG as above and analyzed for LHCGR localization (immunocytochemistry), phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 (western immunoblotting), adhesion to trophoblast-like matrices (adhesion assays) and tight junction integrity (trans-epithelial resistance assessment). MAIN RESULTS AND THE ROLE OF CHANCE Endometrial epithelial LHCGR staining was significantly lower in women stimulated with a GnRH agonist protocol who did not become pregnant in that cycle versus the natural menstrual cycle (P < 0.05). Chronic low-dose hCG exposure in vitro mediated a down-regulation and internalization of the LHCGR in endometrial epithelial cells. Prolonged exposure to chronic low-dose hCG (3-5 days) abrogated ERK 1/2 phosphorylation, adhesion to extracellular matrices and changes in tight junction integrity in response to a subsequent acute high dose (20 IU) of hCG. LIMITATIONS, REASONS FOR CAUTION Studies using cell lines and primary cultures of cells in vitro are not fully representative of the complex endometrial milieu in vivo. WIDER IMPLICATIONS OF THE FINDINGS These data reinforce the clinical observations that precocious or prolonged hCG exposure may detrimentally affect endometrial receptivity and provide a mechanistic basis for these clinical findings. The data appear to support the notion that in women for whom ART has not succeeded, a different, minimally stimulated approach without exposure to exogenous hCG may improve outcomes.

Menstruation has many of the features of an inflammatory process. The complexity and sequence of inflammatory-type events leading to the final tissue breakdown and bleeding are slowly being unravelled. Progesterone has anti-inflammatory properties, and its rapidly declining levels (along with those of estrogen) in the late secretory phase of each non-conception cycle, initiates a sequence of interdependent events of an inflammatory nature involving local inter-cellular interactions within the endometrium. Intracellular responses to loss of progesterone (in decidualized stromal, vascular and epithelial cells) lead to decreased prostaglandin metabolism and loss of protection from reactive oxygen species (ROS). Increased ROS results in release of NFκB from suppression with activation of target gene transcription and increased synthesis of pro-inflammatory prostaglandins, cytokines, chemokines and matrix metalloproteinases (MMP). The resultant leukocyte recruitment, with changing phenotypes and activation, provide further degradative enzymes and MMP activators, which together with a hypoxic environment induced by prostaglandin actions, lead to the tissue breakdown and bleeding characteristic of menstruation. In parallel, at sites where shedding is complete, microenvironmentally-induced changes in phenotypes of neutrophils and macrophages from pro- to anti-inflammatory, in addition to induction of growth factors, contribute to the very rapid re-epithelialization and restoration of tissue integrity.

Endometriosis is a chronic disorder affecting approximately 10% of women in whom endometrial tissue forms painful lesions outside the uterus. It has a major impact on their physical, mental and social well-being but has no known cure, and there is no nonsurgical means of diagnosis. We have used a proteomic approach to identify proteins with altered abundance in the eutopic endometrium of endometriosis patients in the midsecretory phase of the menstrual cycle. 2D-differential in gel electrophoresis (DIGE) and mass spectrometry identified 20 proteins that were present at different levels in endometriosis patients (p0.05), many of which have not previously been associated with endometriosis. Protein abundance changes did not correlate well with published gene array data, emphasizing the extensive post-translational modification that occurs in this tissue. Abundance or localization changes in endometrial tissue were validated by immunohistochemistry and Western blotting for three proteins, vimentin (VIM), peroxiredoxin 6 (PRDX6), and ribonuclease/angiogenin inhibitor 1 (RNH1), while observed changes could not be confirmed for coronin 1A (CORO1A) or transgelin (TAGLN2). In addition, multiple charge and size isoforms were observed for PDRX6 and vimentin (VIM), and an additional PDRX6 isoform was observed in endometriosis patients that was below the level of detection in healthy women. Biological pathway analysis identified that cytoskeletal remodeling via keratin intermediate filaments, processing of the cystic fibrosis transmembrane receptor (CFTR), the glucocorticoid receptor subunit alpha (GCR), and heat shock factor 1 (HSF1) were all significantly over-represented features in endometriosis patients. This study highlights the highly dynamic nature of endometrial tissue and suggests that considerable post-translational modification of proteins is a key factor in the pathology of endometriosis.

Proprotein convertase 5/6 (PC6), a member of the proprotein convertase (PC) family, is a critical regulator in the uterus for embryo implantation. In particular, PC6 is essential for the differentiation of uterine stromal fibroblasts into decidual cells (decidualization). Knockdown of PC6 in the mouse uterus leads to complete failure of decidualization and implantation. It has been envisaged that PC6 functions by proteolytically activating a number of important growth factors and other precursor proteins. However, to date, the precise mechanism of PC6 action in the uterus, particularly during decidualization, is unknown. In this study, we aimed to investigate the mechanisms of PC6 action in decidualization by identifying its physiological substrates using a proteomic approach. Primary human endometrial stromal cells were decidualized and treated with or without recombinant human PC6 (rhPC6). The proteins cleaved by rhPC6 were identified by 2-dimensional fluorescent differential gel electrophoresis. The candidate proteins were validated as PC6 substrates by a number of approaches, including determining their coexpression with PC6 in vivo in decidual cells in the human uterus. A total of 18 protein spots were significantly altered by rhPC6 treatment, 8 of which were assigned clear identities by mass spectrometry. One of these was confirmed to be caldesmon, a key protein involved in organizing the actin microfilaments and regulating cytoskeletal structure. This study demonstrates a novel approach to identify PC-regulated proteins of physiological relevance, and provides important insight into the mechanism by which PC6 regulates decidualization.

Endometriosis is an estrogen-dependent disorder where endometrial tissue forms lesions outside the uterus. Endometriosis affects an estimated 10% of women in the reproductive-age group, rising to 30% to 50% in patients with infertility and/or pain, with significant impact on their physical, mental, and social well-being. There is no known cure, and most current medical treatments are not suitable long term due to their side-effect profiles. Endometriosis has an estimated annual cost in the United States of $18.8 to $22 billion (2002 figures). Although endometriosis was first described more than 100 years ago, current knowledge of its pathogenesis, spontaneous evolution, and the pathophysiology of the related infertility and pelvic pain, remain unclear. A consensus workshop was convened following the 10th World Congress on Endometriosis to establish recommendations for priorities in endometriosis research. One major issue identified as impacting on the capacity to undertake endometriosis research is the need for multidisciplinary expertise. A total of 25 recommendations for research have been developed, grouped under 5 subheadings: (1) diagnosis, (2) classification and prognosis, (3) treatment and outcome, (4) epidemiology, and (5) pathophysiology. Endometriosis research is underfunded relative to other diseases with high health care burdens. This may be due to the practical difficulties of developing competitive research proposals on a complex and poorly understood disease, which affects only women. By producing this consensus international research priorities statement it is the hope of the workshop participants that researchers will be encouraged to develop new interdisciplinary research proposals that will attract increased funding support for work on endometriosis.

PURPOSE OF REVIEW: Chemokines are well known for their roles in the immune system; convincing evidence has emerged demonstrating a broader role for chemokines in the endometrium, particularly during embryo implantation. This review highlights the evidence on newly defined roles for chemokines in the endometrium during embryo implantation, with particular focus on those chemokines expressed by the endometrium. RECENT FINDINGS: The highly regulated temporal and spatial expression of chemokines in the endometrium leads not only to specific recruitment and activation of appropriate leucocytes but also coordinates the precisely orchestrated invasion of trophoblasts through the decidua and maternal vasculature. Results to date implicate chemokine signalling at the maternal-foetal interface in important processes during implantation and placentation, such as leucocyte recruitment and controlled trophoblast invasion. Unravelling such actions of chemokines in the endometrium has provided new insights into these complex processes. SUMMARY: Disturbances of chemokine production, processing, or actions are likely to contribute to dysfunction of implantation and placentation, with implications for early pregnancy loss and disturbed placental and foetal development. More research into altered chemokine function in such conditions may provide leads for new clinical interventions.

Ghrelin acting via the growth hormone secretagogue receptor (GHS-R) stimulates GH secretion from pituitary glands. Both ligand and receptor are present in the pituitary, hypothalamus and many peripheral tissues including the uterus. This study demonstrates the cyclical expression of GHS-R and ghrelin in human endometrium. mRNA and protein for ghrelin and GHS-R were examined using RT-PCR and immunohistochemistry. Both ghrelin and GHS-R mRNA levels were highest in the secretory phase, with lower levels in the mid-proliferative phase and even lower expression in the menstrual phase. Immunoreactive ghrelin and GHS-R were confined predominantly to glandular epithelial and stromal cells with the greatest intensity of staining in secretory phase samples, consistent with the RT-PCR data. Additionally, we examined ghrelins effect on the decidualization of human endometrial stromal cells (HESCs) combined with sex steroid and cAMP treatments using prolactin (PRL) and insulin-like growth factor binding protein-1 (IGFBP-1) production as markers of decidualization. Ghrelin administered in combination with sex steroids to HESC, resulted in an increase in PRL and IGFBP-1 production above that obtained with cAMP, or sex steroids alone (P

Blastocyst implantation is dependent on the differentiation of endometrial stromal cells (ESC) into decidual cells. Decidualization of human ESC in vitro is enhanced by interleukin 11 (IL11), with associated changes in gene expression. Genes downstream of IL11 may provide targets for the treatment of implantation failure or the development of non-hormonal contraceptives. This study aimed to examine the effect of IL11 on interleukin 1 beta (IL1B) mRNA and protein expression during in vitro decidualization of ESC. Cells were decidualized with 17beta-estradiol and medroxyprogesterone acetate in the presence or absence of exogenous IL11, and IL1B mRNA was quantified by real-time RT-PCR. Inactive proIL1B and bioactive IL1B in cell lysates and conditioned media were measured using specific immunoassays. Secretion of bioactive IL1B from decidualizing ESC was investigated by in vitro stimulation of decidualizing cells with lipopolysaccharide, interferon gamma or human chorionic gonadotropin. Immunohistochemistry was carried out on cycling and pregnant decidua using an antibody specific for bioactive IL1B. Exogenous IL11 increased by 28-fold the abundance of IL1B mRNA in decidualizing ESC, and total immunoreactive IL1B was also increased. However, this was not reflected in bioactive IL1B secretion from these cells, and none of the tested stimuli were able to induce its release. Bioactive IL1B was detected in vivo at very low levels and at discrete foci in late secretory phase and first trimester decidua. This regulation of latent and bioactive IL1B at the fetal-maternal interface may prime decidual cells to respond rapidly to immunological challenge or to signals from the blastocyst during implantation.

Mammalian embryos cannot survive without the placenta. Development of the human placenta requires trophoblast proliferation, differentiation, and invasion as well as highly coordinated modulation of the maternal uterus. HtrA1 is a member of the recently identified mammalian HtrA (high temperature requirement factor A) serine protease family with a high level of expression in the placenta. In this study, we examined whether HtrA1 expression (mRNA and protein) is associated with placental development in the human. HtrA1 is up-regulated in both endometrial glands and decidual cells during endometrial preparation for embryo implantation and during first-trimester pregnancy at placentation. HtrA1 expression was also detected in certain trophoblast subtypes during early pregnancy. The villous syncytiotrophoblast and cytotrophoblast showed the strongest expression while the interstitial extravillous trophoblast showed the lowest or no expression of HtrA1. The distinct distribution of HtrA1 at the maternal-trophoblast interface suggests that HtrA1 may play a role in placental development.

Hemochorial placentation involves highly regulated interactions between fetal- and maternal-derived cells. HtrA3, a novel serine protease containing an insulin-like growth factor (IGF) binding domain, was previously shown to increase during early pregnancy in the mouse uterus, being dramatically upregulated post-implantation. The present study examined the regulation of HtrA3 gene in the mouse uterus from post-implantation to late gestation. Both mRNA and protein of HtrA3 were localized specifically in the maternal decidua. In contrast, HtrA3 expression was below detection in trophoblasts, including the giant cells that are in direct contact with the decidua. This pattern persisted from the early stages of placentation to near term. The level of decidual HtrA3 mRNA and its protein gradually decreased as the placenta matured. In the decidua, only the maternal decidual cells, but not blood vessels or uterine NK cells that are present in large numbers, were positive for HtrA3. The specific localization of a protease possessing an IGF-binding domain at the maternal-fetal interface suggests that HtrA3 plays a critical role in mediating maternal decidual remodelling and maintenance, likely in association with the IGF system, in placental development and function.

Interleukin (IL)-11 is essential for embryo implantation in the mouse and evidence suggests it has a role in implantation in humans. This study has evaluated immunoreactive IL-11, IL-11 receptor (R) alpha and leukemia inhibitory factor (LIF) in endometrium of infertile women with endometriosis (I/E) and normal fertile women (controls) during the implantation window. Endometrial biopsies from I/E (N = 7) were timed from the LH surge and were post-ovulatory days (POD) 5-10. Control biopsies (N = 8) from women were between days 19 and 24 of the menstrual cycle. Staining intensity of IL-11, IL-11Ralpha and LIF evaluated using semi-quantitative immunohistochemistry scores. Immunoreactive IL-11, IL-11Ralpha and LIF were present predominantly in glandular epithelium, while luminal epithelium showed patchy staining. All controls stained positively for IL-11, IL-11Ralpha and LIF in glandular epithelium. IL-11 and IL-11Ralpha staining was absent from glandular epithelium in cohorts of I/E. LIF staining intensity in glandular epithelium was significantly lower in I/E compared to controls. The results suggest that reduced endometrial IL-11 and/or LIF may contribute to infertility in some endometriotic women.

CONTEXT: Irregular bleeding affects many users of combined menopausal hormone therapy (HT) and commonly leads to invasive and expensive investigations to exclude underlying malignancy. In most cases no abnormality is found. OBJECTIVE: The main objective of this study was to explore the role of uterine natural killer (uNK) cells and their regulatory cytokine IL-15 in irregular bleeding in HT users. DESIGN: This was a prospective observational study conducted between 2002 and 2004. SETTING: The study was conducted in a tertiary referral menopause clinic at King Edward Memorial Hospital, Western Australia. PATIENTS: Patients included 117 postmenopausal women taking combined HT. INTERVENTIONS: Outpatient endometrial biopsies were taken during and outside bleeding episodes. MAIN OUTCOME MEASURES: The relationship between endometrial uNK cells (CD56+) and bleeding patterns was measured. We also addressed the impact of HT exposure on uNK cell populations, the relationship between endometrial IL-15 expression and uNK cell populations, and killer Ig like receptor genotype in subjects with irregular bleeding. RESULTS: Endometrial CD56+ uNK cells were significantly increased in biopsies obtained during bleeding episodes (P < 0.001), compared with HT users with no bleeding. The highest level of IL-15 expression was also seen in biopsies taken during bleeding. No clear relationship between killer Ig like receptor genotype and bleeding on HT was observed. CONCLUSIONS: Little is known about the mechanisms underlying irregular bleeding in HT users. This is the first report of uNK cells and their association with regulating cytokines in postmenopausal endometrium and demonstrates a possible mechanism by which HT may induce irregular bleeding.

The complexity of the events of embryo implantation and placentation is exemplified by the number and range of cytokines with demonstrated roles in these processes. Disturbance of the normal expression or action of these cytokines results in complete or partial failure of implantation and abnormal placental formation in mice or humans. Of known importance are members of the gp130 family such as interleukin-11 (IL-11) and leukaemia inhibitory factor (LIF), the transforming growth factor beta (TGFbeta) superfamily including the activins, the colony-stimulating factors (CSF), the IL-1 system and IL-15 system. New data are also emerging for roles for a number of chemokines (chemoattractive cytokines) both in recruiting specific cohorts of leukocytes to implantation sites and in trophoblast differentiation and trafficking. This review focuses on those cytokines and chemokines whose expression pattern in the human endometrium is consistent with a potential role in implantation and placentation and for which some relevant actions are known. It examines what is known of their regulation and action along with alterations in clinically relevant situations.

Development of a hemochorial placenta involves trophoblast proliferation, differentiation, and invasion into the uterus to promote blood flow to the embryo. Trophoblast invasion is tightly controlled by expression of specific proteases in the trophoblast and highly coordinated activities in the uterus. One uterine event essential for placentation is the developmentally regulated formation and regression of the decidua. In mice, decidual regression takes place in a temporal- and spatial-specific manner that is coordinated with placental development. In this study, we identified that the serine protease HtrA1 (high temperature requirement factor A1) was specifically expressed in differentiated trophoblast cells, especially the giant cells, during the early stages of placental development. A high level of HtrA1 expression was also detected in decidua capsularis specifically at the decidual-trophoblast interface where active involution occurs. Thus, we have identified a previously unknown role for HtrA1 as a protease potentially important for trophoblast differentiation/invasion and uterine decidual regression during placental development.

This paper characterizes a novel gene, previously identified as uniquely regulated at implantation in mouse uterus. We cloned its full mRNA sequence encoding a serine protease possessing an IGF-binding domain and named it pregnancy-related serine protease (PRSP). PRSP is structurally similar to mammalian HtrA1 (56% amino acid similarity). Northern analysis revealed that the expression of PRSP mRNA was low before pregnancy, but it was increased at implantation and markedly up-regulated post-implantation. In-situ hybridization localized low levels of mRNA expression to the epithelium and stroma during very early pregnancy, but high expression to the decidual cells on day 8.5, primarily at the mesometrial pole where the placenta was forming. By day 10.5, PRSP mRNA was detected in the placenta. We also cloned an alternatively spliced PRSP mRNA that is expressed at a very low level. We located PRSP gene on chromosome 5 and established its intron/exon structure, which unambiguously explains how the two mRNA variants are produced through alternative splicing. Based on PRSP protein domain structure and its unique expression during pregnancy, we propose that PRSP plays an important role in the formation/function of the placenta.

Objectives Endometrial carcinoma (EC) is one of the few cancers where there is a clear relationship between excessive hormone stimulation and malignant transformation. In this study we have analyzed the effects of the female sex steroids estrogen and progesterone on matrix metalloproteinases (MMP-9 and -2) production in primary EC cells and EC cell lines. MMPs are implicated in cancer invasion via mechanisms including extracellular matrix degradation and the processing of a range of molecules, including growth factors and cytokines. Methods Cells were isolated from biopsies collected from three cancer patients undergoing hysterectomy for grade 1 endometrial adenocarcinoma and two patients undergoing procedures unrelated to EC. These cells plus the EC cell lines Ishikawa and HEC-1A were cultured without hormones or with medroxyprogesterone acetate (MPA), estradiol (E 2 ), or these hormones in combination. Gelatin and reverse zymography were used to analyze MMPs and TIMPs, respectively, in culture medium. RT–PCR was used to characterize steroid receptor expression. Results Cell lines differed from primary cells in the range and abundance of MMPs secreted. Treatment with MPA significantly reduced proMMP-9, proMMP-2, and MMP-2 release from primary EC cancer and stromal cells. Treatment with E 2 alone or MPA + E 2 had no significant effect on MMP expression. Primary EC and stromal cells also showed a loss of the progesterone B receptor isoform. Conclusion EC cells retain the suppression of MMPs by progesterone, seen in normal endometrial cells. These data provide a rationale for the use of progestin therapy in the treatment of early stage grade 1 endometrial carcinomas.

Members of the interleukin-6 family of cytokines include leukaemia inhibitory factor (LIF), interleukin-6, interleukin-11, cardiotrophin, ciliary neurotropic growth factor, oncostatin M and the recently discovered cardiotropin-like cytokine (NNT-1). These ligands signal via heterodimeric receptors composed of ligand-specific alpha chains and the common signal-transducing subunit gp130. Gene targeting in mice provided the first indication of a role for interleukin 6 family cytokines in implantation with the generation of mice with a null mutation of the gene encoding LIF. LIF null female mice were infertile because of failure of blastocyst implantation. More recently, interleukin-11 signalling has been shown to be required for the uterine decidualization response. This review describes the insights into the role of interleukin-6 family cytokines in female fertility that have come from gene targeting experiments in mice.

The ovine endometrium is subjected to cyclic oscillations of estrogen and progesterone in preparation for implantation. One response to fluctuating hormonal levels is the degree of hydration of the tissue, suggesting cyclical alterations in glycosaminoglycan/proteoglycan content. The aim of the present study was to quantitate and characterize glycosaminoglycans in the ovine endometrium during estrogen and progesterone dominant stages. Endogenous endometrial glycosaminoglycan content was determined by chemical analysis and characterized by enzyme specific or chemical degradation. [(35)S]-sulphate and [(3)H]-glucosamine labeled proteoglycans/glycosaminoglycans were extracted by cell lysis or with 4M guanidine-HCl. Extracts were purified by anion exchange and gel chromatography and characterized as above. Estrogen and progesterone dominant endometrium contained 3.2 +/- 0.1 and 2.1 +/- 0.1 mg endogenous glycosaminoglycan/g dehydrated tissue, respectively. Characterization of endogenous glycosaminoglycan showed chondroitin sulphate and hyaluronan contributing over 80%. The major difference between hormonal dominant tissue was a higher estrogenic hyaluronan percentage and a higher progestational keratan sulphate percentage (p0.001). Estrogen dominant tissue incorporated 1.6-1.9 fold more radiolabeled proteoglycans/glycosaminoglycans (p0.001). Analysis of newly synthesized proteoglycans/glycosaminoglycans revealed a heparan/chondroitin sulphate ratio of 1:2.2-2.5. Keratan sulphate was not detected. Estrogenic hyaluronan was 1.6 fold greater in [(3)H]-labeled tissue. Analysis of labeled proteoglycans/glycosaminoglycans revealed two size classes with apparent molecular weights2.0 x 10(6) and 0.8-1.1 x 10(5) and a charge class eluting between 0.1-0.5 M NaCl. The greater glycosaminoglycan content (particularly hyaluronan) and synthesis in estrogen dominant tissue supports a role for steroid hormones in endometrial glycosaminoglycan/proteoglycan regulation and consequent tissue hydration. It also suggests a role for these macromolecules in endometrial function and possibly the implantation process.

The human endometrium is a remarkably dynamic tissue, undergoing cycles of proliferation, differentiation and breakdown every 28 days. In preparation for embryo implantation, the endometrium differentiates or decidualizes, involving widespread morphological and functional differentiation of endometrial stromal cells. If pregnancy occurs, the decidua regulates trophoblast invasion and forms the maternal component of the placenta. Uterine remodeling has long been known to be regulated by the ovarian steroid hormones 17beta-estradiol and progesterone; however, only recently has the importance of paracrine factors in mediating the cellular and biochemical changes been recognized. Many growth factors and cytokines, such as inhibins and activins, whose expression is generally limited to developmental and pathological states, are produced by actively remodeling endometrial cells, and play crucial roles in regulating endometrial cell function. Here, we present evidence for integral roles for the inhibin and activin family in the paracrine regulation of endometrial receptivity, decidualization and implantation.

Decidualization of the human endometrium is critical for implantation, but the mechanisms involved are largely unknown. Activin subunits are expressed in endometrium during decidualization. From its known actions in cell differentiation and tissue remodelling, we hypothesized that activin A is involved in the paracrine regulation of decidualization. We examined the expression of activin receptors (ActRs) by semi-quantitative and real-time RT-PCR. mRNA for all ActR subtypes (Ia, Ib, IIa and IIb) was detected in endometrium, with maximal expression in the early secretory phase and in early pregnancy. ActR protein was localized exclusively to stromal and endothelial cells. This expression pattern was confirmed by in-situ hybridization. Activin bioavailability is locally regulated by its binding protein, follistatin, and also by the antagonist, inhibin. Inhibin competition for ActRII binding is enhanced by the binding protein, betaglycan. Follistatin and betaglycan were also detected in the endometrium, localized to stromal and epithelial cells. This co-expression of activin subunits, receptors and binding proteins indicates that stromal cells are capable of responding to activin, and that there is tight local regulation of activin action within the endometrium. As activin production is up-regulated in decidual cells, this provides further evidence for an involvement of activins during stromal cell decidualization.

Hyaluronan is a molecule with many known roles in cellular physiology and is often associated with tissue remodeling. The human endometrium undergoes dramatic remodeling during the course of the normal menstrual cycle but its regulation is not well understood. This study examined the levels of deposition of hyaluronan in human cycling endometrium by histochemical localization, using a highly specific hyaluronan-binding peptide. This was facilitated by avoiding conventional formalin fixation, which results in serious loss of the water-soluble hyaluronan. Peaks of hyaluronan deposition were observed in the stromal compartment during the mid-proliferative (days 5-10) and the mid-secretory phase (days 19-23) of the cycle. In physiological terms, the first phase corresponds to the time of rapid cellular proliferation of undifferentiated cells, whereas the second phase coincides with the time when the implantation of the conceptus would be initiated in a fertile cycle. By the time menstruation starts, very little hyaluronan remains in the stroma. In contrast with the stromal staining, hyaluronan deposition around blood vessels was constant throughout the cycle. The dramatic changes in hyaluronan deposition and their correlation with the cyclic growth and remodeling in the human endometrium suggest a major role for hyaluronan in the physiology of this tissue.

Progestin-only contraceptives are associated with menstrual bleeding disturbances; a major reason why these agents are discontinued. The pathogenesis of abnormal uterine bleeding associated with progestin-only contraceptives remains ill-defined. Matrix metalloproteinases (MMPs) and leukocytes are postulated to be involved in the process of normal menstruation. Immunolocalization of MMPs and leukocytes in (Norplant), and injectable depot medroxyprogesendometrium from women using the progestinterone acetate (DMPA), are widely used, safe and only contraceptives, Norplant or depot medroxyprogesterone acetate (DMPA) compared with normal controls, revealed foci of positive MMP-1 and -3 immunostaining in stromal cells and adjacent extracellular matrix, the presence of MMP-9 in various subtypes of leukocytes and alterations in mast cell phenotype. In women using progestin-only contraceptives, extent of endometrial MMP, neutrophil and eosinophil immunolocalization and the mast cell activation state was similar to or greater than that observed in perimenstrual control women. However, differences in MMP immunostaining were observed in endometrial samples from women using different progestin-only contraceptive agents; in particular, significantly higher MMP-1 immunostaining was observed associated with the use of Norplant compared with DMPA. No correlation was observed with the number of bleeding days recorded. These results suggest that MMP and leukocytes may be involved in endometrial breakdown in women using progestin-only contraceptives.

1. Endothelin (ET) and its mRNA are present in endometrium. Expression of ET varies across the menstrual cycle, reaching maximal levels in the premenstrual phase, suggesting a paracrine role in endometrial bleeding and/or repair. 2. The major cellular source of ET is the epithelium, although endothelium and decidualized stroma are additional sites of production. Epithelial ET is the ET-1 isoform and this is able to contract rat thoracic aortic rings ex vivo. 3. Endothelin-1 production by cultured endometrial epithelial cells is markedly increased by serum and, to a lesser extent, by transforming growth factor-β and interleukin-1α, but not by epidermal growth factor, oxytocin, arginine vasopressin, thrombin or angiotensin II, which stimulate ET production in other tissues. 4. Endothelin-1 has mitogenic actions on endometrial stromal cells; it stimulates the uptake of [3H]-thymidine, acting via the AP-1 cis element c-jun. 5. Neutral endopeptidase (NEP), a membrane-bound ectoenzyme that is capable of degrading ET, is localized principally in endometrial stroma and immunoreactivity is maximal in the secretory phase of the cycle. 6. A potential role for ET in regulating endometrial bleeding is suggested by studies on endometrium from two groups of women who were experiencing abnormal uterine bleeding: users of the contraceptive Norplant® (Leiras Co., Turku, Finland) and subjects with documented menorrhagia. In both groups, ET-1 immunoreactivity in endometrial epithelium was markedly reduced compared with the normal menstrual cycle and did not vary cyclically, while NEP immunoreactivity, particularly in the epithelium, was increased. Thus, ET may be involved in endometrial bleeding, as a vasoconstrictor before the onset of menstruation when vasoconstriction is intense and, subsequently, when it may be required in the cessation of menstrual bleeding. Furthermore, the mitogenic actions of ET may play a role in endometrial regeneration and remodelling during the menstrual cycle, particularly following menstruation.

Current data support the contention that menstruation is the consequence of the actions of matrix metalloproteinases (MMPs) in the endometrium, and that these enzymes are stimulated by the products of an inflammatory process. A number of MMPs, capable of degrading both interstitial matrix and basement membrane components, have been localized to perimenstrual endometrium, and the focal nature of their production suggests local regulation. Emphasis is placed on important relationships between cells of the immune system (specifically, mast cells, eosinophils, neutrophils and macrophages), and the local production and activation of MMPs, whose degradative actions lead to loss of integrity of blood vessels, destruction of endometrial interstitial matrix and the resultant bleeding characteristic of menstruation.

Synthesis of both mRNA and peptide for gastrin-releasing peptide (GRP) has been demonstrated in the pregnant endometrium of sheep and women. However, it is not known whether GRP is synthesized in the sheep uterus during the oestrous cycle. Furthermore the cellular site of GRP mRNA synthesis in the uterus has not been determined. Therefore we examined the synthesis of GRP and determined the cellular location of GRP peptide and mRNA in sheep uterus taken at different times during the oestrous cycle (duration 17 days) and pregnancy (duration 145 days). Northern blot analysis of RNA isolated from ovine endometrium revealed low or no GRP mRNA at days 4, 10, 12 and 14 of the oestrous cycle and a 24-fold rise in GRP mRNA (normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA) between days 14 and 16. A similar pattern was observed during early pregnancy, with a 12-fold rise in GRP mRNA:GAPDH mRNA between days 17 and 20 of pregnancy. Levels of GRP peptide were determined by RIA and found to be low in endometrium isolated at days 4, 10, 12 and 14 of the oestrous cycle (1.0-1.6 pmol/g) and 4 to 5-fold higher at day 16. In situ hybridization localized GRP synthesis to the epithelial cells of the uterine glands at day 16 of the oestrous cycle and at days 17, 20, 40 and 50 of pregnancy. At day 140 of pregnancy diffuse hybridization to cells of the myometrium was also observed. Immunohistochemistry localized GRP peptide to the apical cytoplasm of uterine glandular epithelial cells at day 16 of the oestrous cycle. For samples obtained at day 20 of pregnancy, the area surrounding the glands also showed moderately strong staining. Further staining in the glandular lumen and the stromal tissue surrounding the glands was apparent at day 140 of pregnancy. No GRP immunoreactivity could be detected in the peripheral plasma during the oestrous cycle or the first 20 days of pregnancy. Sizing chromatography of GRP immunoreactivity extracted from endometrial tissue taken at day 10 of the oestrous cycle revealed two peaks that co-eluted with GRP(1-27) and GRP(18-27). However, during luteolysis and oestrus the major peak of GRP immunoreactivity extracted from endometrial tissue was larger than GRP(1-27) and similar to that seen previously in the gravid ovine endometrium. These studies demonstrate that a peptide similar to, but larger than, GRP is a major product of the glandular epithelium of the ovine uterus during the luteal regression phase of the oestrous cycle and post-blastocyst implantation in pregnancy and provide further evidence that GRP-related peptides have important regulatory roles in uterine function.

Endothelin-1 (ET-1) is present in ovine endometrium, primarily in epithelial cells, and increases around the time of implantation. We examined the cell type expressing ET-binding sites in vitro and whether ET-1 has mitogenic actions in the endometrium, alone or in synergy with other growth factors. Purified epithelial and stromal cells were prepared from luteal-phase endometrium. Specific receptors were demonstrated by binding of 125I-ET-1 and proliferative effects of ET-1 and/or other growth factors determined by uptake of [3H]thymidine by cells in serum-free culture. 125I-ET-1 bound to both epithelial (2516 ± 820 c.p.m./well) and stromal (6368 ± 1350 c.p.m./well) cells and was displaced by ET-1 (1 μmol l−1). There were no proliferative effects of ET on epithelial cells. ET-1 (10 nmol l−1) stimulated uptake of [3H]thymidine by stromal cells under serum-free conditions in 13/20 individual cell preparations, to 149 ± 13% of control (untreated=100%) with dose-dependence between the range of 1 to 100 nmol l−1. Stimulation by fetal calf serum was to 377 ± 126% of control. The effects on proliferation by other growth factors (dose; % of control ± s.e.m., number of positives/total number of cell preparations) were: IGF-I (13 nmol l−1; 182 ± 14, 4/4), epidermal growth factor (EGF; 4·8 nmol l−1; 132 ± 5%, 7/7), platelet-derived growth factor-BB (0·4 nmol l−1; 146 ± 3, 2/2) and leukaemia inhibitory factor (0·4 nmol l−1; 110 ± 2, 3/3). All stimulations except that of EGF were significant and dose-responsive but only insulin was additive with ET (350 ± 35, 5/5). ET-1 also stimulated expression of the the AP-1 cis element c-jun, this being maximal at 60 min of exposure to mitogen. ET-1, along with other growth factors has a likely paracrine role in cellular proliferation in the endometrium, possibly in association with blastocyst implantation. Journal of Endocrinology (1997) 152, 283–290

Matrix metalloproteinases (MMP) are strongly implicated in menstruation. Messenger RNA for proMMP-1 and -3 was detectable in normal cycle endometrium only peri-menstrually and menstrually, although mRNA for their tissue inhibitors, TIMP-1 and TIMP-2, was present throughout the cycle. MMP-1, -3 and -9 were demonstrated immunohistochemically to be specifically associated with degraded tissue in menstrual endometrium. Activated mast cells and eosinophils, which release regulators of MMP expression and activators of latent enzymes, were also a marked feature of menstrual endometrium. Cultured endometrial stromal cells released MMP-1, -2, -3 and -9 and TIMP-1 and -2, whereas production by epithelial cells was minimal. Progesterone withdrawal from stromal cell cultures (for the final 4 days of a 10 day culture) increased the release of all four enzymes: all but MMP-2 were also stimulated by interleukin-1 or tumour necrosis factor alpha added to short-term stromal cultures. We postulate that an alteration in the balance of MMP and their inhibitors and the activation of MMP are prerequisites for tissue degradation at menstruation, and that this is regulated by a combination of progesterone withdrawal and paracrine factors from epithelial and stromal cells and from mast cells and eosinophils.

The architecture of the human endometrium is extensively remodelled during the course of each normal menstrual cycle, unlike most other tissues and organs which undergo very little change during adult life. During menstruation, when loss of most of the functionalis layer occurs, there is concomitant epithelial regrowth; repair of the luminal surface is complete almost as bleeding ceases. During the proliferative phase of the cycle and under the influence of rising oestrogen levels, the stromal cells, glands and blood vessels undergo rapid proliferation which results in tissue thickening. Following ovulation (around day 14 of the idealized 28-day cycle), the secretory phase of the cycle is characterized by increasing tortuosity of the spiral arterioles and glands and increased glandular secretory activity. After about day 22, decidualization of many of the stromal fibroblasts also occurs, the resultant decidual cells having many characteristics typical of epithelial cells. Periods of tissue oedema are apparent both in mid-proliferative (days 8–11) and mid-secretory (days 20–23) endometrium. Late in the cycle, there is regression of the tissue as menstruation is initiated.

The endometrium undergoes substantial morphological and functional changes to become receptive to embryo implantation and to enable establishment of a successful pregnancy. Reduced Delta-like ligand 1 (DLL1, Notch ligand) in the endometrium is associated with infertility. DLL1 can be cleaved by ‘a disintegrin and metalloprotease’ (ADAM) proteases to produce a soluble ligand that may act to inhibit Notch signalling. We used an enzyme-linked immunosorbent assay to quantify soluble DLL1 in uterine lavages from fertile and infertile women in the secretory phase of the menstrual cycle. We also determined the cellular location and immunostaining intensity of ADAM12 and 17 in human endometrium throughout the cycle. Functional effects of soluble DLL1 in receptivity were analysed using in vitro adhesion and proliferation assays and gene expression analysis of Notch signalling targets. Soluble DLL1 was significantly increased in uterine lavage samples of infertile women compared with fertile women in the secretory phase of the menstrual cycle. This coincided with significantly increased ADAM17 immunostaining detected in the endometrial luminal epithelium in the mid-secretory phase in infertile women. Soluble DLL1 significantly inhibited the adhesive capacity of endometrial epithelial cells via downregulation of helix–loop–helix and hairy/enhancer of split family member HES1 mRNA. Thus, soluble DLL1 may serve as a suitable target or potential biomarker for receptivity.

Background Synchronous development of the endometrium (to achieve a receptive state) and of the embryo is essential for successful implantation and ongoing pregnancy. Endometrial receptivity exists only for a finite time in a menstrual cycle and the endometrium is refractory to embryo implantation outside of this window. Administration of hormones to stimulate multifollicular development within the ovary, integral to the majority of assisted reproduction (ART) protocols, dramatically alters the hormonal milieu to which the endometrium is exposed versus normal menstrual cycles. Endometrial maturation may be profoundly affected by this altered endocrine environment. Aim Compare endometrial histology in fertile women, fertile women undergoing hormonal stimulation for oocyte donation and infertile women undergoing fresh embryo transfers in an ART cycle with further comparisons between women who did or did not become pregnant. Examine the presence of leukocytes and markers of endometrial maturation. Methods Endometrial histology was examined by hematoxylin and eosin staining with a semi quantitative scoring method developed to compare histological appearance of tissues. The presence of leukocytes and developmental markers was examined by immunohistochemistry and scored. Results Endometrial histology was dramatically altered upon stimulation for ART. However, those women who became pregnant presented with significantly less alterations in histological endometrial maturation. Numbers and activation status of leukocyte populations were also altered within the endometria stimulated for ART, with neutrophils undergoing degranulation, usually observed only pre-menstrually. Conclusion We propose that such developmental changes render the endometrium hostile to the embryo and that modifications to ART protocols should be considered to take account of the requirement for endometrial receptivity and hence increase pregnancy rates.

Background Understanding the pathophysiology of chemokine secretion in endometriosis may offer a novel area of therapeutic intervention. This study aimed to identify chemokines differentially expressed in epithelial glands in eutopic endometrium from normal women and those with endometriosis, and to establish the expression profiles of key chemokines in endometriotic lesions. Methods Laser capture microdissection isolated epithelial glands from endometrial eutopic tissue from women with and without endometriosis in the mid-secretory phase of their menstrual cycles. Gene profiling of the excised glands used a human chemokine and receptor cDNA array. Selected chemokines were further examined using real-time PCR and immunohistochemistry. Results 22 chemokine/receptor genes were upregulated and two downregulated in pooled endometrial epithelium of women with endometriosis compared with controls. CCL16 and CCL21 mRNA was confirmed as elevated in some women with endometriosis compared to controls on individual samples. Immunoreactive CCL16 and CCL21 were predominantly confined to glands in eutopic and ectopic endometrium: leukocytes also stained. Immunoreactive CCL16 was overall higher in glands in ectopic vs. eutopic endometrium from the same woman (P < 0.05). Staining for CCL16 and CCL21 was highly correlated in individual tissues. Conclusion This study provides novel candidate molecules and suggests a potential local role for CCL16 and CCL21 as mediators contributing to the inflammatory events associated with endometriosis.

OBJECTIVE: The objective was to assess endometrial chemokines in users of the levonorgestrel-releasing intrauterine system (LNG-IUS) and correlate them with leucocyte populations, uterine natural killer cells (uNK) and mast cells (MCs). MATERIALS AND METHODS: Endometrium was obtained from two groups of women who had been using LNG-IUS for 3 years or more: 11 amenorrhoeic women formed the non-bleeding group and 15 women who maintained some form of cyclic bleeding comprised the bleeding group. Specific antibodies were used for the assessment of neutrophils, uNK cells and MCs. Immunohistochemistry was performed to locate the chemokines 6Ckine and interleukin-8 (IL-8). RESULTS: Neutrophils were few and without differences between the two groups. uNK cells were significantly higher in the bleeding group (P < 0.0001). There was no difference between the total number of MCs and activated MCs, but there was a greater extracellular area stained for MC tryptase (P < 0.05). Chemokines 6CKine and IL-8 were abundant in the stroma and in the epithelium, and there was no difference between the groups. CONCLUSIONS: We observed more uNK cells in users with bleeding and a greater extracellular area stained for MC tryptase, although there were no differences between the number of MCs and activated MCs or the chemokines 6CKine and IL-8. uNK cells and MC products may play a role in provoking breakthrough bleeding in long-term users of the LNG-IUS.

Background Breakthrough bleeding (BTB) is the most common reason for discontinuation of progestin-only (p-only) contraceptives, yet the causes are unknown. Use of p-only contraceptives is associated with elevated influx of endometrial leukocytes, similar to that observed perimenstrually or within decidualized endometrium. We hypothesized that chemokine expression is altered in women using p-only contraceptives, leading to abnormal leukocyte recruitment and BTB. Methods Expression of eight highly abundant endometrial chemokines was examined using immunohistochemistry and real-time PCR, in endometria from women using subdermal and intrauterine levonorgestrel and correlated to leukocyte subpopulations. Results Macrophage-derived chemokine (MDC), hemofiltrate CC chemokine-1 (HCC-1), monocyte chemoattractant protein-3 (MCP-3), interleukin-8 (IL-8) and eotaxin were strongly produced by epithelial glands, comparable to levels in premenstrual phase endometrium. Stromal cells were negative for chemokines in atrophic/shedding endometria, but intensely positive in highly decidualized tissues for MDC, MCP-3, HCC-1 and 6Ckine. Macrophage inflammatory protein-1beta (MIP-1b) and HCC-4 were suppressed in all p-exposed endometria. Conclusions These data demonstrate that chemokine expression is dysregulated in p-exposed endometria, consistent with the morphological appearance of the endometrium and the leukocyte subsets present. This reinforces a potential role for chemokines in the elevated leukocyte recruitment that contributes to endometrial fragility and BTB.

Considerable evidence supports a role for matrix metalloproteinases (MMP) in menstruation, but their focal pattern of expression within perimenstrual and menstrual endometrium suggests local rather than hormonal regulation. Menstruation shares a number of features with inflammatory responses, with leukocyte infiltration, proliferation and activation, occurring in the endometrium prior to menstruation. We propose that the leukocytes release MMP at this time and also that interactions between leukocytes and the stromal and epithelial cells of the endometrium induce and activate MMP. Co-culture studies using mast cells or neutrophils with endometrial stromal cells support this hypothesis. How leukocytes enter the endometrium is not understood but a role for chemokines has been proposed. The expression patterns of eotaxin and its receptor CCR3 in endometrium support a role in chemoattraction of eosinophils but expression of monocyte chemotactic proteins 1 and 2 does not correlate with macrophage numbers. Nothing is known of how the leukocytes become activated. Nevertheless, the overall result is a tissue in which an inflammatory-type reaction occurs with release of a myriad of potent regulators. These induce production and activation of MMP and alter the ratio between these and their tissue inhibitors, resulting in tissue breakdown.

The extensive remodelling of the human endometrium throughout the menstrual cycle is accompanied by changes in production of matrix metalloproteinases, the activity of which can be inhibited by specific tissue inhibitors or by tissue inhibitors of metalloproteinases (TIMP)s with a 1:1 stoichiometry. This study immunolocalized TIMP-1, TIMP-2 and TIMP-3 in dated normal human endometrium across the menstrual cycle and examined cultured endometrial cells for their production. All three TIMPs were present in the major cellular compartments, luminal epithelium, glands, stroma, endothelial cells and vascular smooth muscle cells with the most intense immunoreactivity in the luminal epithelium. TIMP-1 and -3 were lower in the mid-to-late proliferative phase with a nadir of TIMP-3 particularly in the late proliferative phase. Decidualized stromal cells stained strongly positive for TIMP-1, -2 and -3. Cells of haematopoietic origin never stained. Monensin treatment of tissue resulted in accumulation of TIMPs in all cellular compartments but particularly of TIMP-1 in epithelium. Cultured endometrial stromal cells released more TIMP-1 than TIMP-2 or TIMP-3 into culture medium and all were increased following decidualization in vitro. Epithelial cells in culture produced less TIMPs than stromal cells, and only a few epithelial cells in each culture were immunopositive for TIMP-1. The ubiquitous distribution of TIMPs implicates them in maintenance of endometrial integrity, with changes in the matrix metalloproteinases without concomitant changes in TIMPs determining endometrial matrix degradation.

The endometrial changes that occur in both fertile and nonfertile menstrual cycles are regulated by ovarian steroid hormones, but there is mounting evidence favoring local regulation of these changes. The differentiation or decidualization of stromal cells that precede implantation is an example. Another is the initiation of endometrial breakdown and shedding at focal points throughout the endometrium. Infiltrating immune cells may well be involved in these processes. Large numbers of macrophages and uterine-specific natural killer cells infiltrate the implantation site. In the nonpregnant state, there is a marked influx of neutrophils, eosinophils, and macrophages that may well contribute to focal endometrial inflammation. Because migration of leukocytes is regulated by chemokines, this study attempted to identify the critical chemokines for human endometrium. The gene array technique served to estimate the expression of 21 chemokines in endometrial tissue taken from healthy women with regular menstrual cycles who had used no hormones in the past 3 months. Patterns of expression and cell sources were examined immunohistochemically and by the real-time polymerase chain reaction (RT-PCR) technique. Nine of the 21 chemokines evaluated were very abundant in the endometrium. They included monocyte chemotactic protein-3 (MCP-3), eotaxin, fractalkine (FKN), macrophage inflammatory protein-1β (MIP-1β), secondary lymphoid chemokine (6Ckine), interleukin-8 (IL-8), hemofiltrate CC chemokine-1 (HCC-1), HCC-4, and monocyte chemotactic protein (MDC). Chemokines were most numerous in the menstrual phase of the cycle, followed by the midsecretory, proliferative, and premenstrual phases in that order. In all stages, the most abundant chemokines were MCP-3, eotaxin, FKN, and MIP-1β. MCP-3 was very highly expressed in all endometrial samples. RT-PCR confirmed that all 9 chemokines were expressed by endometrium. Expression of mRNA for most of the chemokines was, in general, maintained at an elevated level in decidual samples from early pregnancy. Immunohistochemical studies showed the epithelial component of the endometrium to be the major cellular source of all the chemokines. Both luminal and glandular epithelium were involved. Immunostaining of chemokines was most marked in epithelial and decidual cells in early pregnancy. A functional grouping of the chemokines associated with implantation, menstruation, and proliferation is shown in Figure 6. The highly regulated expression of chemokines can create a specialized immunologic environment for pregnancy, and in the nonpregnant state, ensure a controlled, localized inflammatory response that resolves rapidly.

Endothelin (ET) and its mRNA are present in human endometrium. Its expression varies across the menstrual cycle, reaching maximal levels in the pre-menstrual phase. Human endometrial epithelial cells are the major in-vitro source of ET, and its release is affected by the stage of the menstrual cycle from which the endometrium is taken and by the cytokines transforming growth factor-beta 1 and interleukin-1 alpha. The cyclical variation in both ET mRNA and protein expression across the normal menstrual cycle, and the differences observed in abnormal uterine bleeding (specifically reduced immuno-reactivity in luminal and glandular epithelium in the endometrium of Norplant users), are consistent with a role for ET in the control of menstrual bleeding. The role of epithelial cell ET is not yet understood. Whether this source of ET is important in endometrial regeneration and repair following menstruation or in vaso-constriction to cease menstrual bleeding remains to be determined.

Background Interleukin (IL) 11 is produced by human endometrium and endometrial cancer tissue. It has roles in endometrial epithelial cell adhesion and trophoblast cell invasion, two important processes in cancer progression. This study aimed to determine the levels of IL11 in uterine lavage fluid in women with endometrial cancer and postmenopausal women. It further aimed to determine the levels of IL11 protein and its signaling molecules in human endometrial cancer of varying grades, and endometrium from postmenopausal women and IL11 signalling mechanisms in endometrial cancer cell lines. Methods IL11 levels in uterine lavage were measured by ELISA. IL11, IL11 receptor(R) α, phosphorylated (p) STAT3 and SOCS3 were examined by immunohistochemistry in endometrial carcinomas and in control endometrium from postmenopausal women and normal cycling women. The effect of IL11 on pSTAT3/STAT3 and SOCS3 protein abundance in endometrial cancer cell lines and non-cancer endometrial epithelial cells was determined by Western blot. Results IL11 was present in uterine flushings and was significantly higher in women with Grade 1 carcinomas compared to postmenopausal women (p < 0.05). IL11 immunostaining was significantly elevated in the endometrial tumour epithelial cells from Grade 1 and 3 compared to endometrial epithelium from postmenopausal and cycling women. IL11Rα immunostaining intensity was increased in cancer epithelium in the Grades 1 and 2 tumours compared to epithelium from postmenopausal women. Both IL11 and IL11Rα localized to vascular endothelial and smooth muscle cells while IL11 also localized to subsets of leucocytes in the cancer tissues. pSTAT3 was found in both the tumour epithelial and stromal compartments but was maximal in the tumour epithelial cells, while SOCS3 was predominantly found in the tumour epithelial cells. pSTAT3 staining intensity was significantly higher in Grade 1 and 2 tumour epithelial cells compared to epithelial cells from cycling and postmenopausal women. SOCS3 staining intensity did not differ between between each tumour and postmenopausal endometrial epithelium but SOCS3 in cycling endometrium was significantly higher compared to postmonopausal and Tumour Grades 2 and 3. IL11 increased pSTAT3/STAT3 in all tumour cell lines, while SOCS3 abundance was increased only in one tumour cell line. Conclusions The present study suggests that IL11 in uterine washings may be useful as a diagnostic marker for early stage endometrial cancer. It indicates that IL11, along with its specific receptor, IL11Rα, and downstream signalling molecules, STAT3 and SOCS3, are likely to play a role in the progression of endometrial carcinoma. The precise role of IL11 in endometrial cancer remains to be elucidated.