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A series of cyclical events within the uterus are crucial for pregnancy establishment. These include endometrial regeneration following menses, under the influence of estrogen (proliferative phase), then endometrial differentiation driven by estrogen/progesterone (secretory phase), to provide a microenvironment enabling attachment of embryo (as a hatched blastocyst) to the endometrial epithelium. This is followed by invasion of trophectodermal cells (the outer layer of the blastocyst) into the endometrium tissue to facilitate intrauterine development. Small extracellular vesicles (sEVs) released by endometrial epithelial cells during the secretory phase have been shown to facilitate trophoblast invasion; however, the molecular mechanisms that underline this process remain poorly understood. Here, we show that density gradient purified sEVs (1.06–1.11 g/ml, Alix+ and TSG101+, ∼180 nm) from human endometrial epithelial cells (hormonally primed with estrogen and progesterone vs. estrogen alone) are readily internalized by a human trophectodermal stem cell line and promote their invasion into Matrigel matrix. Mass spectrometry-based proteome analysis revealed that sEVs reprogrammed trophectoderm cell proteome and their cell surface proteome (surfaceome) to support this invasive phenotype through upregulation of pro-invasive regulators associated with focal adhesions (NRP1, PTPRK, ROCK2, TEK), embryo implantation (FBLN1, NIBAN2, BSG), and kinase receptors (EPHB4/B2, ERBB2, STRAP). Kinase substrate prediction highlighted a central role of MAPK3 as an upstream kinase regulating target cell proteome reprogramming. Phosphoproteome analysis pinpointed upregulation of MAPK3 T204/T202 phosphosites in hTSCs following sEV delivery, and that their pharmacological inhibition significantly abrogated invasion. This study provides novel molecular insights into endometrial sEVs orchestrating trophoblast invasion, highlighting the microenvironmental regulation of hTSCs during embryo implantation.

Implantation success relies on intricate interplay between the developing embryo and the maternal endometrium. Extracellular vesicles (EVs) represent an important player of this intercellular signalling through delivery of functional cargo (proteins and RNAs) that reprogram the target cells protein and RNA landscape. Functionally, the signalling reciprocity of endometrial and embryo EVs regulates the site of implantation, preimplantation embryo development and hatching, antioxidative activity, embryo attachment, trophoblast invasion, arterial remodelling, and immune tolerance. Omics technologies including mass spectrometry have been instrumental in dissecting EV cargo that regulate these processes as well as molecular changes in embryo and endometrium to facilitate implantation. This has also led to discovery of potential cargo in EVs in human uterine fluid and embryo spent media of diagnostic and therapeutic value in implantation success, fertility, and pregnancy outcome. This review discusses the contribution of EVs in functional hallmarks of embryo implantation, and how the integration of various omics technologies is enabling design of EV-based diagnostic and therapeutic platforms in reproductive medicine. This article is protected by copyright. All rights reserved.

STUDY QUESTION Does NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3) inflammasome activation within decidualized endometrial stromal cells accompany menstruation and is this reflected systemically? SUMMARY ANSWER Components of the NLRP3 inflammasome immunolocalize to decidualized endometrial stromal cells immediately prior to menstruation, and are activated in an in vitro model of menstruation, as evidenced by downstream interleukin (IL)-1beta and IL-18 release, this being reflected systemically in vivo. WHAT IS KNOWN ALREADY Menstruation is a highly inflammatory event associated with activation of NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells), local release of chemokines and cytokines and inflammatory leukocyte influx. Systemically, chemokines and cytokines fluctuate across the menstrual cycle. STUDY DESIGN, SIZE, DURATION This study examined the NLRP3 inflammasome and activation of downstream IL-1beta and IL-18 in endometrial tissues from women of known fertility (≥1 previous parous pregnancy) across the menstrual cycle (n ≥ 8 per cycle phase), serum from women during the proliferative, secretory and menstrual phases (≥9 per cycle phase) of the cycle and menstrual fluid collected on Day 2 of menses (n = 18). Endometrial stromal cells isolated from endometrial tissue biopsies (n = 10 in total) were used for an in vitro model of pre-menstrual hormone withdrawal. PARTICIPANTS/MATERIALS, SETTING, METHODS Expression and localization of components of the NLRP3 inflammasome (NLRP3 & apoptosis-associated speck–caspase recruit domain [ASC]) in endometrial tissues was performed by immunohistochemistry. Unbiased digital quantification of immunohistochemical staining allowed determination of different patterns of expression across the menstrual cycle. Serum from women across the menstrual cycle was examined for IL-1beta and IL-18 concentrations by ELISA. An in vitro model of hormone withdrawal from estrogen/progestin decidualized endometrial stromal cells was used to more carefully examine activation of the NLRP3 inflammasome. Endometrial stromal cells isolated from endometrial tissue biopsies (n = 10) were treated with estrogen/medroxyprogesterone acetate for 12 days to induce decidualization (assessed by release of prolactin) followed by withdrawal of steroid hormone support. Activation of NLRP3, & ASC in these cells was examined on Days 0–3 after hormone withdrawal by Western immunoblotting. Release of IL-1beta and IL-18 examined during decidualization and across the same time course of hormone withdrawal by ELISA. Specific involvement of NLRP3 inflammasome activation in IL-1beta and IL-18 release after hormone withdrawal was investigated via application of the NLRP3 inflammasome inhibitor MCC950 at the time of hormone withdrawal. MAIN RESULTS AND THE ROLE OF CHANCE Critical components of the NLRP3 inflammasome (NLRP3, ASC) were increased in menstrual phase endometrial tissues versus early secretory phase tissues (P LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION This study uses descriptive and semi-quantitative measures of NLRP3 inflammasome activation within endometrial tissues. Further, the in vitro model of pre-menstrual hormone withdrawal may not accurately recapitulate the in vivo environment as only one cell type is present and medroxyprogesterone acetate replaced natural progesterone due to its longer stability. WIDER IMPLICATIONS OF THE FINDINGS We provide novel evidence that the NLRP3 inflammasome is activated within decidualized endometrial stromal cells immediately prior to menses and that local activation of the inflammasome within the endometrium appears to be reflected systemically in by activation of downstream IL-1beta and IL-18. Given the prevalence of menstrual disorders associated with inflammation including dysmenorrhoea and aspects of pre-menstrual syndrome, the inflammasome could be a novel target for ameliorating such burdens. STUDY FUNDING/COMPETING INTEREST(S) The authors have no competing interests. J.E. was supported by a Fielding Foundation fellowship, NHMRC project grants (#1139489 and #1141946) and The Hudson Institute of Medical Research. L.A.S. was supported by The Hudson Institute of Medical Research and J.H. by an Australian Government Research Training Program Scholarship. We acknowledge the Victorian Government’s Operating Infrastructure funding to the Hudson Institute. TRIAL REGISTRATION NUMBER N/A

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.

Adequate endometrial stromal cell (ESC) decidualization is vital for endometrial health. Given the importance of extracellular vesicles (EVs) in intercellular communication, we investigated how their protein landscape is reprogrammed and dysregulated during decidual response. Small EVs (sEVs) from human ESC-conditioned media at Day-2 and -14 following decidual stimuli were grouped as well- (WD) or poorly decidualized (PD) based on their prolactin secretion and subjected to mass spectrometry-based quantitative proteomics. On Day 2, in PD- versus WD-ESC-sEVs, 17 sEV- proteins were down-regulated (C5, C6; complement/coagulation cascades, and SERPING1, HRG; platelet degranulation and fibrinolysis) and 39 up-regulated (FLNA, COL1A1; focal adhesion, ENO1, PKM; glycolysis/gluconeogenesis, and RAP1B, MSN; leukocyte transendothelial migration). On Day 14, in PD- versus WD-ESC-sEVs, FLNA was down-regulated while 21 proteins were up-regulated involved in complement/coagulation cascades (C3, C6), platelet degranulation (SERPINA4, ITIH4), B-cell receptor signalling and innate immune response (immunoglobulins). Changes from Days 2 to 14 suggested a subsequent response in PD-ESC-sEVs with 89 differentially expressed proteins mostly involved in complement and coagulation cascades (C3, C6, C5), but no change in WD-ESC-sEVs ESC. Poor decidualization was also associated with loss of crucial sEV-proteins for cell adhesion and invasion (ITGA5, PFN1), glycolysis (ALDOA, PGK1) and cytoskeletal reorganization (VCL, RAC1). Overall, this study indicates varied ESC response even prior to decidualization and provides insight into sEVs-proteomes as a benchmark of well-decidualized ESC. It shows distinct variation in sEV-protein composition depending on the ESC decidual response that is critical for embryo implantation, enabling and limiting trophoblast invasion during placentation and sensing a healthy embryo.

Uniquely among adult tissues, the human endometrium undergoes cyclical shedding, scar-free repair and regeneration during a woman's reproductive life. Therefore, it presents an outstanding model for study of such processes. This Review examines what is known of endometrial repair and regeneration following menstruation and parturition, including comparisons with wound repair and the influence of menstrual fluid components. We also discuss the contribution of endometrial stem/progenitor cells to endometrial regeneration, including the importance of the stem cell niche and stem cell-derived extracellular vesicles. Finally, we comment on the value of endometrial epithelial organoids to extend our understanding of endometrial development and regeneration, as well as therapeutic applications.

OBJECTIVE: To establish a model of human implantation that responds to hormonal stimuli and can differentiate between endometrium from fertile women and those with idiopathic infertility. DESIGN: A trophoblast stem cell (trophectodermal) line (TSC; derived from human pre-implantation embryo) was used to form trophectodermal spheroids (TS). TS attachment to monolayers of endometrial epithelial cell lines or primary endometrial epithelial cells (pHEECs) was determined. SETTING: Independent Medical Research Institute with close clinical linkages INTERVENTIONS: Spheroid attachment and outgrowth was determined with added hormones (estradiol 17β (E), E + medroxyprogesterone acetate (MPA) or E + MPA + human chorionic gonadotropin (hCG)). Spheroid attachment to E/MPA treated pHEEC prepared from fertile women or those with idiopathic infertility tested. MAIN OUTCOME MEASURE: Firmly attached spheroids counted after co-culture for 6 h. Outgrowth was determined by quantitation of area covered by spheroid after firm adhesion. RESULTS: Functional adhesion of TS to two endometrial epithelial cell lines, Ishikawa and ECC-1 cells, was hormonally responsive, with adhesion/outgrowth increased by E/MPA (ECC-1; p < 0.01, Ishikawa; p < 0.01) and E/MPA/hCG (ECC-1; p < 0.001, Ishikawa p < 0.01) versus E alone. The same pattern of hormone responsiveness was observed in pHEEC obtained from fertile women (E vs, E/MPA; p < 0.01, E vs. E/MPA/hCG; p < 0.001). TS adhered to 85% of pHEEC obtained from fertile women (11/13) and 11% of pHEEC obtained from women with unexplained infertility (2/18, p < 0.001). CONCLUSION: This new model of “embryo” implantation largely discriminates between endometrial epithelial cells obtained from fertile vs. infertile women based on adhesion; this holds potential as an in vitro “diagnostic” tool of endometrial infertility.

text Globally, 39% of the adult population is overweight or obese, with the prevalence of obesity following an upward trajectory over the recent decades (WHO). Up to 30% of women of reproductive age in Western countries are obese before conception, and obese women experience higher rates of infertility and pregnancy complications than lean women; however, the mechanisms underpinning obesity-related infertility are poorly understood. Advanced Glycation Endproducts (AGEs) are a proinflammatory modification of proteins exposed to sugars, formed through the Maillard reaction. AGEs are elevated four-fold in the uterine fluid of obese, infertile women, compared to lean. AGEs equimolar to those in the obese microenvironment negatively impact the functions of endometrial epithelial and stromal cells, and adhesion and invasion of trophoblast cells, reducing the potential for successful maternal-fetal interactions (Antoniotti et al., 2018). This research further investigated preimplantation embryo development and endometrial cell functions in the presence of AGEs equimolar to those in obese uterine fluid. Altered local environments in very early life can set offspring up for a lifetime of health or disease (DoHAD); thus, uterine AGEs may contribute to the prevalence of non-communicable disease in children of obese parents. Preimplantation mouse embryos were cultured in vitro with AGEs equimolar with uterine fluid concentrations from lean and obese women, and their development and implantation potential assessed. “Obese” AGEs did not impact the proportion of embryos reaching blastocyst stage by day 4, but significantly reduced the proportion of blastocysts hatching by day 5 (P < 0.01). AGEs equimolar with the obese uterine environment detrimentally impacted trophectoderm formation and function: reduced trophectoderm cell number (P < 0.01), reduced outgrowth on fibronectin (indicative of reduced implantation potential, (P < 0.01), but did not increase cell apoptosis (TUNEL assay). RAGE antagonism, but neither metformin nor antioxidants, improved trophectoderm cell number. Thus, obesity-associated AGEs link obesity and reduced fertility through poor placentation potential of embryos (Hutchison et al, 2020). Endometrial epithelial cell function was examined in the presence of lean and obese concentrations of AGEs. Obese AGEs significantly reduced the rate of proliferation (xCelligence real time cell analysis) of the endometrial epithelial cell line ECC-1 versus lean AGEs (P = 0.04). Antioxidants successfully restored the rate of proliferation in the presence of obese AGEs (P = 0.7 versus lean AGEs). Subsequently, human endometrial epithelial organoid culture was utilised as a more physiologically relevant experimental paradigm. When cultured as organoids, primary endometrial epithelial cells were functionally responsive to obesity-associated AGEs, expressing both RAGE and TLR4. The morphology of organoids in culture was not impacted by the presence of obese AGEs versus lean; however, the proliferation of epithelial cells retrieved from organoid culture was altered by obese AGEs versus lean. Obese AGEs also increased the secretion of proinflammatory CXCL16 versus vehicle control (P = 0.04) while increased secretion of other proinflammatory cytokines and chemokines including TNFa approached significance in the presence of obese AGEs. As the inflammatory milieu is altered in the uterine fluid of infertile women, elevated AGEs may promote an infertile endometrial inflammatory environment. AGEs link obesity and reduced fertility, being detrimental to preimplantation embryo development and endometrial cell function when present at concentrations equal to those in obese uterine fluid. Antioxidants and RAGE antagonism provide beneficial effects to cell function in the presence of obesity-associated AGEs. This research provides evidence supporting AGEs as a factor contributing to obesity related infertility, and as an emerging frontier for reproductive health. Clinically, reduction of uterine AGEs may improve fertility for obese couples wishing to conceive. Antoniotti et al (2018). Hum Rep. 33(4), 654-665. PMID: 29471449 Hutchison et al (2020). RBMO. 41(5), 757-766. PMID: 32972872

Dialogue between an appropriately developed embryo and hormonally-primed endometrium is essential to achieve implantation and establish pregnancy. Importantly, the point-of-first-contact between the embryo and the maternal endometrium occurs at the endometrial luminal epithelium (LE). Implantation events occur within the uterine cavity microenvironment regulated by local factors. Defects in embryo-endometrial communication likely underlie unexplained infertility; enhanced knowledge of this communication, specifically at initial maternal-fetal contact may reveal targets to improve fertility. Using a human endometrial luminal-epithelial (LE) cell line (ECC1), this targeted proteomic study reveals unique protein changes in both cellular (98% unique identifications) and secreted (96% unique identifications) proteins in the transition to the progesterone-dominated secretory (receptive) phase and subsequently to pregnancy, mediated by embryo-derived human chorionic gonadotropin (hCG). This analysis identified 157 progesterone-regulated cellular proteins, with further 193 significantly altered in response to hCG. Cellular changes were associated with metabolism, basement membrane and cell connectivity, proliferation and differentiation. Secretome analysis identified 1059 proteins; 123 significantly altered by progesterone, and 43 proteins altered by hCG, including proteins associated with cellular adhesion, extracellular-matrix organization, developmental growth, growth factor regulation, and cell signaling. Collectively, our findings reveal dynamic intracellular and secreted protein changes in the endometrium that may modulate successful establishment of pregnancy. Biological significance This study provides unique insights into the developmental biology of embryo implantation using targeted proteomics by identifying endometrial epithelial cellular and secreted protein changes in response to ovarian steroid hormones and pregnancy hormones that are essential for receptivity and implantation.

This chapter initially provides a brief overview of current knowledge of neutrophil biology, providing background knowledge. It then examines neutrophil contributions to endometrial remodeling during the normal menstrual cycle, menstruation, and subsequent endometrial repair. This is followed by discussion of what is known of neutrophil contributions to placental development, parturition, and maternal immunotolerance. Finally, it examines how disturbances in neutrophil trafficking and phenotype may contribute to disorders such as abnormal uterine bleeding, pre-eclampsia, antiphospholipid syndrome and preterm birth. Importantly, the chapter highlights that while neutrophils predominantly act in concert with other immune cell types as part of the inflammatory cascade, neutrophils also perform specific independent physiological processes within the female reproductive tract. These independent processes are facilitated via release of inflammatory mediators, proteases, extracellular vesicles, and neutrophil traps released into the local tissue microenvironment, which interact with a variety of nonimmune cell types within the local milieu.

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.

Repair after damage is essential for tissue homeostasis. Postmenstrual endometrial repair is a cyclical manifestation of rapid, scar-free, tissue repair taking ∼3-5 d. Skin repair after wounding is slower (∼2 wk). In the case of chronic wounds, it takes months to years to restore integrity. Herein, the unique "rapid-repair" endometrial environment is translated to the "slower repair" skin environment. Menstrual fluid (MF), the milieu of postmenstrual endometrial repair, facilitates healing of endometrial and keratinocyte "wounds" in vitro, promoting cellular adhesion and migration, stimulates keratinocyte migration in an ex vivo human skin reconstruct model, and promotes re-epithelialization in an in vivo porcine wound model. Proteomic analysis of MF identified a large number of proteins: migration inhibitory factor, neutrophil gelatinase-associated lipocalin, follistatin like-1, chemokine ligand-20, and secretory leukocyte protease inhibitor were selected for further investigation. Functionally, they promote repair of endometrial and keratinocyte wounds by promoting migration. Translation of these and other MF factors into a migration-inducing treatment paradigm could provide novel treatments for tissue repair.-Evans, J., Infusini, G., McGovern, J., Cuttle, L., Webb, A., Nebl, T., Milla, L., Kimble, R., Kempf, M., Andrews, C. J., Leavesley, D., Salamonsen, L. A. Menstrual fluid factors facilitate tissue repair: identification and functional action in endometrial and skin repair.

The regenerative, proliferative phase of a woman's menstrual cycle is a critical period which lays the foundation for the subsequent, receptive secretory phase. Although endometrial glands and their secretions are essential for embryo implantation and survival, the proliferative phase, when these glands form, has been rarely examined. We hypothesized that alterations in the secreted proteome of the endometrium of idiopathic infertile women would reflect a disturbance in proliferative phase endometrial regeneration. Our aim was to compare the proteomic profile of proliferative phase uterine fluid from fertile (n = 9) and idiopathic infertile (n = 10) women. Proteins with ≥2-fold change (P < 0.05) were considered significantly altered between fertile and infertile groups. Immunohistochemistry examined the endometrial localization of identified proteins. Western immunoblotting defined the forms of extracellular matrix protein 1 (ECM1) in uterine lavage fluid. Proteomic analysis identified four proteins significantly downregulated in infertile women compared to fertile women, including secreted frizzled-related protein 4 (SFRP4), CD44, and ECM1: two proteins were upregulated. Seven proteins were unique to the fertile group and six (including isoaspartyl peptidase/L-asparaginase [ASRGL1]) were unique to the infertile group. Identified proteins were classified into biological processes of tissue regeneration and regulatory processes. ASRGL1, SFRP4, and ECM1 localized to glandular epithelium and stroma, cluster of differentiation 44 (CD44) to stroma and immune cells. ECM1 was present in two main molecular weight forms in uterine fluid. Our results indicate a disturbance in endometrial development during the proliferative phase among infertile women, providing insights into human endometrial development and potential therapeutic targets for infertility.Summary SentenceProteomic analysis of proliferative phase uterine fluid in both fertile and infertile women showed significant differences in the secreted proteins including ECM1 which was further studied for its cellular location and hormonal regulation.

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

A major cause of infertility in normal and assisted reproduction cycles is failure of the endometrium to undergo appropriate changes during the secretory phase of the menstrual cycle as it acquires receptivity for an implanting blastocyst. Current dogma states that loss of epithelial polarity in the luminal epithelial cells, the point of first contact between maternal endometrium and blastocyst, may facilitate embryo implantation. Loss of polarity is likely an important change during the secretory phase to overcome mutual repulsion between otherwise polarized epithelial surfaces. Although "plasma membrane transformation" describes morphological/molecular alterations associated with loss of polarity, direct measures of polarity have not been investigated. Transepithelial resistance, a proxy measure of polarity, was downregulated in endometrial epithelial (ECC-1) cells by combined estrogen/progestin, mimicking the hormonal milieu of the secretory phase. Examination of defined polarity markers within human endometrium throughout the menstrual cycle identified downregulation of atypical protein kinase C, Stardust, Crumbs, and Scribble within the luminal-epithelial layer, with upregulation of Scribble within the stromal compartment as the menstrual cycle progressed from the estrogen-dominated proliferative to progesterone-dominated secretory phase. Epithelial (ECC-1) Scribble expression was downregulated in vitro by combined estrogen/progestin and estrogen/progestin/human chorionic gonadotropin treatment, whereas knockdown of Scribble in these cells enhanced "embryo" (trophectodermal spheroid) adhesion. In contrast, Scribble was upregulated within decidualized primary human endometrial stromal cells, with decidualization downregulated upon Scribble knockdown. These data highlight an important contribution of polarity modulation within the human endometrium, likely important for receptivity. Clinical investigations examining how polarity may be modulated in the infertile endometrium may facilitate fertility.

// Sophea Heng 1, 2, 3 , Jemma Evans 1, 2, 4 , Lois A. Salamonsen 1, 2, 4 , Tom W. Jobling 4, 5 and Guiying Nie 1, 2, 3 1 Centre for Reproductive Health, Hudson Institute of Medical Research, Clayton, Victoria, Australia 2 Department of Molecular and Translational Sciences, Monash University, Clayton, Victoria, Australia 3 Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia 4 Department of Obstetrics and Gynaecology, Monash University, Clayton, Victoria, Australia 5 Epworth Research Institute, Epworth Health Care, Richmond, Victoria, Australia Correspondence to: Guiying Nie, email: guiying.nie@hudson.org.au Keywords: endometrial cancer, dystroglycan, tight junction, cell polarity, estrogen Received: October 25, 2016 Accepted: April 11, 2017 Published: April 20, 2017 ABSTRACT Endometrial cancer is one of the most common gynecological malignancies affecting post-menopausal women, yet the underlying mechanisms are not well understood. Dystroglycan (DG) is a large glycoprotein, consisting of α- and β-subunits that are non-covalently associated with each other. Modifications to α-DG have been linked to a variety of cancers, where the N-terminus of α-DG (α-DG-N) is post-translationally removed by a furin-like enzyme. However, the functional significance of α-DG-N removal is unknown. Our previous studies have established that the α-DG cleavage enzyme furin is significantly up-regulated in endometrial cancer. This study aimed to investigate the importance of α-DG-N removal in post-menopausal endometrial cancer. We demonstrated that α-DG-N removal predominantly occurred in early stage endometrial cancer tissues, and that the cleaved α-DG-N was significantly elevated in the uterine lavage of early grade endometrial cancer patients. Furthermore, α-DG-N removal significantly decreased the tight junction integrity and polarity of the endometrial epithelial cells, promoting the loss of polarity markers scribble and atypical protein kinase C (aPKC) and reducing the trans-epithelial electrical resistance. The removal of α-DG-N also sensitized the cells for estrogen-dependent proliferation. These results strongly suggest that α-DG-N removal plays an important role in early stage development of endometrial cancer, and that the elevated levels of α-DG-N in uterine fluid may provide a biomarker for early detection of endometrial cancer.

Current treatment options for uterine fibroids are limited to hormonal manipulation or surgical intervention. We aimed to develop an in vitro model to mirror collagen deposition and extracellular matrix (ECM) formation, the principal features of uterine fibroids, to enable testing of novel therapeutics. Macromolecular crowding with Ficoll 400 and Ficoll 70 in cultures of human uterine myometrial smooth muscle cells containing ascorbic acid, provided the basis for this model. These culture conditions mimic the ‘crowded’ nature of the in vivo extracellular environment by incorporating neutral, space-filling macromolecules into conventional cell cultures. This method of culture facilitates appropriate ECM deposition, thus closely representing the in vivo fibrotic phenotype of uterine fibroids. Macromolecular crowding in Ficoll cultures containing ascorbic acid reduced myometrial smooth muscle cell proliferation and promoted collagen production. Under these conditions, collagen was processed for extracellular deposition as demonstrated by C-propeptide cleavage from secreted procollagen. The fibrosis marker activin was increased relative to its natural inhibitor, follistatin, in crowded culture conditions while addition of exogenous follistatin reduced collagen (Col1A1) gene expression. This in vitro model represents a promising development for the testing of therapeutic interventions for uterine fibroids. However, it does not recapitulate the full in vivo pathology which can include specific genetic and epigenetic alterations that have not been identified in the myometrial smooth muscle (hTERT-HM) cell line. Following screening of potential therapeutics using the model, the most promising compounds will require further assessment in the context of individual subjects including those with genetic changes implicated in fibroid pathogenesis.

The yin and yang of female fertility is a complicated issue; large numbers of women/couples desire fertility and seek assisted reproduction intervention to achieve conception, while others seek to prevent pregnancy. Understanding specific molecules which control endometrial-embryo interactions is essential for both facilitating and preventing pregnancy. SOX17 has recently emerged as an important transcription factor involved in endometrial receptivity and embryo implantation. However, studies to date have examined mouse models of pregnancy which do not necessarily translate to the human. Demonstration of a role for ‘implantation factors’ in a human system is critical to provide a rationale for in depth clinical investigation and targeting of such factors. We demonstrate that SOX17is present within the receptive human endometrium and is up-regulated within human endometrial epithelial cells by combined estrogen & progesterone, the hormonal milieu during the receptive window. SOX17 localizes to the point of adhesive contact between human endometrial epithelial cells and a human ‘embryo mimic’ model (trophectodermal spheroid). Targeting SOX17 in endometrial epithelial cells using CRISPR/Cas9 knockdown or a SOX-F family inhibitor, MCC177, significantly inhibited adhesion of an trophectodermal spheroids to the epithelial cells thereby preventing ‘implantation’. These data confirm the important role of endometrial SOX17 in human endometrial receptivity and embryo implantation.

Trophoblast invasiveness in ruminants is limited to fusion of migrating binucleate cells with uterine epithelium, but considerable tissue remodelling and angiogenesis occurs within the endometrium at implantation. Such processes are elsewhere associated with an altered balance of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). ProMMPs-1, -2, -3 are secreted by cultured ovine endometrial stromal, but not epithelial, cells and expression of proMMP-1 and proMMP-3 is inhibited by interferon tau, the dominant preimplantation trophoblast product, independently of its effects on prostaglandin production. Messenger RNA for TIMP-1 and TIMP-2 is expressed in endometrium throughout the oestrous cycle and early pregnancy. Both TIMP-1 mRNA (0.9 kb transcript) and TIMP-2 mRNA (1.0 kb transcript) expression increase at day 12 through to day 20, suggesting a role in preventing trophoblast invasion. Expression of the 3.5 kb transcript for TIMP-2 decreases from day 16 and is undetectable on day 20. Uterine flushings from both nonpregnant and pregnant ewes on day 16 after oestrus contain proMMP-2, possibly transudated from plasma, and proMMP-9, but concentrations are higher in pregnancy. These enzymes are also detected in conditioned medium following culture of trophoblasts at days 16-20. Whether the trophoblast MMPs have a role in implantation in sheep remains to be established. The production of MMPs and TIMPs in the endometrium and the changes associated with implantation events suggest that they contribute to the marked edometrial remodelling associated with early placentation.

Primary cultures of ovine epithelial and stromal cells have been used to examine paracrine interactions between the endometrium and the preimplantation sheep blastocyst, and in particular the actions of the blastocyst alpha-interferon, ovine trophoblast protein-1 (oTP-1), on endometrial cell metabolism. The synthesis and secretion of several 'pregnancy-related' acidic proteins with molecular weights in the range 70,000-120,000 can be induced by addition of oTP-1 or human recombinant interferon (IFN) to cultured enriched epithelial endometrial cells. Consistent with the antiluteolytic role of oTP-1, dose-dependent attenuation of both PGE and PGF-2 alpha release has been demonstrated. Arachidonic acid added to the cells increased overall PG release but the inhibitory effects of interferons were still apparent. Immunocytochemical analysis of PG synthase demonstrated marked cyclic variation of its localization within the endometrium, but no differences in distribution or intensity of staining were apparent in endometrium of early pregnancy (Day 15) compared with that of the cycle (Day 15). It appears that conceptus-induced changes in PG release do not occur via changes in concentration or localization of PG synthase but rather by modifying its activity. Highly purified epithelial cells cultured on matrigel-coated millicell inserts retain important morphological features seen in vivo. Under such conditions, PGF-2 alpha and PGE release into the basal compartment was greater than that into the apical compartment. Stromal fibroblasts cultured under similar conditions secreted less PGF-2 alpha but more PGE than epithelial cells. Whilst the limitations of in-vitro studies are acknowledged, these findings are compatible with and markedly extend what is known of the action of embryonic interferons in vivo in the establishment of pregnancy in sheep.

Embryo implantation into the receptive endometrium is critical in pregnancy establishment, initially requiring reciprocal signalling between outer layer of the blastocyst (trophectoderm cells) and endometrial epithelium; however, factors regulating this crosstalk remain poorly understood. Although endometrial extracellular vesicles (EVs) are known to signal to the embryo during implantation, the role of embryo-derived EVs remains largely unknown. Here, we provide a comprehensive proteomic characterisation of a major class of EVs, termed small EVs (sEVs), released by human trophectoderm cells (Tsc-sEVs) and their capacity to reprogram protein landscape of endometrial epithelium in vitro. Highly purified Tsc-sEVs (30-200 nm, ALIX+ , TSG101+ , CD9/63/81+ ) were enriched in known players of implantation (LIFR, ICAM1, TAGLN2, WNT5A, FZD7, ROR2, PRICKLE2), antioxidant activity (SOD1, PRDX1/4/6), tissue integrity (EZR, RAC1, RHOA, TNC), and focal adhesions (FAK, ITGA2/V, ITGB1/3). Functionally, Tsc-sEVs were taken up by endometrial cells, altered transepithelial electrical resistance, and upregulated proteins implicated in embryo attachment (ITGA2/V, ITGB1/3), immune regulation (CD59, CD276, LGALS3), and antioxidant activity (GPX1/3/4, PRDX1/2/4/5/6): processes that are critical for successful implantation. Collectively, we provide critical insights into Tsc-sEV-mediated regulation of endometrial function that contributes to our understanding of the molecular basis of implantation.

Evidence is presented for expression of the insulin receptor on the surface of mammalian spermatozoa as well as transcripts for the receptor substrate adaptor proteins (IRS1-4) needed to mediate insulin action. Exposure to this hormone resulted in insulin receptor phosphorylation (pTyr972), activation of AKT (pSer473) and the stimulation of sperm motility. Intriguingly, the male germ line is also shown to be capable of generating insulin, possessing the relevant mRNA transcript and expressing strong immunocytochemical signals for both insulin and C-peptide. Insulin could be released from the spermatozoa by sonication in a concentration-dependent manner but was not secreted in response to glucose, fructose or stimulation with progesterone. However, insulin release could be induced by factors present in human uterine lavages. Furthermore, the endometrium was also shown to possess the machinery for insulin production and action (mRNA, insulin, C-peptide, proprotein convertase and insulin receptor), releasing insulin into the uterine lumen prior to ovulation. These studies emphasize the fundamental importance of extra-pancreatic insulin in regulating the reproductive process, particularly in the support of spermatozoa on their perilous voyage to the site of fertilization.

Endometrial extracellular vesicles (EVs) are emerging as important players in reproductive biology. However, how their proteome is regulated throughout the menstrual cycle is not known. Such information can provide novel insights into biological processes critical for embryo development, implantation, and successful pregnancy. Using mass spectrometry-based quantitative proteomics, we show that small EVs (sEVs) isolated from uterine lavage of fertile women (UL-sEV), compared to infertile women, are laden with proteins implicated in antioxidant activity (SOD1, GSTO1, MPO, CAT). Functionally, sEVs derived from endometrial cells enhance antioxidant function in trophectoderm cells. Moreover, there was striking enrichment of invasion-related proteins (LGALS1/3, S100A4/11) in fertile UL-sEVs in the secretory (estrogen plus progesterone-driven, EP) versus proliferative (estrogen-driven, E) phase, with several players downregulated in infertile UL-sEVs. Consistent with this, sEVs from EP- versus E-primed endometrial epithelial cells promote invasion of trophectoderm cells. Interestingly, UL-sEVs from fertile versus infertile women carry known players/predictors of embryo implantation (PRDX2, IDHC), endometrial receptivity (S100A4, FGB, SERPING1, CLU, ANXA2), and implantation success (CAT, YWHAE, PPIA), highlighting their potential to inform regarding endometrial status/pregnancy outcomes. Thus, this study provides novel insights into proteome reprograming of sEVs and soluble secretome in uterine fluid, with potential to enhance embryo implantation and hence fertility.

Successful implantation requires synchronous development of embryo and endometrium. Endometrial receptivity results from progesterone-induced differentiation of endometrial cells, generally achieved during the mid-secretory phase of the cycle. Failure to properly develop receptivity results in failed or inadequate implantation and hence no ongoing pregnancy. The blastocyst undergoes final development, apposition, attachment and initiates invasion of the endometrial epithelium within the uterine cavity. Thus, the microenvironment provided by uterine fluid, particularly glandular secretions, is essential for implantation. Analysis of endometrial fluid has identified cytokines, chemokines, proteases, antiproteases and other factors that modulate blastocyst functions relevant to implantation. Exosomes/microvesicular bodies released from the endometrium (and likely also the embryo) are present in uterine fluid. These can transfer miRNA, proteins and lipids between cells, thus providing endometrial-embryo communication in the peri-implantation period. Understanding the uterine microenvironment, and its effects on endometrial-embryo interactions, will provide opportunities to modify current infertility treatments to improve success rates.

Menstruation is the process by which the functionalis layer of the endometrium is shed and restored during each menstrual cycle in women, without scarring or loss of function. The trigger for menstruation is withdrawal of hormonal support in a nonconception cycle, which induces highly controlled inflammatory mechanisms starting with influx and activation of specific leukocyte subsets and release of bioactive mediators, including degradative enzymes. Tissue breakdown and repair occur simultaneously in different/adjacent areas of the tissue. Re -epithelialization rapidly covers the degraded surface, independently of hormone (estrogen) support: full regeneration of the endometrial functionalis follows with estrogen support during the ensuing proliferative phase.

STUDY QUESTION Do obese levels of advanced glycation end products (AGEs) within the uterine cavity detrimentally alter tissue function in embryo implantation and placental development? SUMMARY ANSWER Obese levels of AGEs activate inflammatory signaling (p65 NFκB) within endometrial epithelial cells and alter their function, cause endoplasmic reticulum (ER) stress in endometrial stromal cells and impair decidualization, compromise implantation of blastocyst mimics and inhibit trophoblast invasion. WHAT IS KNOWN ALREADY Obese women experience a higher incidence of infertility, recurrent miscarriage and pregnancy complications compared with lean women. Oocyte donation cycles suggest a detrimental uterine environment plays a role in these outcomes. STUDY DESIGN, SIZE, DURATION Uterine lavage and tissues from lean (BMI 19.5-24.9, n = 17) and obese (BMI > 30, n = 16) women examined. Cell culture experiments utilizing human endometrial epithelial, trophectoderm and trophoblast cell lines and primary human stromal cells used to examine the functional impact of obese levels of AGEs. PARTICIPANTS/MATERIALS, SETTING, METHODS Levels of AGEs examined within uterine lavage assessed by ELISA to determine differences between lean and obese women. Expression and localization of AGEs, receptor for AGEs (RAGE) and NFκB within endometrial tissues obtained from lean and obese women determined by immunohistochemistry. Endometrial epithelial cells (ECC-1), primary human stromal cells and trophoblast cells (HTR8-SVneo) treated with lean (2000 nmol/mol lysine) or obese (8000 nmol/mol lysine) uterine levels of AGEs and p65 NFκB (western immunoblot), real-time adhesion, proliferation migration and invasion (xCelligence real-time cell function analysis), decidualization (cell morphology and prolactin release), ER stress (western immunoblot for p-PERK) determined. Co-cultures of endometrial epithelial cells and blastocyst mimics (trophectoderm spheroids) similarly treated with lean or obese uterine levels of AGEs to determine their impact on embryo implantation. MAIN RESULTS AND THE ROLE OF CHANCE AGEs were significantly elevated (P = 0.004) within the obese (6503.59 μmol/mol lysine) versus lean (2165.88 μmol/mol lysine) uterine cavity (uterine lavage) with increased immunostaining for AGEs, RAGE and NFkB within obese endometrial tissues during the proliferative phase of the menstrual cycle. Obese uterine levels of AGEs inhibited adhesion and proliferation of endometrial epithelial (ECC-1) cells compared to treatment with lean uterine levels of AGEs. Obese uterine AGE levels impacted primary human endometrial stromal cell decidualization and activated ER stress within these cells. Obese uterine levels of AGEs also inhibited trophectodermal spheroid adhesion to hormonally primed endometrial epithelial cells and trophoblast cell line HTR8/SV-neo invasion. LARGE SCALE DATA N/A. LIMITATIONS REASONS FOR CAUTION Mechanistic studies are performed in vitro and may not completely recapitulate cell function in vivo. WIDER IMPLICATIONS OF THE FINDINGS These data corroborate clinical data suggesting the presence of an altered uterine environment in obese women and demonstrate that elevated uterine levels of AGEs within these women may detrimentally impact endometrial function, embryo implantation and placental development. Uterine AGE assessment in infertility work up may prove useful in determining underlying causes of infertility. AGEs can be targeted pharmacologically and such treatments may prove effective in improving reproductive complications experience by obese women. STUDY FUNDING/COMPETING INTEREST(S) Supported by NHMRC Fellowship (#1002028 to L.A.S.), and the Victorian Government's Operational Infrastructure Support Program. MTC is supported by a JDRF Australia Clinical Research Network Career Development Award. The authors have declared that no conflict of interest exists.

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.

Extracellular vesicles (EVs), including exosomes (30-150 nm) and microvesicles (100-1500 nm), play important roles in mediating cell-cell communication. Such particles package distinct cargo elements, including lipids, proteins, mRNAs, microRNAs, and DNA, that vary depending on the cell of origin and its phenotype. This cargo can be horizontally transferred to target cells where its components can reprogram the recipient cell to modify its function. EVs have been identified within the uterine cavity of women, sheep, and mice, where they contribute to the microenvironment of sperm transport, and of blastocyst and endometrial preparation for implantation. It is likely that exosomes and microvesicles carry different cargo and coordinate different roles in this intrauterine environment. Understanding and defining these subtypes of EVs is important for future functional studies and clinical translation. Here we critically review the various purification and validation procedures for extracellular vesicle analysis and discuss what is known of endometrial-derived exosome cargo and of their hormonal regulation. The current knowledge of the functions of uterine exosomes, with respect to sperm transport and function, and of their actions on trophectodermal cells to promote implantation are summarized and evaluated in their physiological context. Given the potential importance of this form of cell-cell interactions within the reproductive tract, the critical issues discussed will guide new insights in this rapidly expanding field.

Embryo implantation into receptive endometrium requires synergistic endometrial-blastocyst interactions within the uterine cavity and is essential for establishing pregnancy. We demonstrate that exosomes (40-150 nm nanovesicles) released from endometrial epithelial cells are an important component of these interactions. We defined the proteome of purified endometrial epithelial-derived exosomes (Exos) influenced by menstrual cycle hormones estrogen (E; proliferative phase) and estrogen plus progesterone (EP; receptive phase) and examined their potential to modify trophoblast function. E-/EP-Exos were uniquely enriched with 254 and 126 proteins, respectively, with 35% newly identified proteins not previously reported in exosome databases. Importantly, EP-Exos protein cargo was related to fundamental changes in implantation: adhesion, migration, invasion, and extracellular matrix remodeling. These findings from hormonally treated ECC1 endometrial cancer cells were validated in human primary uterine epithelial cell-derived exosomes. Functionally, exosomes were internalized by human trophoblast cells and enhanced their adhesive capacity, a response mediated partially through active focal adhesion kinase (FAK) signaling. Thus, exosomes contribute to the endometrial-embryo interactions within the human uterine microenvironment essential for successful implantation.

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.

Immune tolerance at the fetomaternal interface must be established during the processes of implantation and pregnancy. Monoclonal nonspecific suppressor factor beta (MNSFβ) is a secreted protein that possesses antigen-nonspecific immune-suppressive function. It was previously reported that intrauterine immunoneutralization of MNSFβ significantly inhibited embryo implantation in mice. In the present study, MNSFβ protein expression was up- or downregulated by overexpression or RNA interference, respectively, in HCC-94 cells and the culture supernatants used to determine effects of MNSFβ on the secretion of IL-4 and TNFα from mouse lymphocytes as detected by ELISA. A coculture model of mouse embryos and endometrial stromal cells was also utilized to determine the effects of a specific anti-MNSFβ antibody on hatching and growth of embryos in vitro. The results show that MNSFβ induced secretion of IL-4 and inhibited secretion of TNFα from mouse lymphocytes. Following immunoneutralization of MNSFβ protein in the HCC-94 supernatant, the stimulatory effect of MNSFβ on IL-4 secretion from mouse lymphocytes was reduced, while the inhibitory effect on secretion of TNFα was abrogated. Expression of MNSFβ was detected in both embryonic and endometrial stromal cells, and its immunoneutralization inhibited the hatching and spreading of embryos in an in vitro coculture model. These results indicated that MNSFβ may play critical roles during the peri-implantation process by regulating cytokine secretion of lymphocytes and by mediating the crosstalk between embryonic cells and endometrial stromal cells.

Repair of the postmenstrual endometrium presents a unique opportunity to examine nonscarring repair in an adult tissue. We aimed to characterize and determine the importance of extracellular matrix (ECM) dynamics in cell migration during endometrial repair. Utilizing an in vivo mouse model of postmenstrual repair and an in vitro model of human endometrial re-epithelialization, we determined the dynamic changes in expression of ECM and related factors in both models by array analysis of repairing areas. We also validated expression of integrins, growth factors, protease inhibitors, basement membrane, and adhesion molecules in vitro and in both mouse and human repairing endometrium by quantitative RT-PCR and immunohistochemical studies. Finally, we determined the functional importance of integrin-fibronectin interactions and matrix metalloprotease (MMP)-facilitated cell movement during re-epithelialization and propose a model for cell locomotion during postmenstrual repair. These data demonstrated the dynamic expression and functional importance of ECM interactions in endometrial repair, which may be important for scar-free repair.

Over the past decade, high-throughput proteomics technologies have evolved considerably and have become increasingly more commonly applied to the investigation of female reproductive diseases. Proteomic approaches facilitate the identification of new disease biomarkers by comparing the abundance of hundreds of proteins simultaneously to find those specific to a particular clinical condition. Some of the best studied areas of female reproductive biology applying proteomics include gynaecological cancers, endometriosis and endometrial infertility. This review will discuss the progress that has been made in these areas and will highlight some of the emerging technologies that promise to contribute to better understanding of the female reproductive disease.

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.

Hormonal contraceptives are unsuitable for many women; thus, the development of new, nonhormonal contraceptives is of great interest. In women, uterine epithelial expression of interleukin 11 (IL11) and its receptor (IL11RA) suggests IL11 is critical for blastocyst attachment during implantation. Il11ra-deficient mice are infertile due to a defective decidualization response to the blastocyst, leading to total pregnancy loss. We examined the effect of administering a PEGylated IL11 antagonist, PEGIL11A (where PEG is polyethylene glycol), on pregnancy outcomes in mice and IL11 signaling in human endometrial epithelial cells (HES). PEGIL11A was detected in sera up to 72 h after intraperitoneal (IP) injection versus up to 2 h for the non-PEGylated antagonist. Following IP injection, PEGIL11A localized to uterine decidual cells and reduced immunoreactive cyclin D3 (IL11 decidual target). To inhibit IL11 action during early decidualization, PEGIL11A or control were administered IP on Days 3-6 (beginning just prior to maximal decidual Il11 expression). On Day 6, mesometrial decidualization was disturbed in PEGIL11A-treated animals with regions of hemorrhage visible in the mesometrial decidua. On Day 10, severe decidual destruction was visible: implantation sites contained significant hemorrhage, and the uterine luminal epithelium had reformed, suggesting a return to estrous cycling. These results demonstrate that PEGIL11A blocked IL11 action in the decidua during early decidualization, which totally abolished pregnancy and which is equivalent to the Il11ra(-/-) mouse. PEGIL11A significantly diminished STAT3 phosphorylation in HES cells in vitro (Por = 0.05). This study provides valuable information for PEGIL11A that could lead to the development of this protein as a nonhormonal contraceptive.

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.

Abnormal uterine bleeding can severely affect the quality of life for women. After menstruation, the endometrium must adequately repair to limit and stop bleeding. Abnormal uterine bleeding may result from incorrect or inadequate endometrial repair after menstruation. Previous studies have shown an important contribution of activin to skin wound healing, with severely delayed wound repair observed in animals transgenically induced to overexpress activin’s natural inhibitor, follistatin. Activin subunits have also been identified within human endometrium; however, their role in endometrial repair is unknown. We assessed the contribution of activin to endometrial repair after menses using a human in vitro cell wounding method and our well-characterized mouse model of endometrial breakdown and repair applied to mice overexpressing follistatin. Endometrial repair after menses is initiated with reepithelialization of the uterine surface. To mimic this repair, we utilized a human endometrial epithelial cell line (ECC-1) and demonstrated significant stimulation of wound closure after activin A administration, and attenuation of this response by addition of follistatin. Immunolocalization of activin subunits, βA and βB, in control endometrium from the mouse model demonstrated specific epithelial and stromal localization and some leukocyte staining (βA) around sites of endometrial repair, suggestive of a role for activin in this process. Follistatin-overexpressing animals had significantly higher circulating follistatin levels than wild-type littermates. There was a significant delay in endometrial repair after breakdown in follistatin transgenic animals compared with control animals. This study demonstrates for the first time a functional role for activin in endometrial repair after menses.

Many women using progestogen (P)-only contraceptives experience uterine bleeding problems. In clinical trials, a single low dose of mifepristone, given to Implanon users at the beginning of a bleeding episode reduced the number of bleeding days by ∼50% compared with controls. In this study, a single dose of mifepristone was administered to etonogestrel (ENG)-exposed pseudo-pregnant mice, 5 days after artificial decidualization was induced when the endometrium showed signs of bleeding. Control mice received vehicle alone. Mice were culled 12-, 18-, 24- and 48-h post-treatment. In the continued presence of ENG, a single dose of mifepristone stimulated tissue breakdown followed by very rapid repair: most treated tissues were fully restored to the pre-decidualized state by 48 h post-treatment. During repair, proliferating cells (Ki67 immunostained) were localized to a band of cells around the basal area in breaking down tissues and to the repairing luminal epithelium and glands. Progesterone receptor-positive cells were largely localized to the basal area of the breaking down tissue in treated mice compared with decidual cells in controls. Oestrogen receptor-positive cells were observed in the repairing luminal epithelium and glands compared with the decidua and the basal region in control tissues. It is concluded that mifepristone treatment stimulates rapid restoration of luminal epithelial integrity: such action may be a key event in reducing the number of bleeding days observed in women using Implanon who were treated with a single dose of mifepristone.