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Embryo implantation requires synchronized dialogue between the receptive endometrium and activated blastocyst via locally produced soluble mediators. During the mid-secretory (MS) phase of the menstrual cycle, increased glandular secretion into the uterine lumen provides important mediators that modulate the endometrium and support the conceptus during implantation. Previously we demonstrated the importance of vascular endothelial growth factor (VEGF) in the human uterus, particularly with respect to embryo implantation. In the current study, proteomic analysis of human uterine lavage fluid identified the presence of placental growth factor (PlGF) a homolog of VEGF, that binds the VEGF receptor 1 (VEGFR1). Analysis of immunostaining for PlGF in human endometrial tissue across the menstrual cycle (from both fertile and infertile women) revealed PlGF was predominantly localised to glandular and luminal epithelial cells, with staining in the decidualising stromal cells surrounding the maternal spiral arteries in the secretory phase of the menstrual cycle. Immunoreactive PlGF was also detected in subpopulations of endometrial leukocytes. Functional studies demonstrated that culturing mouse embryos with recombinant human (rh)PlGF enhanced blastocyst cell number and outgrowth. Furthermore, treatment of human endometrial epithelial cells (EEC) with rhPlGF enhanced EEC adhesion. Taken together, these data demonstrate that PlGF is abundant in the human endometrium, and secreted into the uterine lumen where it mediates functional changes in cellular adhesion with important roles in implantation.

Background We hypothesized that genes that are up-regulated in the uterine endometrium at the initiation of conceptus elongation in cattle, and that encode for secreted proteins, contribute to the composition of the uterine luminal fluid (ULF) and ultimately, drive conceptus elongation. The aims of this study were to: 1) screen endometrial transcriptomic data for genes that encode secreted proteins on Day 13; 2) determine temporal changes in the expression of these genes during the estrous cycle/early pregnancy; 3) determine if expression of these genes is affected by altered concentrations of progesterone (P4) in vivo and 4) determine if the protein products of these genes are detectable in ULF. Results Of the fourteen candidate genes examined, quantitative real-time PCR analysis revealed the expression of APOA1 , ARSA , DCN , LCAT , MUC13 , NCDN , NMN , NPNT , NXPH3 , PENK , PLIN2 and TINAGL1 was modulated in the endometrium (P<0.05) as the estrous cycle/early pregnancy progressed. APOA1 , DCN and NPNT expression was higher in cyclic compared to pregnant heifers, and pregnancy increased (P<0.05) the expression of LCAT , NCDN , NMN , PLIN2 and TINAGL1 . The magnitude of the increase in expression of APOA1 , PENK and TINAGL1 on Day 13 was reduced (P<0.05) in heifers with low P4. Furthermore, low P4 decreased (P<0.05) the expression of LCAT and NPNT on Day 7, while an early increase (P<0.05) in the expression of NXPH3 and PLIN2 was observed in heifers with high P4. The protein products of 5 of the candidate genes ( APOA1, ARSA, LCAT, NCDN and PLIN ) were detected in the ULF on either Days 13, 16 or 19 of pregnancy. Conclusion Using a candidate gene approach, we determined that both P4 concentration and the presence of the conceptus alter endometrial expression of PLIN2 , TINAGL1 , NPNT , LCAT , NMN and APOA1 . Comparison of the expression profiles of these genes to proteins detected in ULF during conceptus elongation (i.e., Days 13 through 19) revealed the presence of APOA1, ARSA, LCAT, NCDN as well as members of the PLIN family of proteins that may play roles in driving conceptus elongation in cattle.

In order to determine the effect of endometrial injury (EI) on in vitro fertilization (IVF) outcomes in women with unexplained subfertility and explore the relationship between EI and endometrial inflammatory cytokines, 66 women with unexplained subfertility undergoing IVF treatment were recruited. 38 patients in the EI group underwent EI in the mid-luteal phase of the cycle and 28 patients in the non-EI (NEI) group. According to the pregnancy outcome, the NEI and EI groups were divided into NEI-nonpregnant (NEI-NP), NEI-pregnant (NEI-P), EI-NP, and EI-P. All patients underwent aspiration of endometrial secretions immediately before embryo transfer. The concentrations of ten mediators were measured using Milliplex Magnetic Bead assay. The clinical pregnancy was significantly higher in the EI than in the NEI group. The concentrations of interleukin- (IL-) 6, IL-8, IL-12 (p70), IL-13, interferon- (IFN-) γ, monocyte chemotactic protein- (MCP-) 1, and vascular endothelial growth factor (VEGF) were significantly higher in the EI than the NEI group. The expression of IFN-γ and VEGF in the EI-P was significantly increased compared to the EI-NP group. These findings suggest that, in women with unexplained subfertility, endometrial injury might be a potential method to improve clinical pregnancy rates by promoting the expression of IFN-γ and VEGF.

Purpose Along with comparative investigation of the decidualization potential and IL-6 secretion by fresh and frozen ESCs, we also aimed to evaluate the effectiveness of co-culture systems based on fresh or frozen ESCs in terms of clinical pregnancy rates. Methods Outcome analysis of a total of 215 IVF cycles with co-culture with fresh or frozen ESCs was performed. Endometrial tissue was obtained from 17 healthy donors. Concentrations of secreted prolactin, IGFBP-1, and IL-6 in conditioned media from cultured fresh and frozen ESCs (decidualized or not) were measured using ELISA or ECLIA. Results Embryo co-culture with frozen ESCs resulted in a much lower pregnancy rate compared to the alternative system using fresh ESCs. Furthermore, cultivated frozen ESCs showed considerably decreased release of prolactin, IGFBP-1, and IL-6 compared to fresh ESCs, indicating that cryopreservation negatively affects their decidualization potential and cytokine production. Conclusions Altogether, this data illustrates the need for optimization and improvement of the existing autologous endometrial co-culture systems.

The divergent requirement for tolerance to support conception and protective response against sexually transmitted infections defines the unique immunological dynamics in the female reproductive tract (FRT). In part, these requirements are achieved by the cyclic modulation of cytolytic CD8T cell function in the FRT that underlie the respective immunosuppressive and immunocompetent milieus during the secretory and proliferative phases of the menstrual cycle. The CD8T cell function can be dampened by exposure to indoleamine 2,3-dioxygenase and/or arginase enzymes. Indeed, these 2 enzymes are known as primary inducers of immune suppression in tumor microenvironments. This review discusses the intriguing parallel expression of these 2 enzymes in tumor microenvironments and in the secretory endometrium. We surmise that investigating the underlying natural mechanisms that suppress and restore the immunocompetence of CD8T cells in the FRT each month may provide valuable insights into ways to artificially recapitulate these mechanisms and inhibit immune suppression in tumor microenvironments.

This study describes the novel localization of the antimicrobial peptide human intestinal defensin-5 (HD-5) in female genital tract epithelia. Using a 3' rapid amplification of cDNA ends (RACE) protocol, HD-5 was cloned from a vaginal epithelial cell RNA preparation, and its identity was confirmed by sequencing. Tissue samples from multiple donors were subsequently screened for HD-5 expression by reverse transcription polymerase chain reaction. HD-5 message was invariantly expressed by normal vagina and ectocervix and inflamed fallopian tube, but variably expressed by normal endocervix, endometrium, and fallopian tube (60, 64, and 29% of specimens, respectively). Expression in endometrium was the highest during the early secretory phase of the menstrual cycle. Using immunohistochemistry and confocal microscopy, HD-5 peptide was localized in the upper half of the stratified squamous epithelium of the vagina and ectocervix, with the intensity of cellular staining increasing toward the lumen. In positive endocervix, endometrium, and fallopian tube specimens, HD-5 was located in apically oriented granules and on the apical surface of a proportion of columnar epithelial cells. Using Western blot analysis, secreted HD-5 was detected in cervicovaginal lavages, with the highest concentrations found during the secretory phase of the menstrual cycle. We hypothesize that HD-5 is an intrinsic component of the female urogenital innate immune defense system and that its expression may be modulated by hormonal and proinflammatory factors.

Inhibins (INH) are dimeric glycoproteins, composed of an alpha subunit (INH-alpha) and one of two possible beta subunits (INH-betaA or INH-betaB). They have substantial roles in human reproduction and in endocrine-responsive tumours. Therefore, the aims of this study were to determine the frequency and tissue distribution of INH-alpha, INH-betaA and INH-betaB in normal human endometrium and glandular-cystic endometrial polyps, and polyps caused by tamoxifen use. Tissue samples were obtained from women in the proliferative, early secretory and late secretory phase as well as glandular-cystic polyps and endometrial polyps associated with tamoxifen use (n = 5 each). Immunohistochemistry with specific monoclonal antibodies, a semi-quantitative analysis and statistical evaluation was performed. INH-alpha, INH-betaA and INH-betaB were primarily observed in glandular and luminal epithelial cells, with a variant staining intensity in stromal cells. INH-alpha in glands was significantly higher during the early secretory phase (p < 0.05) and the late secretory phase (p < 0.01) than in the proliferative phase with a significant difference between the early secretory and the late secretory phases (p < 0.01). INH-betaA expression was significantly higher during the late secretory than the proliferative phase (p < 0.05) and the late secretory than the early secretory phase (p < 0.05), with no significant differences for INH-betaB. Glandular-cystic polyps showed significantly lower expression of INH-alpha and INH-betaA than the late secretory endometria (p < 0.05 and p < 0.01 respectively). Additionally, tamoxifen-associated polyps also demonstrated a significantly lower expression of INH-alpha and INH-betaA than late secretory endometria (p < 0.01 and p < 0.01 respectively). No statistical differences were observed between tamoxifen-associated and glandular-cystic polyps. INH-alpha, INH-betaA and INH-betaB were expressed in normal endometrium and endometrial polyps. A cyclical expression of INH-alpha and INH-betaA in normal glands may reflect a functional and hormone-dependent role in human endometrium. Significant differences in staining reaction between the late secretory endometria and polyps suggest that this tissue remains in the proliferating state rather than the secretory state. Therefore, endometrial polyps may be tumours of dysregulation with mainly proliferating characteristics, being unable to synchronise with normal endometrium.

Inhibins are multipotent dimeric glycoproteins, composed of an alpha-subunit and one of two possible beta-subunits (betaA or betaB). Aims of this study were (a): the immunohistochemical characterisation of normal human endometrium for the inhibin-alpha subunit; (b) the assessment of the secretion and metabolism of inhibin, E2 and cortisol; (c) the evaluation of any relationship between these three substances in cell culture medium of isolated and cultivated normal human endometrial glandular cells. Samples of human endometrium were obtained from 34 premenopausal patients. Nineteen endometrial specimen (proliferative [PP] n=8; early secretory [ES] n=7; late secretory phase [LS] n=4) were brought into cell culture. Fifteen endometrial specimen (PP n=5; ES n=5; LS n=5) were paraffin-fixed and used for the immunohistochemical analysis for inhibin-alpha. Stromal and epithelial cells were separated by collagenase digestions, filtrations, sedimentations and Ficoll-gradient centrifugation. E2 and cortisol were measured with radioimmunoassay (RIA) and inhibin with enzyme-immunoassay (EIA). Statistical analysis was performed with the non-parametric Mann-Whitney rank-sum test and linear regression analysis. Inhibin-alpha showed a weak (positive) expression during proliferative phase, which increased significantly as the menstrual cycle continued. In secretory glands the mean inhibin concentration was higher than that from proliferative samples. A significant correlation was observed between inhibin and E2 (p<0.001) as well as cortisol and inhibin (p<0.0001) in glands from proliferative phase. Between inhibin and E2 (p<0.05) as well as inhibin and cortisol (p<0.002) a significant correlation in early secretory glands was also noted. In late secretory phase inhibin and E2 (r2=0.78650; p<0.0001), inhibin and cortisol (r2=0.58326; p<0.001) and E2 and cortisol (r2=0.52880; p<0.001) showed a significant correlation. In conclusion, we found a cyclical expression of inhibin-alpha subunit in the endometrium demonstrated by immunohistochemical means. A higher in vitro secretion of inhibin from secretory glands was also observed. In addition, a significant correlation between inhibin with E2 and cortisol in PP and ES glands and a significant correlation between inhibin, E2 and cortisol in LS glands could also be demonstrated. We conclude that inhibin can be associated with E2 and cortisol metabolism, playing an important role in paracrine/autocrine mechanisms in the endometrium and possibly exerting its function through cortisol and E2. The cortisol concentration also correlates with E2, suggesting a link between these steroids in the endometrial function. The correlation of inhibin, E2 and cortisol suggest complex autocrine/ paracrine mechanisms in human endometrial glands, modulated and controlled by all these three substances.

Secretion into the uterine lumen follows a precise pattern during early pregnancy. Near the end of the second week of pregnancy and coincident with elongation of conceptuses, retinol, retinol binding protein (RBP), estradiol (E2), and prostaglandins E (PGE) and F (PGF) increase in the uterine lumen, and RBP mRNA increases in the endometrium. In the present studies the potential for E2 (0.1 microM) and retinol (10 microM) to regulate RBP and PG production by cultured luminal (LEC) and glandular (GEC) epithelial cells collected from postpubertal females and LEC from prepubertal gilts was examined. Endometrial tissue was collected surgically from cyclic and pregnant females (n = 8) on d 10 and 13 postestrus (first day of estrus = d 0) and from 120- and 150-d-old prepubertal gilts that were treated with progesterone (P4) (2.2 mg x kg(-1) x d(-1), n = 6) or corn oil (n = 6) for 14 d prior to tissue collection. The LEC from postpubertal females responded to retinol with increased (P < 0.05) RBP, PGE, and PGF in culture medium and increased (P < 0.07) RBP mRNA but E2 decreased (P < 0.05) RBP and RBP mRNA and had no effect on prostaglandins. No E2 or retinol effects on secretions of GEC occurred in vitro, but a day x pregnancy status interaction (P < 0.06) affected PGE output by the GEC. Secretion of PGE was greater when GEC were collected on d 10 of pregnancy than from d-10 cyclic or d-13 pregnant or cyclic females. Both E2 and retinol stimulated (P < 0.05) secretion of RBP by LEC isolated from prepubertal gilts, but their effects were not additive. In vivo treatment of prepubertal gilts with P4 increased (P < 0.05) RBP and decreased (P < 0.05) PG production by LEC in vitro. Therefore responses to E2 and retinol differ between pre- and post-pubertal females, and retinol may function in the regulation of endometrial RBP and PG secretion.

We have investigated whether there are any differences in the release of urokinase plasminogen activator (uPA) and its inhibitor (PAI-1) from cultured endometrial and endometriotic stromal cells, and whether the release is regulated by epidermal growth factor (EGF) or transforming growth factor β1 (TGFβ1). The cells were isolated from endometriomas and endometrium from women with and without endometriosis. After treatment with EGF or TGF and in untreated controls, incubated media collected at 0, 24, 48 and 72 h were analyzed by ELISA. Stromal cells from all three types of tissues released uPA and PAI-1, but the soluble receptor of uPA was not measurable in any group. The basal release of uPA and PAI-1 from endometriotic cells was higher than from endometrial cells. The uPA release in endometriotic cells was reduced with and without the addition of EGF (p < 0.05) or TGFβ1 (p < 0.05). EGF increased the release of PAI-1 from stromal cells from women without endometriosis (p < 0.05) but decreased the release of PAI-1 from stromal cells from endometriotic women (p < 0.05). TGFβ1 increased the release of PAI-1 from endometriotic cells (p < 0.05) but had no effect in endometrial cells.