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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

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

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.

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.

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.

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.

Progestin-only contraceptives are associated with menstrual bleeding disturbances; a major reason why these agents are discontinued. The pathogenesis of such abnormal uterine bleeding associated with progestin-only contraceptives remains ill-defined. Matrix metalloproteinases (MMP)s and mast cells (MC)s are postulated to be involved in endometrial breakdown observed in normal menstruation. In this study comparisons were made of the immunolocalization of MMP-1 and -3 and MC in endometrium from women using Norplant or depot medroxyprogesterone acetate (DMPA) with normal controls. Positive MMP immunostaining was observed focally in stromal cells and adjacent extracellular matrix. Quantitative assessment revealed significantly higher MMP-1 immunostaining associated with the use of Norplant compared with DMPA or menstrual phase controls. Endometrial MMP-1 immunostaining in DMPA users was similar to that in menstrual controls. Positive MMP-3 immunolocalization was observed in a minority of endometrial samples. Activated MC, shown by the presence of extracellular MC tryptase, predominated in the endometrium of Norplant and DMPA users as also observed in menstrual phase controls. There was no correlation between MMP immunostaining, number of MC and number of bleeding days reported. These results indicate that in women using progestin-only contraceptives, endometrial MMP-1, -3 and MC demonstrate similarities to menstrual phase controls but also variation with different progestins.

Norplant, subdermally implanted slow-release levonorgestrel, is an effective and widely used contraceptive agent but has a high rate of discontinuation due to unacceptable abnormal uterine bleeding. Matrix metalloproteinases (MMPs) are expressed in normal cycling endometrium and are postulated to be responsible for the tissue breakdown at menstruation. We have compared the immunolocalization of MMP-9 and migratory cells in endometrium from Indonesian women using Norplant with normal controls. Positive MMP-9 immunostaining was observed intracellularly within stromal and intravascular leukocytes and extracellularly in areas of tissue lysis adjacent to these migratory cells. The MMP-9 positive cells were identified as neutrophils, eosinophils, CD3+ T-cells and macrophages. Quantitative assessment revealed that the number of MMP-9 positive cells, neutrophils and eosinophils were significantly increased in those endometrial biopsies from Norplant users displaying a shedding morphology and in normal controls at menstruation. There was no correlation between the number of MMP-9 positive cells and the number of bleeding days reported. Endometrial immunostaining for tissue inhibitor of metalloproteinases was similar in Norplant users and normal controls. These results suggest that MMP-9, an enzyme capable of degrading basement membrane components, may be involved in endometrial breakdown in women using Norplant.

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