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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Glycodelin-A stimulates the conversion of human peripheral blood CD16-CD56bright NK cell to a decidual NK cell-like phenotype.

STUDY QUESTION: Does glycodelin-A (GdA) induce conversion of human peripheral blood CD16-CD56bright natural killer (NK) cells to decidual NK (dNK) cells to facilitate placentation? SUMMARY ANSWER: GdA binds to blood CD16-CD56bright NK cells via its sialylated glycans and converts them to a dNK-like cells, which in turn regulate endothelial cell angiogenesis and trophoblast invasion via vascular endothelial growth factor (VEGF) and insulin-like growth factor-binding protein 1 (IGFBP-1) secretion, respectively. WHAT IS KNOWN ALREADY: dNK cells are the most abundant leucocyte population in the decidua. These cells express CD16-CD56bright phenotype. Peripheral blood CD16-CD56bright NK cells and hematopoietic precursors have been suggested to be capable of differentiating towards dNK cells upon exposure to the decidual microenvironment. These cells regulate trophoblast invasion during spiral arteries remodelling and mediate homoeostasis and functions of the endothelial cells. GdA is an abundant glycoprotein in the human decidua with peak expression between the 6th and 12th week of gestation, suggesting a role in early pregnancy. Indeed, GdA interacts with and modulates functions and differentiation of trophoblast and immune cells in the human feto-maternal interface. Aberrant GdA expression during pregnancy is associated with unexplained infertility, pregnancy loss and pre-eclampsia. STUDY DESIGN, SIZE, DURATION: CD16+CD56dim, CD16-CD56bright and dNK cells were isolated from human peripheral blood and decidua tissue, respectively, by immuno-magnetic beads or fluorescence-activated cell sorting. Human extravillous trophoblasts were isolated from first trimester placental tissue after termination of pregnancy. Biological activities of the cells were studied after treatment with GdA at a physiological dose of 5 µg/mL. GdA was purified from human amniotic fluid by immuno-affinity chromatography. PARTICIPANTS/MATERIALS, SETTING, METHODS: Expression of VEGF, CD9, CD49a, CD151 and CD158a in the cells were determined by flow cytometry. Angiogenic proteins in the spent media of NK cells were determined by cytokine array and ELISA. Blocking antibodies were used to study the functions of the identified angiogenic proteins. Endothelial cell angiogenesis was determined by tube formation and trans-well migration assays. Cell invasion and migration were determined by trans-well invasion/migration assay. Binding of normal and de-sialylated GdA, and expression of L-selectin and siglec-7 on the NK cells were analysed by flow cytometry. The association between GdA and L-selectin on NK cells was confirmed by immunoprecipitation. Extracellular signal-regulated protein kinases (ERK) activation was determined by Western blotting and functional assays. MAIN RESULTS AND THE ROLE OF CHANCE: GdA treatment enhanced the expression of dNK cell markers CD9 and CD49a and the production of the functional dNK secretory product VEGF in the peripheral blood CD16-CD56bright NK cells. The spent media of GdA-treated CD16-CD56bright NK cells promoted tube formation of human umbilical vein endothelial cells and invasiveness of trophoblasts. These stimulatory effects were mediated by the stimulatory activities of GdA on an ERK-activation dependent production of VEGF and IGFBP-1 by the NK cells. GdA had a stronger binding affinity to the CD16-CD56bright NK cells as compared to the CD16+CD56dim NK cells. This GdA-NK cell interaction was reduced by de-sialylation. GdA interacted with L-selectin, expressed only in the CD16-CD56bright NK cells, but not in the CD16+CD56dim NK cells. Anti-L-selectin functional blocking antibody suppressed the binding and biological activities of GdA on the NK cells. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Some of the above findings are based on a small sample size of peripheral blood CD16-CD56bright NK cells. These results need to be confirmed with human primary dNK cells. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study on the biological role of GdA on conversion of CD16-CD56bright NK cells to dNK-like cells. Further investigation on the glycosylation and functions of GdA will enhance our understanding on human placentation and placenta-associated complications with altered NK cell biology. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Hong Kong Research Grant Council Grant 17122415, Sanming Project of Medicine in Shenzhen, the Finnish Cancer Foundation, Sigrid Jusélius Foundation and the Finnish Society of Clinical Chemistry. The authors have no competing interests to declare.

Immunomodulatory activity of glycodelin: implications in allograft rejection.

Glycodelin is an immunomodulator, indispensable for the maintenance of pregnancy in humans. The glycoprotein induces apoptosis in activated CD4+ T cells, monocytes and natural killer (NK) cells, and suppresses the activity of cytotoxic T cells, macrophages and dendritic cells. This study explores the immunosuppressive property of glycodelin for its possible use in preventing graft rejection. Because glycodelin is found only in certain primates, the hypothesis was investigated in an allograft nude mouse model. It is demonstrated that treatment of alloactivated mononuclear cells with glycodelin thwarts graft rejection. Glycodelin decreases the number of activated CD4+ and CD8+ cells and down-regulates the expression of key proteins known to be involved in graft demise such as granzyme-B, eomesodermin (EOMES), interleukin (IL)-2 and proinflammatory cytokines [tumour necrosis factor (TNF)-α and IL-6], resulting in a weakened cell-mediated immune response. Immunosuppressive drugs for treating allograft rejection are associated with severe side effects. Glycodelin, a natural immunomodulator in humans, would be an ideal alternative candidate.

[Effect of GnRHa controlled ovarian hyperstimulation on mouse embryo implantation and its mechanism].

The purpose of the present study was to investigate the effects and underlying mechanism of gonadotropin-releasing hormone agonist (GnRHa) controlled ovarian hyperstimulation (COH) on embryo implantation in mice. Forty female Kunming mice aged 9 weeks were randomly divided into two groups (control and COH groups). The COH group received intraperitoneal (i.p.) injections of aminocyclin acetate (GnRHa), human menopausal gonadotropin (HMG) and human chorionic gonadotropin (hCG), while the control group was given equal amount of physiological saline by i.p. injection. One male mouse and two female mice were put into the same cage at 16:00 on the hCG injection day, and on the fourth day of pregnancy, 10 mice from each group were killed. The levels of serum estradiol (E2) and progesterone (P) were measured by radioimmunoassay; HE staining was used to observe the morphology of ovarian and endometrial tissues. The protein expression levels of endometrial leukemia inhibitory factor (LIF), phosphorylated signal transducer and activator of transcription 3 (p-STAT3), heparin-binding epidermal growth factor-like growth factor (HB-EGF) and glycodelin A were detected by Western blot and immunohistochemistry. Ten mice from each group were sacrificed on the eighth day of pregnancy, and the status of the uterus and the average number of blastocysts were observed. The results showed that, compared with control group, the serum E2 level in COH group was significantly decreased (P < 0.05), while the P level was increased significantly (P < 0.05); the ovarian follicles at different developmental stages were rare, corpus lutea (CL) were visible and multiple, the endometrium was thinned, and the number of endometrial glands was reduced (P < 0.05); the contents of LIF, p-STAT3, HB-EGF and glycodelin A in the endometrium were decreased significantly (P < 0.05) on the fourth day of pregnancy; mouse blastocysts developed slowly and were decreased in number on the eighth day of pregnancy (P < 0.05). The above results suggest that GnRHa COH can affect embryo implantation in mice. The mechanism may be related to the imbalance of gonadal hormone, the changes in the structure of the endometrium and the expressions of LIF, p-STAT3, HB-EGF and glycodelin A in the implantation stage, which may lead to the decrease of endometrial receptivity and the abnormal dialogue between the embryo and the uterus.

Affinity purification of native glycodelin from amniotic fluid for biological investigations and development of a glycodelin ELISA for clinical studies.

INTRODUCTION: Glycodelin is a glycoprotein with different oligosaccharides that are responsible for its diverse biological functions in contraception and immunosuppression. Therefore, it is necessary to have access to adequate amounts of glycodelin with retained carbohydrate structure for functional studies because the carbohydrate part can be lacking or be insufficient in recombinant glycodelin from prokaryotic and eukaryotic cell systems. METHODS AND RESULTS: Native glycodelin was purified from amniotic fluid by a series of affinity chromatography steps and had many glycosylated forms verified by mass spectrometry. About 7.5 mg glycodelin was obtained from 1.5 L amniotic fluid. No high molecular mass forms of glycodelin were found in amniotic fluid. Aliquots of the purified glycodelin were used as an immunogen in rabbits for antibody production against glycodelin and a calibrator in a highly sensitive glycodelin enzyme-linked immunosorbent assay (ELISA) with a detection limit of about 1 µg/L. CONCLUSIONS: Native glycodelin was purified from amniotic fluid and used as an immunogen for raising a rabbit antibody against glycodelin and a calibrator in a highly sensitive glycodelin ELISA. We found no high molecular mass forms of glycodelin in amniotic fluid. Aliquots of the purified glycodelin were set aside for functional studies which are in progress.

Endometrial flushing αVß3 integrin, glycodelin and PGF2α levels for evaluating endometrial receptivity in women with polycystic ovary syndrome, myoma uteri and endometrioma.

The aim of this cross-sectional study is to compare endometrial flushing fluid levels of αVß3 integrin, glycodelin and PGF2α during the midluteal phase of the menstrual cycle of women with polycystic ovary syndrome (PCOS, n = 20), myoma uteri (n = 20) and endometrioma (n = 19) with the healthy controls (n = 20). After collecting samples at the midluteal phase of ovulatory volunteers and storing them at -80 °C, αVß3 integrin, glycodelin and PGF2α levels were analyzed using ELISA. The mean ages of the groups were 28.90 ± 5.45, 37.25 ± 2.73, 32.84 ± 6.62 and 32.15 ± 5.18 in PCOS, myoma uteri, endometrioma and control groups, respectively. The αVß3 integrin level (ng/ml) was statistically significantly higher in endometrioma group (9.70 ± 1.72, p < 0.05) as compared to myoma uteri and control groups. Similarly, glycodelin level (ng/ml) was significantly higher in endometrioma group (341.04 ± 93.32) than PCOS (p < 0.01), myoma uteri (p < 0.001) and healthy subjects (p < 0.001). Moreover, PGF2α level (350.04 ± 464.50 ng/ml) was significantly higher in PCOS group relative to myoma uteri (p < 0.001), endometrioma (p < 0.05) and control (p < 0.05) groups. In conclusion, αVß3 integrin level was significantly higher in endometrioma subjects than those with myoma uteri and control groups; glycodelin level was significantly higher in endometrioma group than other three groups, and lastly, PCOS patients had significantly higher PGF2α levels than those patients with myoma uteri, endometrioma and controls.

Investigation on the ability of first trimester glycodelin and angiopoietin-2 to predict small-for-gestational age pregnancies at delivery.

BACKGROUND: The aim was to investigate whether first trimester glycodelin and angiopoietin-2 can predict small-for-gestational age (SGA) at delivery, individually or in combination. METHODS: In this case-control study we measured glycodelin and angiopoietin-2 on serum from 170 singleton pregnant women delivering SGA neonates and 985 singleton pregnant women delivering normal-weighted neonates. All values were converted to multiples of the medians (MoM). RESULTS: Pregnant women delivering SGA neonates had lower first trimester glycodelin and angiopoietin-2 MoM values [median (interquartile range)] compared with pregnant women delivering normal-weighted neonates for glycodelin: 0.86 (0.58-1.24) vs. 1.03 (0.74-1.45), p<0.001, and for angiopoietin-2: 0.89 (0.69-1.19) vs. 1.01 (0.78-1.31), p<0.001. The prediction performances of the biomarkers showed that the areas under the curve (AUC) were 0.59 (glycodelin), 0.58 (angiopoietin-2), and 0.60 (glycodelin and angiopoietin-2). CONCLUSIONS: We demonstrated that first trimester glycodelin and angiopoietin-2 were associated with SGA, but they were, individually and in combination, poor predictors of SGA at delivery. The AUCs were low which indicate low detection rates and high false positive rates.

The dimeric crystal structure of the human fertility lipocalin glycodelin reveals a protein scaffold for the presentation of complex glycans.

Human glycodelin (Gd) is an abundant glycoprotein from the lipocalin family and is involved in crucial biological processes such as reproduction and immune reaction. In females and males, Gd is found in four distinct glycoforms-A, C, F and S-that arise from different N-linked oligosaccharide side chains at amino acid residues Asn28 and Asn63. We have expressed Gd (carrying two amino acid substitutions to improve solubility) as a non-glycosylated protein in Escherichia coli via periplasmic secretion and determined its X-ray structure at 2.45 Å resolution. Gd reveals a classical lipocalin fold including two disulfide bridges, which is however unusually compact and lacks a pronounced central pocket inside the ß-barrel, in line with its low affinity for hydrophobic ligands. Instead, this lipocalin exhibits a unique homodimeric quaternary structure that appears ideally suited as a scaffold for the presentation of specific glycans. In fact, the four oligosaccharides are presented in close proximity on the same side of the dimer surface, which increases avidity for cellular receptors, e.g. during sperm-egg recognition. A bioinformatic analysis indicated that Gd orthologues exclusively occur in certain suborders of primates that have a menstrual cycle, suggesting that this lipocalin with its role in fertility only recently emerged during evolution.

Glycodelin expression in pregnant patients with cervical intraepithelial neoplasia: a case-control study.

We aimed to evaluate glycodelin immunostaining in pregnant women with a first diagnosis of cervical intraephitelial neoplasia (CIN) and to correlate the expression of CIN with Ki-67 and glycodelin immunostaining. A retrospective case-control study was performed including 20 patients with natural pregnancy and with first time onset of CIN occurring not later than 16 gestational weeks. The control group included 20 non-pregnant patients matched for age, parity, smoking status and number of previous sexual partners. Exclusion criteria included previous cervical treatment, immunocompromised status and chronic hepatitis B and/or C. Staining for Glycodelin and for Ki-67 was expressed using a classification based on the distribution of positivity on a semi-quantitative three-point scale. An inverse relationship was observed between glycodelin immunostaining and CIN grade in pregnant patients (p = 0.01), with a significantly higher expression in CIN1 than in CIN2 and CIN3, but not in non-pregnant patients (p = 0.81). Positivity for Ki-67 was less intense in pregnant than in non-pregnant patients. A significant inverse relationship was observed between glycodelin immunostaining and Ki-67 expression (p = 0.02). We suggest that the higher expression of glycodelin in pregnancy is related to a lower proliferative activity in CIN, which is probably associated to hormonal status of pregnancy. Further clinical studies are needed to support these findings.

Follicular phase serum and follicular fluid glycodelin measurements in gonadotropin-releasing hormone (GnRH)-antagonist assisted reproduction cycles: A prospective cohort study.

PURPOSE: To establish the serum pattern for glycodelin and to investigate the possible correlations of serum and follicular fluid (FF) glycodelin with clinical pregnancy in gonadotropin-releasing hormone (GnRH)-antagonist controlled cycles. MATERIALS AND METHODS: A prospective observational study conducted with 80 infertile couples who received a GnRH-antagonist controlled cycle. Glycodelin levels were measured in FF, day 2-3, and ovarian pick-up (OPU)-day serum samples. RESULTS: There were no significant differences in serum glycodelin concentrations in either the early follicular phase or the preovulatory phase, and in FF glycodelin concentrations between clinically pregnant and non-pregnant patients. OPU-day serum glycodelin was found to be significantly higher than early follicular serum glycodelin level in all patients whether pregnancy occurred or not. CONCLUSION: Although day 2-3 and OPU-day measurements of serum glycodelin levels were not significant in predicting clinical pregnancy, the pattern of serum glycodelin seems different in GnRH-antagonist controlled cycles than natural and GnRH-agonist controlled cycles.

Ulipristal acetate resembles mifepristone in modulating human fallopian tube function.

STUDY QUESTION: Do ulipristal acetate (UPA) and mifepristone have an effect on ciliary beat frequency and muscular contractions in the human Fallopian tube? SUMMARY ANSWER: UPA, in resemblance to mifepristone, inhibits ciliary beat and muscular contraction of the human Fallopian tube, probably through an agonistic effect on the tubal progesterone receptor. WHAT IS KNOWN ALREADY: UPA, like mifepristone, acts as an emergency contraceptive mainly by inhibiting ovulation. Little is known about its effects on tubal function. STUDY DESIGN, SIZE, DURATION: This was an in vitro experimental study using Fallopian tube samples collected from 11 women undergoing hysterectomy for benign non-tubal gynaecological conditions. PARTICIPANTS/MATERIALS, SETTING, METHODS: The tubal epithelium and longitudinal smooth muscle fibres were isolated, cultured and treated with UPA at graded concentrations of 0, 20, 200 and 2000 ng/ml, and mifepristone at graded concentrations of 0, 300, 3000 and 30 000 ng/ml, respectively. After treatment, ciliary beat frequency was determined using a photometric method. Basal tone, amplitude and frequency of muscular contraction were recorded through a force transducer. The mRNA expression of progesterone receptor (total and PR-B isoform), glycodelin and adrenomedullin were determined by real-time quantitative PCR. MAIN RESULTS AND THE ROLE OF CHANCE: There was an overall dose-dependent suppressive effect on ciliary beat frequency (P < 0.0001) after treatment with UPA at all concentrations and with mifepristone at 3000 ng/ml or above. The basal tone, amplitude and frequency of muscular contractions were significantly reduced (P < 0.05) after treatment with UPA at 200 ng/ml or above, and with mifepristone at 3000 ng/ml or above. UPA treatment at 200 ng/ml or above significantly up-regulated the mRNA expression of progesterone receptor and glycodelin and down-regulated the mRNA expression of adrenomedullin in Fallopian tube tissue (P < 0.05). LIMITATIONS, REASONS FOR CAUTION: Whether or not the tubal effect may translate into additional mechanisms for contraceptive action in vivo is uncertain. WIDER IMPLICATIONS OF THE FINDINGS: The clinical relevance of UPA with regard to contraceptive activity is worthy of further exploration. STUDY FUNDING/COMPETING INTERESTS: The study was supported by a Seed Fund from the Centre of Reproduction, Development and Growth, Faculty of Medicine, the University of Hong Kong. All authors have no competing interest to declare.