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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.

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

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.

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.

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.

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.

High temperature requirement factor A3 (HtrA3), a member of the HtrA protease family, is highly expressed in the developing placenta, including the maternal decidual cells in both mice and humans. In this study we deleted the HtrA3 gene in the mouse and crossed females carrying zero, one, or two HtrA3-expressing alleles with HtrA3+/− males to investigate the role of maternal vs fetal HtrA3 in placentation. Although HtrA3−/− mice were phenotypically normal and fertile, HtrA3 deletion in the mother resulted in intra-uterine growth restriction (IUGR). Disorganization of labyrinthine fetal capillaries was the major placental defect when HtrA3 was absent. The IUGR caused by maternal HtrA3 deletion, albeit being mild, significantly altered offspring growth trajectory long after birth. By 8 months of age, mice born to HtrA3-deficient mothers, independent of their own genotype, were significantly heavier and contained a larger mass of white fat. We further demonstrated that in women serum levels of HtrA3 during early pregnancy were significantly lower in IUGR pregnancies, establishing an association between lower HtrA3 levels and placental insufficiency in the human. This study thus revealed the importance of maternal HtrA3 in optimizing placental development and its long-term impact on the offspring well beyond in utero growth.

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.

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.

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.

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.

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.

Embryo implantation is a complex process involving blastocyst attachment to the endometrial epithelium and subsequent trophoblast invasion of the decidua. We have previously shown that the chemokines CX3CL1 and CCL14 are abundant in endometrial vasculature, epithelial, and decidual cells at this time, and that their receptors, CX3CR1 and CCR1, are present on invading human trophoblasts. CX3CL1 and CCL14 promote trophoblast migration. We hypothesized that these endometrial chemokines promote trophoblast migration by regulating adhesion molecules and extracellular matrix (ECM) components on the trophoblast, similar to mechanisms used in leukocyte trafficking. Trophoblast cells (AC1M-88) used previously showed a marked increase in adhesion to fibronectin following treatment with CX3CL1 and CCL14. Alterations in trophoblast adhesion and ECM following chemokine stimulation were examined using pathway-specific oligo-arrays and quantitative real-time RT-PCR. More than 30 genes were affected by CX3CL1 treatment, and 15 genes were found to be regulated by CCL14 treatment. Real-time RT-PCR quantitation revealed significant changes in the mRNA transcripts of alpha-catenin (CTNNA1), extracellular matrix protein 1 (ECM1), osteopontin (SPP1), integrin alpha 6 (ITGA6), matrix metalloproteinase 12 (MMP12), and integrin beta 5 (ITGB5) following chemokine treatment. Several of these genes have previously been implicated in implantation. Immunohistochemistry confirmed the presence of integrin alpha 6 and SPP1 protein in first-trimester human implantation sites. The temporal and spatial expression of chemokines, their receptors, adhesion, and ECM at the maternal-fetal interface emphasizes an important role in the controlled directional migration of trophoblasts through the maternal decidua. For the first time, this study demonstrates the direct effects of CX3CL1 and CCL14 on trophoblast adhesion molecules and ECM, suggesting mechanisms by which trophoblast cells migrate during early pregnancy.

BACKGROUND Successful blastocyst implantation requires the differentiation of human endometrial stromal cells (HESC), a process known as decidualization. Activin A, a transforming growth factor beta (TGFbeta) superfamily member, enhances HESC decidualization and localizes to decidual cells in human endometrium. Other TGFbeta superfamily members, including BMP2, BMP4, BMP7, GDF5, GDF8, GDF11, TGFbetas and Nodal, may also play a role during decidualization. This study aimed to identify these TGFbeta family members in human endometrium, and to determine whether they are involved in human decidualization. METHODS Protein localization of TGFbeta family members was examined in secretory phase human endometrium and first trimester decidua by immunohistochemistry. mRNA expression was examined in HESC. Activin inhibitors (Activin-M108A/SB431542) with differing specificities for the other TGFbeta members under consideration were applied during HESC decidualization in vitro. The secretion levels of potential TGFbeta superfamily members were measured during decidualization, and recombinant proteins added to examine their effect. RESULTS This study has identified BMP2, BMP4, BMP7, GDF5, GDF8 and GDF11 but not Nodal in secretory phase human endometrium, but only BMP2, GDF5 and TGFbeta1 protein were detected in decidual cells. All ligands except Nodal were expressed by cultured HESC. Both inhibitors significantly reduced decidualization validating the role of activin, but potentially also other TGFbeta members, during decidualization. BMP2 and TGFbeta1 secretion increased during HESC decidualisation and exogenous administration of these proteins significantly enhanced decidualization in vitro. CONCLUSIONS Like activin, BMP2 and TGFbeta1 are likely to be involved in HESC decidualization. This is the first study to identify and localize BMP4, BMP7, GDF5, GDF8 and GDF11 in secretory phase human endometrium. Understanding the factors critical for the implantation process is needed for improving fertility and pregnancy outcomes.

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.

Pro-protein convertases (PCs) are a family of serine proteases (furin, PC1/3, PC2, PACE4, PC4, PC5/6, PC7/8) responsible for post-translational processing and activation of inactive precursors of many regulatory proteins. Endometrial PC6 is critical for implantation in mice and for decidualization of human endometrial stromal cells (ESCs). This study investigated the endometrial expression of other PCs during the menstrual cycle and early pregnancy to elucidate potential redundancies. Furin, PC4, PACE4, and PC7 along with PC6 transcripts were detected in total endometrial RNA, whereas PC1 and PC2 transcription levels were negligible. Quantitative RT-PCR demonstrated highest levels of furin mRNA during menstruation and lowest levels during the proliferative phase. Furin protein was immunolocalized in endometrial luminal and glandular epithelia, stromal fibroblasts, endothelia, and leukocytes. PACE4 and PC7 proteins were also immunodetected in endometrial stroma and glands. Total furin, PC7, and PACE4 proteins were constitutive in both stromal and glandular compartments throughout the cycle and during first trimester pregnancy. Furthermore, Furin and PC7 transcription was unaltered during decidualization of ESCsin vitroin contrast to PC6 which is significantly up-regulated during decidualization. Thus, whereas PC6 is tightly regulated during endometrial preparation for implantation, furin, PACE4, and PC7 are constitutively expressed in human endometrium, but must be considered if PC6 is to be targeted for manipulation of fertility.

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.

The endometrium (the mucosal lining of the uterus) is a dynamic tissue that undergoes extensive remodeling, secretory transformation in preparation for implantation of an embryo, inflammatory and proteolytic activity during menstruation, and rapid postmenstrual repair. A plethora of local factors influence these processes. Recently, a cysteine-rich protein, CRISP3, a clade of the CRISP, antigen 5, pathogenesis-related (CAP) protein superfamily, has been implicated in uterine function. The localization, regulation, and potential function of CRISP3 in both the human and mouse endometrium is described. CRISP3 localizes to the luminal and glandular epithelium of the endometrium within both species, with increased immunoreactivity during the proliferative phase of the human cycle. CRISP3 also localizes to neutrophils, particularly within the premenstrual human endometrium and during the postbreakdown repair phase of a mouse model of endometrial breakdown and repair. Endometrial CRISP3 is produced by primary human endometrial epithelial cells and secreted in vivo to accumulate in the uterine cavity. Secreted CRISP3 is more abundant in uterine lavage fluid during the proliferative phase of the menstrual cycle. Human endometrial epithelial CRISP3 is present in both a glycosylated and a nonglycosylated form in vitro and in vivo. Treatment of endometrial epithelial cells in vitro with recombinant CRISP3 enhances both adhesion and proliferation. These data suggest roles for epithelial and neutrophil-derived CRISP3 in postmenstrual endometrial repair and regeneration.

Maternal-fetal communications are critical for the establishment of pregnancy. Embryonic growth and differentiation factors produced by the oviduct and uterus play essential roles during the pre- and early post-implantation phases. Although several studies indicate roles for activin in embryonic development, gene-knockout studies have failed to identify a critical role in mammalian embryogenesis. We hypothesized that activin is produced by maternal tissues during the establishment of pregnancy, and thus maternally derived activin could compensate for the absence of embryonic activin in null homozygotes during critical developmental stages. We investigated the expression of inhibin alpha, activin betaA, and betaB subunits in the mouse oviduct and uterus during the estrous cycle and early pregnancy, and in the early conceptus. Inhibin alpha subunit was weakly expressed, while activin betaA and betaB subunits were strongly expressed in oviduct and uterus at estrous, and dramatically upregulated in the uterus on each day of pregnancy between days 3.5 and 8.5 post coitum. Prior to implantation, activin betaA and betaB subunits were immunolocalized to oviductal and uterine epithelial cells; following implantation they were expressed in the stroma, in a wave preceding decidualization. Later in pregnancy, activin betaA and betaB subunits were present in decidua basalis, trophoblast giant cells, and labyrinth zone of the developing placenta. Expression of activin betaA subunit was also detected in blastocysts and early post-implantation embryos. These data are consistent with a role for maternally derived activins in the support of the pre-implantation embryo, and during gastrulation and embryogenesis.

Transforming growth factor β (TGFβ) superfamily members are closely associated with tissue remodelling events and reproductive processes. This review summarises the current state of knowledge regarding the expression and actions of TGFβ superfamily members in the uterus, during the menstrual cycle and establishment of pregnancy. TGFβs and activin β subunits are abundantly expressed in the endometrium, where roles in preparation events for implantation have been delineated, particularly in promoting decidualisation of endometrial stroma. These growth factors are also expressed by epithelial glands and secreted into uterine fluid, where interactions with preimplantation embryos are anticipated. Knockout models and embryo culture experiments implicate activins, TGFβs, nodal and bone morphogenetic proteins (BMPs) in promoting pre- and post-implantation embryo development. TGFβ superfamily members may therefore be important in the maternal support of embryo development. Following implantation, invasion of the decidua by fetal trophoblasts is tightly modulated. Activin promotes, whilst TGFβ and macrophage inhibitory cytokine-1 (MIC-1) inhibit, trophoblast migration in vitro, suggesting the relative balance of TGFβ superfamily members participate in modulating the extent of decidual invasion. Activins and TGFβs have similar opposing actions in regulating placental hormone production. Inhibins and activins are produced by the placenta throughout pregnancy, and have explored as a potential markers in maternal serum for pregnancy and placental pathologies, including miscarriage, Down’s syndrome and pre-eclampsia. Finally, additional roles in immunomodulation at the materno-fetal interface, and in endometrial inflammatory events associated with menstruation and repair, are discussed.

Human embryo implantation is a complex process involving blastocyst attachment to the endometrial epithelium and subsequent trophoblast invasion of the decidua. Chemokines, critical regulators of leukocyte migration, are abundant in endometrial epithelial and decidual cells at this time. We hypothesized that endometrial chemokines stimulate trophoblast invasion. Chemokine receptors CX3CR1 and CCR1 were immunolocalized in human first-trimester implantation sites, specifically to endovascular extravillous trophoblasts, but not to the invading interstitial EVTs (iEVTs), with weak staining also on syncytium. CCR3 was localized to invading iEVTs and to microvilli on the syncytial surface. Expression of CX3CL1 (fractalkine), CCL7 (MCP-3), and their receptors (CX3CR1, CCR1, CCR2, CCR3, and CCR5) mRNA was examined in cellular components of the maternal-embryonic interface by RT-PCR. Both chemokines were abundant in entire endometrium and placenta, endometrial cells (primary cultures and HES, a human endometrial epithelial cell line) and trophoblast cell lines (JEG-3, ACIM-88, and ACIM-32). Chemokine receptor mRNA was expressed by placenta and trophoblast cell lines: CCR1 by all trophoblast cell types, whereas CCR2, CCR3, and CX3CR1 were more variable. CX3CR1, CCR1, CCR2, and CCR5 were also expressed by endometrial cells. Migration assays used the trophoblast cell line most closely resembling extravillous cytotrophoblast (AC1M-88). Trophoblast migration occurred in response to CX3CL1, CCL14, and CCL4, but not CCL7. Endometrial cell-conditioned media also stimulated trophoblast migration; this was attenuated by neutralizing antibodies to CX3CL1 and CCL4. Thus, chemokines are expressed by maternal and embryonic cells during implantation, whereas corresponding receptors are on trophoblast cells. Promotion of trophoblast migration by chemokines and endometrial cell conditioned medium indicates an important involvement of chemokines in maternal-fetal communication.

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.

HTRA3 is a newly identified serine peptidase of the mammalian HTRA (high-temperature requirement factor A) family, that is upregulated dramatically during mouse placental development. The current study determined whether HTRA3 was involved in human placentation. During the menstrual cycle, HTRA3 was expressed primarily in the endometrial glands, being significantly upregulated toward the mid- to late secretory phases; prominent expression in the stroma detected only in the decidual cells in the late secretory phase. Thus, overall endometrial HTRA3 expression was highest in the late secretory phase, when the endometrium is prepared for maternal-trophoblast interaction. During the first trimester of pregnancy, both glandular and decidual HTRA3 expression increased further with the decidual upregulation being highly significant. The strong link between HTRA3 expression and endometrial stromal cell decidualization was further established in an in vitro model using primary endometrial stromal cells. HTRA3 was also expressed by certain trophoblast subtypes in the first-trimester placenta: strongly in the villous syncytiotrophoblast, trophoblast shell, and endovascular trophoblast and weakly in the distal portion of the trophoblast cell columns but not in villous cytotrophoblast, the proximal region of the cell columns, or interstitial trophoblast. Upregulation of HTRA3 expression in association with placental development was revealed by a significant elevation of this protein in the maternal serum during the first trimester. We thus propose that HTRA3 is a previously unrecognized factor closely associated with and potentially important for human placentation. This study established crucial groundwork for future investigations toward establishing the physiological roles of HTRA3 in human placentation.

Considerable correlative evidence suggests an important role for matrix metalloproteinases (MMPs) in menstruation, a process which occurs naturally in very few species. In this study, MMP expression was examined in a mouse model of endometrial breakdown and repair and the functional importance of MMPs determined. In the model, progesterone support was withdrawn from mice in which endometrial decidualization had been induced; 24 h later, endometrial breakdown was complete, and the entire decidual zone had been shed. Re-epithelialization had occurred by 36 h, and the endometrium had undergone extensive restoration toward a predecidualized state by 48 h. Immunoreactive MMP9 and MMP7 colocalized with leukocyte subsets, particularly neutrophils, whereas MMP13 staining was always extracellular. MMP3 and MMP7 were abundant during re-epithelialization in close proximity to newly reforming epithelium. The functional importance of MMPs in these processes was examined using two MMP inhibitors, doxycycline and batimistat. Both inhibitors effectively reduced MMP activity, as assessed by in situ zymography, but did not have significant effects on endometrial breakdown or repair. This study demonstrates that although MMPs are present in abundance during endometrial breakdown and repair in this mouse model, they are not the key mediators of these processes.

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.

Successful embryo implantation involves complex interactions between the embryo and the uterus and is critical in establishing pregnancy. Proprotein convertase (PC) 6 (PC6) is one of the PC endoproteases regulating protein function through posttranslational activation of precursor proteins, including growth and differentiation factors. Here we show that PC6 protein is induced in the uterine stromal cells specifically at the site of embryo attachment during early pregnancy in mice. In vivo blocking of uterine production of PC6 protein using morpholino antisense oligonucleotides in mice resulted in total inhibition of implantation, revealing a vital role for PC6 in modulating the uterus for embryo implantation. Studies in primates (rhesus monkey and human) showed a dramatic upregulation of endometrial PC6 during the phase of uterine receptivity and at implantation, particularly during a critical uterine cell differentiation process termed decidualization. Thus, the current studies have demonstrated that PC6 is an essential molecule in modulating uterine function to support the establishment of embryo implantation. Interestingly, PC6 is one of the PCs identified to be important in processing the coat protein of HIV; inhibition of PCs has been suggested to be an effective approach to reduce HIV transmission. We therefore propose the novel concept that PC6 could be a potential nonhormonal target in the female reproductive tract for dual protection for women, both in preventing pregnancy and reducing HIV infection.

The endometrium is hostile to embryo implantation except during the ‘window of receptivity’. A change in endometrial gene expression is required for the development of receptivity. Calbindin-d9k (CaBP-d9k) and calbindin-d28k (CaBP-d28k) are proteins possessing EF-hand motifs which have high affinity for Ca2+ions. Previously, it has been demonstrated that, in mouse endometrium, the expression of both calbindins is highly regulated during implantation and that both proteins play critical but functionally redundant roles at implantation. This study was the first to determine the expression of these two calbindins in the human and rhesus monkey endometrium. Initial RT-PCR analysis demonstrated that CaBP-d28k but not CaBP-d9k mRNA expression is detectable in the endometrium of both species. Western blot analysis confirmed the presence of immuno-reactive CaBP-d28k protein in the primate endometrium. Furthermore, the endometrial expression pattern of CaBP-d28k mRNA and protein was examined by Northern blot analysis and immunohistochemistry respectively in both species across the menstrual cycle and during early pregnancy. Semi-quantitative statistical analysis of the immunohistochemistry results revealed that, in the human, CaBP-d28k protein expression was maximal in luminal and glandular epithelium during the mid-secretory phase, coinciding with the time when the endometrium is receptive to embryo implantation. Expression in rhesus monkey showed a similar trend. These results suggest that, in the primate endometrium, only CaBP-d28k is expressed and that the specific regulation of this calbindin is potentially important for the establishment of uterine receptivity.

Successful ovulation and implantation processes play a crucial role in female fertility. Adamts-1, a matrix metalloproteinase with disintegrin and thrombospondin motifs, has been suggested to be regulated by the progesterone receptor in the hormonal pathway leading to ovulation. With the primary aim of investigating the role of Adamts-1 in female fertility, we generated Adamts-1 null mice. Forty-five percent of the newborn Adamts-1 null mice die, with death most likely caused by a kidney malformation that becomes apparent at birth. Surviving female null mice were subfertile, whereas males reproduced normally. Ovulation in null females was impaired because of mature oocytes remaining trapped in ovarian follicles. No uterine phenotype was apparent in Adamts-1 null animals. Embryo implantation occurred normally, the uteri were capable of undergoing decidualization, and no morphological changes were observed. These results demonstrate that a functional Adamts-1 is required for normal ovulation to occur, and hence the Adamts-1 gene plays an important role in female fertility, primarily during the tissue remodeling process of ovulation.

Menstruation and endometrial regeneration occur during every normal reproductive cycle in women and some Old World primates. Many of the cellular and molecular events of menstruation have been identified by correlative or in vitro studies, but the lack of a convenient model for menstruation in a laboratory animal has restricted functional studies. In this study, a mouse model for menstruation first described by Finn in the 1980s has been modified for use in a commonly used inbred strain of mouse. A decidual stimulus was applied into the uterine lumen of appropriately primed mice and leukocyte numbers and apoptosis were examined over time following progesterone withdrawal. Endometrial tissue breakdown was initiated after 12-16 h, and by 24 h, the entire decidual zone had been shed. Re-epithelialization was nearly complete by 36 h and the endometrium was fully restored by 48 h. Leukocyte numbers increased significantly in the basal zone by 12 h after progesterone withdrawal, preceding stromal destruction. Stromal apoptosis was detected by TUNEL staining at 0 and 12 h but decreased by 16 h after progesterone withdrawal. This mouse model thus mimics many of the events of human menstruation and has the potential to assist in elucidation of the functional roles of a variety of factors thought to be important in both menstruation and endometrial repair.

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.

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.

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.

The relationships between activities of matrix metalloproteinases (MMPs) in the canine uterus and the occurrence of degeneration of the luminal epithelium, cystic endometrial hyperplasia, pyometra and uterine remodelling post partum were determined. Mature bitches (n = 10) were ovariectomized, treated with hormones (oestradiol benzoate, progestagen) and investigated at stages simulating pro-oestrus (n = 2), oestrus (n = 2), dioestrus (n = 2), and mid- (n = 2) and late (n = 2) anoestrus (3 and 9 weeks, respectively, after cessation of treatment with progestagen). Untreated bitches (n = 1 per group) served as controls (Expt 1). An additional 10 ovariectomized bitches, at the end of treatment-induced simulated dioestrus, were treated each day for a further 3 weeks either with the same dose (standard dose, n = 3), a decreased dose (n = 3) or an increased dose (n = 3) of progestagen, or no treatment (withdrawal dose, n = 1). These bitches were then investigated (Expt 2). A suture was placed in the lumen of one uterine horn of another five bitches at ovariectomy. Three of these bitches were treated to induce simulated dioestrus and two bitches served as untreated controls. In the hormone-treated bitches, the suture resulted in cystic endometrial hyperplasia in one bitch and in cystic endometrial hyperplasia with pyometra in two bitches. The control bitches showed no cystic endometrial hyperplasia or pyometra (Expt 3). Four intact bitches were studied at 2 (n = 1), 3 (n = 2) and 11 (n = 1) weeks post partum. Uterine tissues were also collected from two bitches with naturally occurring cystic endometrial hyperplasia with pyometra (Expt 4). All uteri were examined histologically and the activities of MMP-2, -7 and -9 (latent and active forms) were assessed using zymography of extracts of endometrium. In Expts 1 and 2, marked degeneration of the luminal epithelium in six of 25 bitches (simulated mid-anoestrus, withdrawal dose and decreased dose groups) was not associated with changes in MMP activities. Markedly increased activities of MMP-2 (active form), -7 (latent form) and -9 (active and latent forms) were observed in the bitches with cystic endometrial hyperplasia with pyometra (but not with cystic endometrial hyperplasia alone) and in the bitches at 2 and 3 weeks post partum (but not at 11 weeks post partum). These results indicate that MMPs are not involved with degeneration of the luminal epithelium, but are involved with uterine remodelling in the postpartum bitch and with cystic endometrial hyperplasia with pyometra.

BACKGROUND: Classical studies in monkeys suggested that menstruation results from vasoconstriction, hypoxia and necrosis. The heterodimeric hypoxia-inducible factor (HIF) complex is critical in oxygen homeostasis via increased stability of HIF-1α/2α monomers, and these act as markers of hypoxia. We hypothesized that focal hypoxia in perimenstrual endometrium results in locally increased matrix metalloproteinases (MMP), leading to tissue destruction. METHODS: HIF-1α, HIF-2α and HIF-1β were immunolocalized in cycling endometrium. Endometrial stromal cells were cultured under hypoxic and normoxic conditions and MMP measured by zymography and Western blots. RESULTS: HIF-1α and HIF-2α were detected in only some endometrial samples, and not confined to the perimenstrual tissue. Where present, they were primarily cytoplasmic, not nuclear. HIF-1β was localized in epithelium, leukocytes and some decidual cells. Cultured endometrial stromal cells responded to hypoxia with increased cellular HIF-1α and secreted vascular endothelial growth factor. ProMMP-1 and proMMP-3 production was reduced in response to hypoxia regardless of the steroidal milieu (no added steroids, estrogen or estrogen plus progesterone). Active MMP-2 and membrane type 1 MMP but not proMMP-2 or proMMP-9 production were also inhibited by hypoxia. CONCLUSIONS: These results do not support a role for hypoxia in the focally increased production and activation of MMP observed prior to and during menstruation.

The mammalian uterus is normally not receptive to embryo implantation except during the very limited ‘window of implantation’. To identify genes that may be responsible for this phenomenon the technique of RNA differential display (DD-PCR) was applied to implantation and inter-implantation sites on day 4.5 of pregnancy in the mouse, the time at which the blastocyst becomes attached to the endometrium. Three of these genes were identified as splicing factor SC35, calbindin-D9k and monoclonal non-specific suppressor factorβ (MNSFβ). Expression of SC35 mRNA, which is responsible for removal of introns from pre-mRNA, is much higher in implantation than in interimplantation sites during pregnancy. Expression of alternatively spliced mRNAs for SC35 is differentially regulated by early pregnancy and steroid hormones. By contrast, calbindin-D9k, a regulator of calcium, is upregulated by progesterone and its mRNA increases in the uterus during early pregnancy compared with during the cycle, although it is significantly lower in implantation sites than in interimplantation sites on days 4.5–5.5 of pregnancy, but subsequently becomes barely detectable in both sites. The mRNA for calbindin-D9k is predominantly in endometrial luminal epithelium. MNSFβ, a cytokine involved in regulation of the immune system, showed lower expression at implantation sites than interimplantation sites on day 4.5 of pregnancy, when embryos first attach to the uterus and initiate implantation, and on day 5.5 when implantation has advanced. Immunohistochemically, the protein was localized to endometrial stromal cells in the non-pregnant uterus, but disappeared as decidualization progressed. The precise roles of these three proteins in the process of embryo implantation remains to be determined. Homologues of the proteins may contribute to the development of the ‘window of implantation’ in the human and hence be appropriate targets for new post-coital contraceptives or may be manipulated to improve fertility.

Menstruation involves the shedding of the functional layer of the endometrium in the absence of pregnancy. At sites where tissue shedding is complete, re-epithelialization of the tissue is essential for repair and termination of bleeding. The complement of growth factors that mediate post-menstrual endometrial repair are yet to be completely elucidated. Galectins regulate many cell functions important for post-menstrual repair, such as cell adhesion and migration. Galectin-7 has a well characterized role in re-epithelialization and wound healing. We hypothesized that galectin-7 would be important in re-epithelialization during post-menstrual repair. We aimed to identify endometrial expression of galectin-7 in women undergoing normal endometrial repair and in women with amenorrhoea who do not experience endometrial breakdown and repair, and to determine whether galectin-7 enhances endometrial re-epithelialization in vitro. Galectin-7 immunolocalized to the endometrial luminal and glandular epithelium during the late secretory and menstrual phases, and to decidualized stroma in regions exhibiting tissue breakdown. Immunostaining intensity was significantly reduced in the endometrium of women with amenorrhoea compared with normally cycling woman. ELISA identified galectin-7 in menstrual fluid at significantly elevated levels compared with matched peripheral plasma. Exogenous galectin-7 (2.5 µg/ml) significantly enhanced endometrial epithelial wound repair in vitro; this was abrogated by inhibition of integrin binding. Galectin-7 elevated epithelial expression of extracellular matrix-related molecules likely involved in repair including β-catenin, contactin and TGF-β1. In conclusion, galectin-7 is produced by the premenstrual and menstrual endometrium, where it accumulates in menstrual fluid and likely acts as a paracrine factor to facilitate post-menstrual endometrial re-epithelialization.

Significant numbers of neutrophils are found extravascularly within the endometrium only during the immediate premenstrual and menstrual phases of the cycle. In this study we investigated the effect of neutrophil products on the synthesis and activation of matrix metalloproteinases (MMP), enzymes considered to play a crucial role in the degradation of endometrial tissue that occurs at menstruation. Latent MMP-2, MMP-3 and MMP-9 released by endometrial stromal fibroblasts and peripheral blood neutrophils were activated when the two cell types were cultured together. Tissue inhibitors of metalloproteinases (TIMP) 1 and 2 were also degraded in this system. Neutralization studies identified a role for the serine protease, elastase, in the observed activation of MMP. Although cultured endometrial neutrophils behaved similarly to peripheral blood neutrophils in their ability to release latent MMP-9 and elastase, no active forms of MMP-2. MMP-3 and MMP-9 were detected in supernatant from co-cultures containing endometrial neutrophils and stromal fibroblasts. This appeared to be due to an alteration in the neutrophil production of elastase and inhibitors. e.g. alpha1-antitrypsin, in these cultures so that active elastase was not available. Our results demonstrate that any involvement of neutrophils in the tissue destruction occurring at menstruation may be tightly regulated by the focal concentration of degradative enzymes and their respective inhibitors.

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.