Detrimental actions of obesity-associated advanced glycation end-products on endometrial epithelial cell proliferation are alleviated by antioxidants.

RESEARCH QUESTION: Advanced glycation end-products (AGE) are elevated in the uterine environment of obese infertile women. Can the detrimental effects of AGE on endometrial epithelial cells be mitigated with therapeutics, and recapitulated in a more physiologically relevant primary model (organoids)? DESIGN: Human endometrial epithelial cells (ECC-1) were exposed to AGE at concentrations physiologically representative of uterine fluid in lean or obese individuals, and three potential therapeutics: 25 nmol/l receptor for AGE (RAGE) antagonist FPS-ZM1, 100 µmol/l metformin, or a combination of antioxidants (10 µmol/l N-acetyl-l-cysteine, 10 µmol/l N-acetyl-l-carnitine and 5 µmol/l α-lipoic acid). Real-time cell analysis (xCELLigence, ACEA Biosciences) determined the rate of adhesion and proliferation. The proliferation of organoid-derived cells and secretion of cytokines from organoids was characterized in the presence of AGE (n = 5). The uterine fluid of women undergoing assisted reproduction was profiled for AGE-associated inflammatory markers (n = 77). RESULTS: ECC-1 proliferation was reduced by AGE from obese versus lean conditions and vehicle control (P = 0.04 and P < 0.001, respectively), and restored to a proliferation corresponding to lean conditions by antioxidants. AGE influenced organoid derived primary endometrial epithelial cell proliferation in a donor-dependent manner. AGE increased the organoid secretion of the proinflammatory cytokine CXCL16 (P = 0.006). Clinically, CXCL16 correlated positively to maternal body mass index (R = 0.264, P = 0.021) and intrauterine glucose concentration (R = 0.736, P < 0.0001). CONCLUSIONS: Physiologically relevant concentrations of AGE alter endometrial epithelial cell function. Antioxidants restore the rate of proliferation of AGE-treated endometrial epithelial (ECC-1) cells. Primary endometrial epithelial cells, cultured as organoids, demonstrate altered proliferation and CXCL16 secretion in the presence of AGE equimolar with the uterine fluid from obese individuals.

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

Introduction: A healthy pregnancy requires successful blastocyst implantation into an adequately prepared or 'receptive' endometrium. Decidualization of uterine endometrial stromal fibroblast cells (hESF) is critical for the establishment of a healthy pregnancy. microRNAs (miRs) are critical regulators of cellular function that can be released by a donor cell to influence the physiological state of recipient cells. We aimed to determine how decidualization affects hESF miR release and investigated the function of one decidualization regulated miR, miR-19b-3p, previously shown to be associated with recurrent pregnancy loss. Method: miR release by hESF was determined by miR microarray on culture media from hESF decidualized in vitro for 3 and 14 days by treatment with oestradiol and medroxyprogesterone acetate. Cellular and whole endometrial/decidual tissue miR expression was quantified by qPCR and localized by in situ hybridization. The function of miR-19b-3p in HTR8/Svneo trophoblast cells was investigated using real time cell analysis (xCELLigence) and gene expression qPCR. Results: From our miR screen we found that essentially all hESF miR release was reduced following in vitro decidualization, significantly so for miR-17-5p, miR-21-3p, miR-34c-3p, miR-106b-5p, miR-138-5p, miR-296-5p, miR-323a-3p, miR-342-3p, miR-491-5p, miR-503-5p and miR-542-5p. qPCR demonstrated that miR-19b-3p, 181a-2-3p and miR-409-5p likewise showed a significant reduction in culture media following decidualization but no change was found in cellular miR expression following decidualization. In situ hybridization localized miR-19b-3p to epithelial and stromal cells in the endometrium and qPCR identified that miR-19b-3p was significantly elevated in the cycling endometrium of patients with a history of early pregnancy loss compared to normally fertile controls. Functionally, overexpression of miR-19b-3p significantly reduced HTR8/Svneo trophoblast proliferation and increased HOXA9 expression. Discussion: Our data demonstrates that decidualization represses miR release by hESFs and overexpression of miR-19b-3p was found in endometrial tissue from patients with a history of early pregnancy loss. miR-19b-3p impaired HTR8/Svneo proliferation implying a role in trophoblast function. Overall we speculate that miR release by hESF may regulate other cell types within the decidua and that appropriate release of miRs by decidualized hESF is essential for healthy implantation and placentation.

Assessment of potential biomarkers of pre-receptive and receptive endometrium in uterine fluid and a functional evaluation of the potential role of CSF3 in fertility.

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

Idiopathic infertility in women is associated with distinct changes in proliferative phase uterine fluid proteins.

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.

Soluble Delta-like ligand 1 alters human endometrial epithelial cell adhesive capacity.

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

The proliferative phase underpins endometrial development: Altered cytokine profiles in uterine lavage fluid of women with idiopathic infertility.

Endometrial gland development occurs during the proliferative phase of a woman's menstrual cycle, laying the foundation for the subsequent receptive, secretory phase when pregnancy is established. Idiopathic infertility has been rarely investigated with respect to the proliferative phase endometrium. We investigated whether gland development and/or altered secretion of cytokines during the proliferative phase is associated with infertility. Area of the glandular epithelium (GE) was measured in proliferative phase endometrial tissue collected from fertile (n=18) and infertile (n=14) women. Cytokines were measured in proliferative phase uterine lavage of fertile (n=15) and infertile (n=15) women. Immunohistochemistry determined cellular localisation of transforming growth factor alpha (TGFα) and interferon gamma (IFNγ) in proliferative phase endometrial tissue. For statistical analysis the cohort was divided into women <35years and ⩾35years. There were no significant differences in GE area of infertile and fertile women. C-C motif chemokine 11 (P=0.048), TGFα (P=0.049), IFNγ (P=0.033) and interleukin-1 alpha (P=0.047) were significantly elevated in uterine lavage from infertile women <35years compared to fertile but not in women ⩾35years. TGFα and IFNγ localised predominantly to GE in both the fertile and infertile endometrium. The potential impact of this altered proliferative phase environment on subsequent receptivity is discussed.

The Microenvironment of Human Implantation: Determinant of Reproductive Success.

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.

Development of a high-throughput assay for human proprotein convertase 5/6 for detecting uterine receptivity.

Embryo implantation requires a healthy embryo and a receptive uterus. In women, the inner lining of the uterus, the endometrium, remains in a hostile state and becomes receptive for embryo implantation for only a short period during each menstrual cycle. Determining endometrial receptivity is vital in in vitro fertilization (IVF) treatment because the timing of embryo transfer needs to be synchronized with endometrial receptivity. We have previously demonstrated that proprotein convertase 5/6A (PC6) is highly expressed in the receptive endometrium and that PC6 is critical for receptivity establishment in women. Furthermore, endometrial PC6 is secreted into the uterine fluid, and levels correlate with receptivity status. Detection of PC6 in uterine fluids, therefore, would provide a nonsurgical assessment of endometrial receptivity. However, to date no assays are available for human PC6. In this study, we produced three PC6 monoclonal antibodies (mAbs) and developed a sandwich enzyme-linked immunosorbent assay (ELISA) for PC6 detection in human uterine fluids. The PC6 mAbs were confirmed to be highly specific to PC6, and the ELISA detected PC6 in human uterine fluids with a significantly higher level during the receptive phase. This newly established PC6 ELISA provides an important tool in the development of noninvasive strategies to detect endometrial receptivity in women.

Fresh versus frozen embryo transfer: backing clinical decisions with scientific and clinical evidence.

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.

Assessing receptivity in the endometrium: the need for a rapid, non-invasive test.

Successful implantation of an embryo into the uterus requires synchrony between the blastocyst and the endometrium. Endometrial preparedness, or receptivity, occurs only for a very short time during the mid-secretory phase of the menstrual cycle in fertile women. Failure to achieve receptivity results in infertility and is a rate-limiting step for IVF success. Frozen embryo transfer in non-stimulation cycles is already improving live birth rates. However, an important tool that is missing in the armoury of reproductive specialists is a means to rapidly assess endometrial receptivity, either during initial assessment or immediately prior to embryo transfer. The development of a wealth of omics technologies now opens the way for identifying potential receptivity markers, although validation of these is still a major issue. This review assesses the current state of the field and the requirements to proceed to a valid clinical test.

Proteomics of the human endometrium and uterine fluid: a pathway to biomarker discovery.

Failure of the endometrium to achieve receptivity results in infertility, and it is also a rate-limiting step in in vitro fertilization (IVF) success. The microenvironments provided by the endometrium during the receptive phase and that support implantation are highly complex and constantly changing as implantation progresses. Although a number of gene array studies have defined mRNA changes across the cycle, with infertility, and in IVF cycles, these have not generally been informative due in part to the subsequent regulation of transcription and posttranslational modifications of the proteins. State-of-the-art proteomic technologies now enable analysis of changes in the endometrium and its secretome related to cycle phase and associated with infertility. These techniques include two-dimensional differential in-gel electrophoresis, isobaric tags for relative and absolute quantitation, and multiplex analyses of selected panels of markers. Subsequent definition of cellular location, timing of production of identified proteins, and their regulation by steroid hormones and blastocyst-derived factors provide indications of their functions and their relationship to the establishment of pregnancy. Proteins discovered by proteomic analyses and fully evaluated will provide the differentiative profiles necessary to inform clinical practice and serve as an end point for optimizing stimulation cycles in IVF clinics as well as more clearly defining the molecular mechanisms underlying successful implantation.

Evaluation of midkine and anterior gradient 2 in a multimarker panel for the detection of ovarian cancer.

The aims of this study were: to characterise and compare plasma concentrations of midkine (MDK) in normal healthy women with concentrations observed in women with ovarian cancer; and to establish and compare the performance of MDK with that of anterior gradient 2 protein (AGR2) and CA125 in the development of multi-analyte classification algorithms for ovarian cancer. Median plasma concentrations of immunoreactive MDK, AGR2 and CA125 were significantly greater in the case cohort (909 pg/ml, 765 pg/ml and 502 U/ml, respectively n = 46) than in the control cohort (383 pg/ml, 188 pg/ml and 13 U/ml, respectively n = 61) (p < 0.001). The area under the receiver operator characteristic curve (AUC) for MDK and AGR2 was not significantly different (0.734 +/- 0.046 and 0.784 +/- 0.049, respectively, mean +/- SE) but were both significantly less than the AUC for CA125 (0.934 +/- 0.030, p < 0.003). When subjected to stochastic gradient boosted logistic regression modelling, the AUC of the multi-analyte panel (MDK, AGR2 and CA125, 0.988 +/- 0.010) was significantly greater than that of CA125 alone (0.934 +/- 0.030, p = 0.035). The sensitivity and specificity of the multi-analyte algorithm were 95.2 and 97.7%, respectively. Within the study cohort, CA125 displayed a sensitivity and specificity of 87.0 and 94.6%, respectively. The data obtained in this study confirm that both MDK and AGR2 individually display utility as biomarkers for ovarian cancer and that in a multi-analyte panel significantly improve the diagnostic utility of CA125 in symptomatic women.

Increased plasma concentrations of anterior gradient 2 protein are positively associated with ovarian cancer.

Ovarian cancer is often asymptomatic and is diagnosed at an advanced stage with poor survival rates, thus there is an urgent need to develop biomarkers for earlier detection of ovarian cancer. In the present study, we demonstrate for the first time that the previously reported metastasis-inducing protein AGR2 (anterior gradient protein 2) can be detected in the blood of ovarian cancer patients. Using a newly developed ELISA, we show significantly increased concentrations of AGR2 protein in plasma from cancer patients relative to normal controls. Plasma AGR2 concentrations were highest in stages II and III ovarian cancer patients and were similarly elevated in patients with both serous and non-serous tumours. The identification of elevated plasma concentrations of AGR2 may provide a useful biomarker to aid in the discrimination of normal and ovarian cancer patients particularly when used in combination with CA125.

Phase II biomarker trial of a multimarker diagnostic for ovarian cancer.

PURPOSE: The primary hypothesis to be tested in this study was that the diagnostic performance (as assessed by the area under the receiver operator characteristic curve, AUC) of a multianalyte panel to correctly identify women with ovarian cancer was significantly greater than that for CA-125 alone. METHODS: A retrospective, case-control study (phase II biomarker trial) was conducted that involved 362 plasma samples obtained from women with ovarian cancer (n = 150) and healthy controls (n = 212). A multivariate classification model was developed that incorporated five biomarkers of ovarian cancer, CA-125; C-reactive protein (CRP); serum amyloid A (SAA); interleukin 6 (IL-6); and interleukin 8 (IL-8) from a modelling cohort (n = 179). The performance of the model was evaluated using an independent validation cohort (n = 183) and compared with of CA-125 alone. RESULTS: The AUC for the biomarker panel was significantly greater than the AUC for CA-125 alone for a validation cohort (p < 0.01) and an early stage disease cohort (i.e. Stages I and II; p < 0.01). At a threshold of 0.3, the sensitivity and specificity of the multianalyte panel were 94.1 and 91.3%, respectively, for the validation cohort and 92.3 and 91.3%, respectively for an early stage disease cohort. CONCLUSIONS: The use of a panel of plasma biomarkers for the identification of women with ovarian cancer delivers a significant increase in diagnostic performance when compared to the performance of CA-125 alone.