A Pilot Prospective Study Evaluating the Effect of Curcuma-Based Herbal Food Supplement on the Outcome of In Vitro Fertilization in Patients Testing Positive for Four Immunological Biomarkers.

Background and Objectives: Inflammation and oxidative stress have been described to reduce the chance for pregnancy instauration and maintenance. NOFLAMOX, a recently developed herbal preparation with recognized antioxidant and anti-inflammatory properties, can represent an interesting treatment to increase the chance of pregnancy, both physiological or after in vitro fertilization (IVF). The aim of this study was to assess NOFLAMOX's effect; a population with unexplained infertility was screened for the recently described IMMUNOX panel based on four immunological biomarkers with a prospective study approach. Materials and Methods: Patients with unexplained infertility and positive for at least one of the biomarkers of the IMMUNOX panel were included in this study and treated with NOFLAMOX for three months prior to an IVF cycle. Results: Eighty-six patients (n = 86) were screened with the IMMUNOX panel and the forty-seven (54.5%) found positive were included in this study. In more detail, 11 were positive for TNFα (23.4%), 18 (38.3%) for glycodelin (GLY), 29 (61.7%) for Total Oxidative Status (TOS), and 32 (68.1%) for Complement Activity Toxic Factor (CATF). After three months of treatment, a significant reduction in the number of IMMUNOX-positive patients was observable, with 26 patients who turned IMMUNOX-negative displaying a quantitative statistically significant variation of 100% (11/11), 38.9% (7/18), 65.5% (18/29), and 75% (24/32), for TNFα, glycodelin, TOS, and CATF, respectively. Followed in the subsequent IVF cycle, this NOFLAMOX-treated population showed a pregnancy rate of 42.3% compared to the 4.7% of the IMMUNOX-positive group of patients. Conclusions: Taken together, the results of this study suggest that NOFLAMOX could represent an interesting option for those patients with unexplained infertility of inflammatory/oxidative origin. Further studies are needed to confirm these results and explore possible strategies to restore fertility in women with immune-mediated sterility.

The dependence between glycodelin and selected metalloproteinases concentrations in bovine placenta during early gestation and parturition with and without retained foetal membranes.

Pregnancy course depends on the appropriate connection between the mother and the developing foetus. Pregnancy is completed when the placenta is timely expelled. Placental retention is one of the possible pregnancy complications. Extracellular matrix, including adhesive proteins and enzymes that can break down collagens, seems to be responsible for it. The aim of the present study was to examine the impact of one of the adhesive proteins - glycodelin (Gd) - on selected metalloproteinases degrading collagens (MMP2, MMP3, MMP7). Placental tissues from healthy pregnant cows collected during early-mid pregnancy (2nd month n = 7, 3rd month n = 8, 4th month n = 6) and in cows that properly released placenta (NR; n = 6) and cows with retained foetal membranes (R; n = 6) were experimental material. The concentrations of glycodelin and protein content of selected metalloproteinases were measured by ELISA in the maternal and foetal placental homogenates as well as in the culture of epithelial cells derived from the maternal part of the placenta. The presence of these protein molecules was confirmed by Western Blotting. In the bovine placenta, the concentrations of examined proteins exhibit significant changes during placental formation. Gd, MMP3 and MMP7 concentrations decrease with pregnancy progress (between the 2nd and 4th month), while MMP2 concentrations were on the same level in this period. During parturition, concentrations of Gd and MMP3 were significantly higher in the R group compared to the NR group. In parallel, MMP2 concentrations did not show significant differences between the groups (NR vs R), and MMP7 concentrations decreased significantly in the maternal part of the placenta in cows with retained foetal membranes (R). Obtained results show correlations between the gestational age and proteins' (Gd, MMP3, MMP7) concentration, both in the maternal and foetal part of the placenta.

Does non-cavity distorting intramural fibroid affect endometrium around the time of embryo implantation?

The effect of the intramural fibroids not distorting the cavity remains controversial on implantation and pregnancy. The aim of this study was to examine the impact of non-cavity distorting intramural fibroids on endometrium. Fifty-six women with non-cavity distorting intramural fibroid were recruited in this study. Paired endometrial specimens, one from beneath the fibroid (ipsilateral endometrium) and the other from the opposite side of uterine cavity, away from the fibroid (contralateral endometrium) were obtained 7-9 days after the luteinizing hormone surge in a natural cycle. Histological dating, Mucin1 and Glycodelin expression and uterine natural killer (uNK) cell density were compared between the paired samples. The median (IQR) H-score of Mucin1 staining in the ipsilateral luminal epithelium was 210% (142-230%), which was significantly (p < 0.05) higher than that of the contralateral luminal endometrium (157%, IQR 114-176%). There was no significant difference in Mucin1 expression in the glandular epithelium. There was no significant difference in Glycodelin expression in luminal and glandular epithelium, uNK cells density or histological dating results between the paired endometrial samples. In conclusion, it is uncertain whether the altered expression of Mucin1 in luminal epithelium alone may have impact on implantation when other markers are not changed.

Biomarkers at 6 weeks' gestation in the prediction of early miscarriage in pregnancy following assisted reproductive technology.

INTRODUCTION: Miscarriage is a major concern in early pregnancy among women having conceived with assisted reproductive treatments. This study aimed to examine potential miscarriage-related biophysical and biochemical markers at 6 weeks' gestation among women with confirmed clinical pregnancy following in vitro fertilization (IVF)/embryo transfer (ET) and evaluate the performance of a model combining maternal factors, biophysical and biochemical markers at 6 weeks' gestation in the prediction of first trimester miscarriage among singleton pregnancies following IVF/ET. MATERIAL AND METHODS: A prospective cohort study was conducted in a teaching hospital between December 2017 and January 2020 including women who conceived through IVF/ET. Maternal mean arterial pressure, ultrasound markers including mean gestational sac diameter, fetal heart activity, crown rump length and mean uterine artery pulsatility index (mUTPI) and biochemical biomarkers including maternal serum soluble fms-like tyrosine kinase-1 (sFlt-1), placental growth factor (PlGF), kisspeptin and glycodelin-A were measured at 6 weeks' gestation. Logistic regression analysis was carried out to determine significant predictors of miscarriage prior to 13 weeks' gestation and performance of screening was estimated by receiver-operating characteristics curve analysis. RESULTS: Among 169 included pregnancies, 145 (85.8%) pregnancies progressed to beyond 13 weeks' gestation and had live births whereas 24 (14.2%) pregnancies resulted in a miscarriage during the first trimester. In the miscarriage group, compared to the live birth group, maternal age, body mass index, and mean arterial pressure were significantly increased; mean gestational sac diameter, crown rump length, mUTPI, serum sFlt-1, glycodelin-A, and the rate of positive fetal heart activity were significantly decreased, while no significant differences were detected in PlGF and kisspeptin. Significant prediction for miscarriage before 13 weeks' gestation was provided by maternal age, fetal heart activity, mUTPI, and serum glycodelin-A. The combination of maternal age, ultrasound (fetal heart activity and mUTPI), and biochemical (glycodelin-A) markers achieved the highest area under the curve (AUC: 0.918, 95% CI 0.866-0.955), with estimated detection rates of 54.2% and 70.8% for miscarriage before 13 weeks' gestation, at fixed false positive rates of 5% and 10%, respectively. CONCLUSIONS: A combination of maternal age, fetal heart activity, mUTPI, and serum glycodelin-A at 6 weeks' gestation could effectively identify IVF/ET pregnancies at risk of first trimester miscarriage.

Is neonatal uterine bleeding responsible for early-onset endometriosis?

BACKGROUND: It has been hypothesized that the origin of early-onset endometriosis could be from endometrial mesenchymal stem cells (eMSCs) in neonatal uterine blood (NUB). There is no information on the possible mechanistic basis linking an association between NUB/neonatal endometrium and development of early-onset endometriosis. In this study we performed a series of experiments to clarify the mechanistic link between NUB and/or neonatal endometrium and development of early-onset endometriosis. METHODS: We retrospectively collected postmortem neonatal endometria (n = 15) and prospectively collected NUB (n = 18) of female babies for the analysis of different biological markers including eMSCs. Immunohistochemical analysis of neonatal endometria was performed to examine the expression patterns of ovarian steroid receptors (ER/PGR), decidualization (prolactin, IGFBP1), pre-decidualization (Glycodelin A, α-SMA), proliferation (Ki-67 index), vascularity (CD31 + cells), immunocompetent CD68+, CD45+, CD56 + cells and some putative markers of eMSCs. Cell transfer method and immunocytochemistry were used to investigate the eMSCs and/or endometrial cells in NUB. RESULTS: Immunohistochemical analysis of postmortem neonatal endometria revealed variable staining response to ER/PGR, decidual markers, and substantial proliferative and angiogenic activity. A moderate to strong immunoexpression of Glycodelin-A was found in both neonatal and adult endometria. The tissue infiltration of CD56+, CD45 + and CD68 + immunocompetent cells was significantly low in neonatal endometria than that in adult endometria (p = 0.0003, p < 0.0001, p = 0.034, respectively). No eMSCs or even endometrial cells were detected in NUB. However, a variable expression of some phenotypes of eMSCs (CD90/CD105) was found in neonatal endometria. CONCLUSIONS: Based on our serial experiments we did not find any supporting evidence for the role of NUB in early-onset endometriosis. Neonatal endometria showed variable expression of ovarian steroid receptors, decidualization, and a substantial amount of proliferative and angiogenic activity. As an alternative mechanism, a significantly less tissue accumulation of immunocompetent cells in neonatal endometria may explain the survival of ER + and PGR + cells should they make entry into the pelvis and consequent development of early endometriosis with the onset of ovarian function. Future study with large sample size and application of modified technological tools is warranted to test the NUB hypothesis and to clarify their biological or clinical significance. TRIAL REGISTRATION: not applicable.

Validation of a multiple marker test for early pregnancy outcome prediction.

PURPOSE: To validate the use of a multiple biomarker test panel for predicting first trimester pregnancy outcome in a multi-center cohort. METHODS: A case-control study of women presenting with pain and bleeding in early pregnancy at 5-10 weeks gestational age was performed at three academic centers. Sera from women with ectopic pregnancy (EP), viable intrauterine pregnancy (IUP), and miscarriage (SAB) were analyzed via immunoassay for Activin A (AA), Progesterone (P4), A Disintegrin And Metalloprotease-12 (ADAM12), pregnancy-associated plasma protein A (PAPP-A), glycodelin (Glyc), and human chorionic gonadotropin (hCG). Biomarkers were assessed for reproducibility using medians, ranges, standard deviations, and area under receiver-operating characteristic curve (AUC) and accuracy in early pregnancy outcome classification compared to a previous derivation population. RESULTS: In 192 pregnancies, the biomarkers demonstrated good reproducibility with similar medians, ranges, and AUCs when compared to the derivation population except glycodelin. Pregnancy location was conclusively classified in 53% (n = 94) of the whole study sample with 78% accuracy. Pregnancy viability was conclusively classified in 58% (n = 112) of the new sample with 89% accuracy. Results were similar with subsequent model revisions where glycodelin was excluded and in the subgroups of subjects with a hCG below 2000 mIU/mL and a gestational age less than 6 weeks. CONCLUSION: The use of a panel of biomarkers to maximize test accuracy of a prediction of pregnancy location and prediction of pregnancy viability was reproducible and validated in an external population from which it was derived, but clinical utility is limited based on the test characteristics obtained.

The Role of Recombinant Glycodelin in the Differentiation of Regulatory T-Lymphocytes.

The effect of recombinant glycodelin (GdA) on the number of T-regulatory lymphocytes (Treg) in a culture of activated CD4+ lymphocytes was investigated, while simultaneously assessing the proliferative status of the cells. Recombinant GdA from E. coli and from HEK293 cells were used in the study at concentrations of 0.2; 2 and 10 µg/mL. It was found that only a low concentration (0.2 µg/mL) of recombinant GdA of bacterial origin reduced the number of proliferating CD4+ lymphocytes as well as the number of Treg (CD4+CD25highCD127-/low) in the experimental system in vitro.

Effect of Glycodelin on the Cytokine Profile of Rats during Allogeneic Bone Marrow Cell Transplantation.

The protein glycodelin (PP14, PAEP) is associated with pregnancy and exhibits pronounced immunotropic properties. We studied the effect of glycodelin on the cytokine profile of blood serum during transplantation a suspension of allogeneic red bone marrow cells to Wistar rats. Recombinant glycodelin was administered to the animals 4 times (14 µg each time). Against the background of bone marrow cell transplantation, the levels of proinflammatory (IL-1α, IL-1ß, and IL-18) and regulatory (IL-7, IL-12) cytokines and CSF (M-CSF) increased in blood serum, which indicates a systemic inflammatory response to the allograft. Glycodelin administration against the background of bone marrow cell allotransplantation led to a significant decrease in the proinflammatory cytokine IL-17A on day 21 of the experiment; the concentrations of the other cytokines remained unchanged. In general, glycodelin can suppress the level of IL-17A, a marker of graft rejection, which opens perspectives for its further investigation as a potential drug.

Serum and cervico-vaginal glycodelin concentrations as predictors of successful implantation after embryo transfer.

OBJECTIVE: Evaluation of glycodelin (Gd) concentrations in serum and cervico-vaginal secretions as a predictor for implantation after ICSI. MATERIALS AND METHODS: Prospective study on 50 women undergoing ICSI where long protocol ovarian stimulation was used. Serum and cervico-vaginal lavage Gd concentrations were measured then rates of biochemical and clinical pregnancy were detected and predictive value was evaluated using logistic regression analysis. RESULTS: Using cut-off values of 2.2 ng/ml and 1.9 ng/ml for serum and cervico-vaginal Gd concentrations respectively for biochemical pregnancy and values of 2.7 ng/ml and 1.3 ng/ml respectively for clinical pregnancy, there was no significant difference regarding sensitivity (72% & 56%, and 72% & 89%, respectively and respectively). Specificity was statistically similar for biochemical pregnancy (72% and 89%, respectively) while specificity was significantly higher for clinical pregnancy using cervico-vaginal Gd concentration of 1.3 ng/ml (88%) compared to serum Gd concentration of 1.9 ng/ml (53%). CONCLUSION: Glycodelin appears to be a promising marker for implantation after IVF/ICSI.

Biomolecular Markers of Recurrent Implantation Failure-A Review.

Currently, infertility affects 8-12% of reproductive age couples worldwide, a problem that also affects women suffering from recurrent implantation failure (RIF). RIF is a complex condition resulting from many physiological and molecular mechanisms involving dynamic endometrium-blastocyst interaction. The most important are the endometrial receptivity process, decidualization, trophoblast invasion, and blastocyst nesting. Although the exact multifactorial pathogenesis of RIF remains unclear, many studies have suggested the association between hormone level imbalance, disturbances of angiogenic and immunomodulatory factors, certain genetic polymorphisms, and occurrence of RIF. These studies were performed in quite small groups. Additionally, the results are inconsistent between ethnicities. The present review briefly summarizes the importance of factors involved in RIF development that could also serve as diagnostic determinants. Moreover, our review could constitute part of a new platform for discovery of novel diagnostic and therapeutic solutions for RIF.

Detection of a novel glycodelin biomarker using electrochemical immunosensor for endometriosis.

Endometriosis is one of the important issues in women worldwide, which decreases the quality of women's lives in their reproductive age. The diagnosis of endometriosis is carried out by the invasive procedure, which is expensive and painful. In the last few decades, researchers have given more attention to constructing a suitable biomarker-based biosensor for semi/non-invasive diagnosis of endometriosis. As a result, glycodelin (GLY) was found as a promising biomarker because of its selectivity and sensitivity. To the best of our knowledge, it was the first study that reported the detection of GLY biomarker using an electrochemical immunosensor. Briefly, a label-free electrochemical immunosensing platform was constructed through in-situ surface modification of cysteamine layer and immobilisation of antibody (anti-GLY) with help of glutaraldehyde. The interaction between antigen and antibody was measured using square wave voltammetry (SWV). The SWV signal could decrease proportionally with the increasing GLY concentration ranging from 1 to 1000 ng mL-1 (R2 = 0.9981) and a detection limit (LOD) of 0.43 ng mL-1. Moreover, an immunosensor could exhibit high sensitivity, selectivity, long-term stability, reproducibility and regeneration. Accuracy of the immunosensor was compared with enzyme-linked immunosorbent assay (ELISA), and satisfying results were obtained. The detection of GLY biomarker may be a new possibility for endometriosis diagnosis.

Pregnancy Zone Protein (PZP) is significantly upregulated in the decidua of recurrent and spontaneous miscarriage and negatively correlated to Glycodelin A (GdA).

BACKGROUND: Pregnancy Zone Protein (PZP) is an immunosuppressive protein that is expressed by the placenta and has also been identified in immune cells. When PZP and Glycodelin A (GdA) are combined, they act synergistically to inhibit Th-1 immune response. Little is known about its combined expression and role in normal and disturbed first trimester pregnancy. PATIENTS AND METHODS: We investigated the expression of PZP and GdA in placental tissue obtained from spontaneous miscarriage (SM) (n = 19) and recurrent miscarriage (RM) (n = 17) at gestational weeks 6-13 by immunohistochemistry and on mRNA-level by either TaqMan PCR or in situ hybridization. Placental tissue from legal terminations of healthy pregnancies (n = 15) served as control group. Immunofluorescence double staining was used to analyse the combined expression of PZP and GdA in decidual tissue. RESULTS: The protein level of PZP was significantly increased in decidual stroma of SM samples compared to the decidua of control specimens and also significantly upregulated in the decidual stroma cells in the RM group. Concerning GdA, the decidual stroma revealed a significantly decreased protein level in the group with spontaneous abortions than in the group with healthy pregnancies. There was also a significant downregulation of GdA in the decidual stroma of RM samples compared to the control group. We observed a significant negative correlation of PZP and GdA in decidual stromal tissue of recurrent abortion. We could confirm the staining results for PZP as well as for GdA on mRNA level. Both proteins are co-localized in decidual stroma as analysed by immunofluorescence double staining. CONCLUSION: A balanced expression of GdA and its carrier protein PZP in the decidua seems crucial for a successful ongoing pregnancy. According to our data, these immunosuppressive proteins are co-localized in the decidual tissue and show a negative correlation only in patients suffering from recurrent abortion.

Decidual glycodelin-A polarizes human monocytes into a decidual macrophage-like phenotype through Siglec-7.

Decidual macrophages constitute 20-30% of the total leukocytes in the uterus of pregnant women, regulating the maternal immune tolerance and placenta development. Abnormal number or activities of decidual macrophages (dMs) are associated with fetal loss and pregnancy complications, such as preeclampsia. Monocytes differentiate into dMs in a decidua-specific microenvironment. Despite their important roles in pregnancy, the exact factors that regulate the differentiation into dMs remain unclear. Glycodelin-A (PAEP, hereafter referred to as GdA) is a glycoprotein that is abundantly present in the decidua, and plays an important role in fetomaternal defense and placental development. It modulates the differentiation and activity of several immune cell types residing in the decidua. In this study, we demonstrated that GdA induces the differentiation of human monocytes into dM-like phenotypes in terms of transcriptome, cell surface marker expression, secretome, and regulation of trophoblast and endothelial cell functions. We found that Sialic acid-binding Ig-like lectin 7 (Siglec-7) mediates the binding and biological actions of GdA in a sialic acid-dependent manner. We, therefore, suggest that GdA, induces the polarization of monocytes into dMs to regulate fetomaternal tolerance and placental development.

Organoids of Human Endometrium: A Powerful In Vitro Model for the Endometrium-Embryo Cross-Talk at the Implantation Site.

Embryo implantation has been defined as the "black box" of human reproduction. Most of the knowledge on mechanisms underlining this process derives from animal models, but they cannot always be translated to humans. Therefore, the development of an in vitro/ex vivo model recapitulating as closely and precisely as possible the fundamental functional features of the human endometrial tissue is very much desirable. Here, we have validated endometrial organoids as a suitable 3D-model to studying epithelial endometrial interface for embryo implantation. Transmission and scanning electron microscopy analyses showed that organoids preserve the glandular organization and cell ultrastructural characteristics. They also retain the responsiveness to hormonal treatment specific to the corresponding phase of the menstrual cycle, mimicking the in vivo glandular-like aspect and functions. Noteworthy, organoids mirroring the early secretive phase show the development of pinopodes, large cytoplasmic apical protrusions of the epithelial cells, traditionally considered as reliable key features of the implantation window. Moreover, organoids express glycodelin A (GdA), a cycle-dependent marker of the endometrial receptivity, with its quantitative and qualitative features accounting well for the profile detected in the endometrium in vivo. Accordingly, organoids deriving from the eutopic endometrium of women with endometriosis show a GdA glycosylation pattern significantly different from healthy organoids, confirming our prior data on endometrial tissues. The present results strongly support the idea that organoids may closely recapitulate the molecular and functional characteristics of their cells/tissue of origin.

Altered glycosylation of glycodelin in endometrial carcinoma.

Glycodelin is a major glycoprotein expressed in reproductive tissues, like secretory and decidualized endometrium. It has several reproduction related functions that are dependent on specific glycosylation, but it has also been found to drive differentiation of endometrial carcinoma cells toward a less malignant phenotype. Here we aimed to elucidate whether the glycosylation and function of glycodelin is altered in endometrial carcinoma as compared with a normal endometrium. We carried out glycan structure analysis of glycodelin expressed in HEC-1B human endometrial carcinoma cells (HEC-1B Gd) by mass spectrometry glycomics strategies. Glycans of HEC-1B Gd were found to comprise a typical mixture of high-mannose, hybrid, and complex-type N-glycans, often containing undecorated LacNAc (Galß1-4GlcNAc) antennae. However, several differences, as compared with previously reported glycan structures of normal human decidualized endometrium-derived glycodelin isoform, glycodelin-A (GdA), were also found. These included a lower level of sialylation and more abundant poly-LacNAc antennae, some of which are fucosylated. This allowed us to select lectins that showed different binding to these classes of glycodelin. Despite the differences in glycosylation between HEC-1B Gd and GdA, both showed similar inhibitory activity on trophoblast cell invasion and peripheral blood mononuclear cell proliferation. For the detection of cancer associated glycodelin, we established a novel in situ proximity-ligation based histochemical staining method using a specific glycodelin antibody and UEAI lectin. We found that the UEAI reactive glycodelin was abundant in endometrial carcinoma, but virtually absent in normal endometrial tissue even when glycodelin was strongly expressed. In conclusion, we established a histochemical staining method for the detection of endometrial carcinoma-associated glycodelin and showed that this specific glycodelin is exclusively expressed in cancer, not in normal endometrium. Similar methods can be used for studies of other glycoproteins.

Glycodelin is internalized by peripheral monocytes.

Glycodelin is produced by the endometrial cells during the luteal phase and first trimester of pregnancy and plays a role in the regulation of the endometrial immunology. However, the molecular connection between glycodelin and the maternal immune system is not clear. To better understand the possible physiological interaction between the endometrium and the maternal immune system, we investigated (1) whether glycodelin binds to mainly peripheral monocytes, and in case (2) whether the binding to the membrane only depends on the protein backbone or a carbohydrate structure is needed, and in case (3) whether glycodelin is internalized after binding to the membrane. We demonstrated that glycodelin - with or without the carbohydrate structure - was preferentially bound and internalized to peripheral monocytes. Surprisingly, we found signals in the nucleus of the monocytes indicating a potential regulating effect of glycodelin may be exerted through the nucleus. However, further studies should be performed to confirm this finding.

Glycodelin regulates the numbers and function of peripheral natural killer cells.

Natural killer (NK) cells comprise of ∼70% of the immune cell population in the maternal decidua and ∼15% of the mononuclear cells in the peripheral blood. The decidual NK cells capable of producing high levels of cytokines are functionally distinct from the peripheral NK cells that exhibit high cytotoxicity. The numbers of peripheral NK cells and their cytotoxicity potential have been correlated with pregnancy outcome. In the same context, glycodelin, an immunomodulatory protein, has been recognized to be essential for the establishment and maintenance of pregnancy, and its' reduced levels are associated with recurrent spontaneous abortions. We investigated the effect of glycodelin on the peripheral NK cells. Our results reveal that glycodelin suppresses the cytotoxicity of peripheral NK cells via downregulating perforin, granzyme B and IFNγ. Glycodelin also induces caspase-dependent death in only activated peripheral NK cells, the effect suggested to be mediated by glycodelin upon engaging with the CD7 cell surface receptor. Thus, during pregnancy, glycodelin modulates the function and the number of cytotoxic NK cells that pose a deleterious effect on the fetus, a semi-allograft. This study provides insights into the mechanism of the regulatory effect of glycodelin on NK cells and could possibly be exploited for the management of miscarriages.

N-acetylgalactosamine-6-sulfatase (GALNS), Similar to Glycodelin, Is a Potential General Biomarker for Multiple Malignancies.

BACKGROUND/AIM: The aim of this study was to evaluate N-acetylgalactosamine-6-sulfatase (GALNS) as a new biomarker candidate for detecting lung cancer. Glycodelin or PAEP, the serum levels of which are known to be elevated in lung and other cancers, served as a benchmark for comparison. PATIENTS AND METHODS: A total of 170 serum samples from healthy controls and patients with pneumonia, lung cancer, breast cancer, colon cancer, liver cancer, and head and neck cancer were analyzed for the levels of GALNS and PAEP by ELISA. RESULTS: The median serum levels of GALNS and PAEP in all cancer types as well as pneumonia patients were significantly higher than those of the healthy controls. CONCLUSION: In addition to previously known cancers, the median serum levels of PAEP were also found to be higher in liver and head and neck cancer patients. GALNS and PAEP are promising general biomarkers for multiple cancers and deserve further evaluation.

Clues to Non-Invasive Implantation Window Monitoring: Isolation and Characterisation of Endometrial Exosomes.

Despite the significant advances in the last decades, low implantation rate per transferred embryo still remains a major concern in assisted reproductive techniques, highlighting a need to better characterize endometrial receptivity also by mean of specific biomarkers. Based on physiology and on the intimate contact with endometrium as the tissue of interest, in this study we developed and validated an optimized protocol that uses extracellular vesicles (EVs) recovered from uterine flushings and from a cervical brush, the latter never used until now as an EVs source, as surrogates for endometrial biopsies. This method combines the safety of sampling with the ability to study the expression profile across the uterine cycle. We have compared the yield and composition of EVs recovered from different biofluids samples and fractions thereof, opting for chemical precipitation as the EV isolation procedure, assuring the highest yield without introducing any bias in specific EV recovery. Moreover, collected EVs, in particular exosome-like vesicles, express putative endometrial markers, such as glycodelin A and receptors for estrogen and progesterone, thus confirming their endometrial origin. We also identified uterine flushing EVs, in particular those recovered from its mucous fraction, as the richest source of endometrial transcripts, likely correlated to cellular (epithelial) origin of these vesicles. Finally, our pilot quantitative assessment of three endometrial gene profiles, in samples collected at different time points along the luteal phase, revealed the fluctuations apparently recapitulating gene expression variability prior reported during the menstrual cycle. Unlike tissue biopsy that is subjected to inter- and intra-sample differences, our data suggest that EVs from liquid biopsies (from uterine flushings and a cervical brush) obtained through less-invasive procedures, can be substrate to detect and track the tissue representative expression profiles, better depicting the total endometrium complexity.

Proteomic analysis reveals the negative modulator of sperm function glycodelin as over-represented in semen exosomes isolated from asthenozoospermic patients.

STUDY QUESTION: Are there differences in the proteomic profile of exosomes isolated from seminal plasma of normozoospermic (NSP) and severe asthenozoospermic (SA) men, potentially contributing to sperm features? SUMMARY ANSWER: A relevant group of proteins known to positively regulate sperm functions were over-represented in seminal exosomes of NSP men, i.e. cysteine-rich secretory protein-1 (CRISP1), while the inhibitory protein glycodelin was enriched in exosomes of SA subjects. WHAT IS KNOWN ALREADY: Exosomes are secreted along the male reproductive tract and are thought to be involved in spermatozoa maturation and function. Ejaculated spermatozoa are still able to capture exosomes; exosomes of NSP individuals improve sperm motility and prompt capacitation, while exosomes of SA men fail to exert similar features. STUDY DESIGN, SIZE, DURATION: Semen samples from NSP and SA men, aged 18 to 55 and registered at a single IVF center, were considered for this study project. Subjects were subdivided into three groups: a discovery cohort (five NSP men and six SA patients), a validation cohort (seven NSP and seven SA men) and the 'glycodelin analysis' cohort (20 NSP and 37 SA men). Exosomes were purified from semen of every participant. PARTICIPANTS/MATERIALS, SETTING, METHODS: Exosomes were characterized by nanoparticle tracking analysis, transmission electron microscopy and western blot. Comprehensive proteomics analysis of the exosomal proteome was performed by nanoscale liquid chromatographic tandem mass spectrometry analysis. Funrich software was used to determine statistical enrichment of pathways, networks and Gene Ontology terms of the identified proteins. Validation of differentially expressed proteins was performed through ELISA and western blot analysis. MAIN RESULTS AND THE ROLE OF CHANCE: The comprehensive proteomic analysis identified a total of 2138 proteins for both groups. There were 89 proteins found to be differentially expressed in exosomes of NSP versus SA subjects, of which 37 were increased in the NSP group and 52 were increased in the SA group. One-third of the exosomes-associated proteins highly expressed in NSP samples were involved in the reproductive process; conversely, the over-expressed proteins in exosomes of SA samples were not functionally specific. Quantitative data were confirmed on seminal exosomes from different cohorts of subjects. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Transfer of the proteins from exosomes to spermatozoa has been only partially demonstrated and up-take mechanisms are still poorly defined. WIDER IMPLICATIONS OF THE FINDINGS: Seminal exosomes carry proteins that are potentially able to either favour or inhibit the reproductive process in humans. A better understanding of these phenomena might pave the way for novel intervention measures in terms of male infertility. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the Italian Ministry of Health through an Institution Seed Grant. None of the authors has any competing interests.