Non-decidualized Endometrium Research Articles

In situ Synthesized Monosodium Urate Crystal Enhances Endometrium Decidualization via Sterile Inflammation During Pregnancy.

High level of uric acid (UA) is the major origin of gout, and is highly associated with various pregnant complications, such as preeclampsia and gestational diabetes. However, UA’s level and role in the very early stage of pregnancy has not been uncovered. This study aims to investigate the relevance of serum UA and decidualization, an essential process for the establishment and maintenance of pregnancy in women and mice during the early stage of pregnancy. In this study, we first proved that expression level of UA synthase xanthine dehydrogenase (XDH) is highly increased along with decidualization of endometrial stromal cells in both in vitro and in vivo models. Furthermore, serum and endometrial levels of UA are higher in mice with decidualized uterin horn and in vitro decidualized stromal cells. The existence of monosodium urate (MSU) crystal was also confirmed by immunostaining. Next, the roles of MSU on decidualization were explored by both in vitro and in vivo models. Our data shows MSU crystal but not UA enhances the decidualization response of endometrial stromal cells, via the upregulation of inflammatory genes such Ptgs2 and Il11. inhibiting of Cox-2 activity abolishes MSU crystal induced higher expression of decidualization marker Prl8a2. At last, in women, we observed enriched expression of XDH in decidua compare to non-decidualized endometrium, the serum level of UA is significantly increased in women in very early stage of pregnancy, and drop down after elective abortion. In summary, we observed an increased serum UA level in the early stage of women’s pregnancy, and proved that the increased level of UA results from the expressed XDH in decidualizing endometrium of both human and mouse, leading to the formation of MSU crystal. MSU crystal can enhance the decidualization response via inflammatory pathways. Our study has uncovered the association between UA, MSU, and decidualization during the early stage of pregnancy.

Matrix metalloproteinase expression and activity in trophoblast-decidual tissues at organogenesis in CF-1 mouse

During early placentation, matrix metalloproteinases (MMPs) play important roles in decidualization, trophoblast migration, invasion, angiogenesis, vascularization and extracellular matrix (ECM) remodeling of the endometrium. The aim of our study was to analyze the localization, distribution and differential expression of MMP-2 and -9 in the organogenic implantation site and to evaluate in vivo and in vitro decidual MMP-2 and -9 activities on day 10 of gestation in CF-1 mouse. Whole extracts for Western blotting of organogenic E10-decidua expressed MMP-2 and -9 isoforms. MMP-2 immunoreactivity was found in a granular and discrete pattern in ECM of mesometrial decidua (MD) near maternal blood vessels and slightly in non-decidualized endometrium (NDE). Immunoexpression of MMP-9 was also detected in NDE, in cytoplasm of decidual cells and ECM of vascular MD, in trophoblastic area and in growing antimesometrial deciduum. Gelatin zymography showed that MMP-9 activity was significantly lower in CM compared to the active form of direct (not cultured) and cultured decidua. The decidual active MMP-9 was significantly higher than the active MMP-2. These results show differential localization, protein expression and enzymatic activation of MMPs, suggesting specific roles for MMP-2 and MMP-9 in decidual and trophoblast tissues related to organogenic ECM remodeling and vascularization during early establishment of mouse placentation.

PreImplantation Factor (PIF) promoting role in embryo implantation: increases endometrial Integrin-α2β3, amphiregulin and epiregulin while reducing betacellulin expression via MAPK in decidua

BackgroundViable embryos secrete preimplantation factor (PIF), a peptide that has autocrine effects where levels correlate with cultured embryos development. sPIF (PIF synthetic analog) promotes implantation by regulating decidual-cells immunity, adhesion, apoptosis and enhances trophoblastic cell invasion. Herein sPIF priming effects on non-decidualized endometrium and decidualized-stroma are investigated, assessing elements critical for effective embryo-maternal cross-talk, prior to and at implantation.MethodsWe tested sPIF effect on human non-pregnant endometrial epithelial and non-decidualized stroma α2β3 integrin expression (IHC and flow cytometry), comparing with scrambled PIF (PIFscr-control). We examined sPIF effect on decidualized non-pregnant human endometrial stromal cells (HESC) determining pro-inflammatory mediators expression and secretion (ELISA) and growth factors (GFs) expression (Affymetrix global gene array). We tested sPIF effect on HESC Phospho-kinases (BioPlex) and isolated kinases activity (FastKinase).ResultssPIF up-regulates α2β3 integrin expression in epithelial cells, (P < 0.05) while PIFscr had no effect. In contrast, in stromal cell cultures sPIF had no effect on the same. In HESC, sPIF up-regulates pro-inflammatory cytokines; IL8, IL1β and IL6 expression. The major increase in GRO-α, ICAM-1 and MCP-3 expression is coupled with same ligands secretion (P < 0.05). sPIF modulates in HESC GFs expression: up-regulates amphiregulin and epiregulin- critical for implantation and enhances several fibroblast growth factors (FGF) relevant for decidual function. In contrast, sPIF down-regulates major pro-proliferative ligands, betacellulin and IGF1 expression. sPIF modulatory effect on GFs is exerted by down-regulating pro-proliferative phospho-activated MAPkinases, p-MEK1 and p-ERK (P < 0.01, P < 0.04, respectively). Stress-induced p-38-MAPK (P = 0.04) and c-Jun kinase signaling involved MAPK8IP2 (−2.1 fold) expression decreased which protects against reactive oxygen species. Although pro-inflammatory p-NFkB (P = 0.06) decrease was mild, its promoter TNFRS11 expression markedly (−25-fold) decreased. In contrast, anti-proliferative phosphatases PTPRZ1 and PPP2R2C expression increased.ConclusionssPIF post-fertilization primes endometrial-epithelium, while during implantation creates a beneficial pro-inflammatory milieu. PIF acts by balancing decidual pro-implantation properties while controlling excessive pro-proliferative and inflammatory signals expression. Overall, PIF influences critical peri-implantation events in a sequential coordinated fashion which facilitates embryo implantation.

T9.3 Decidual cell surface and secreted factors regulate extravillous trophoblast function: important implications in placental development

Integrins are heterodimeric glycoproteins acting as membrane receptors for extracellular matrix components. The specificity of these receptors towards one particular matrix glycoprotein depends on the type of α and β subunit combination. Since integrins are involved in the migratory behaviour of cells and since cytotrophoblastic cells are constitutively invasive, we undertook to immunolocalize the α2, α5 and α6 integrin subunits in normal and hydatidiform molar trophoblast, in an implantation site as well as in decidualized and non-decidualized endometrium. α6 positivity was confined to villous cytotrophoblast and was clearly polarized towards the basement membrane. Extravillous cytotrophoblastic cells were α6-negative but became α5-positive. In contrast to normal trophoblast, villous cytotrophoblast from hydatidiform molar tissue was α5-positive. We conclude that the expression of a α5 integrin subunit on cytotropholastic cell surfaces is correlated with the appearance of an invasive phenotype. α6 and α2 integrin subunits were both localized on the surface and glandular epithelium of the endometrium and their expression was increased during the secretory phase but became low or undetectable after decidualization. In contrast, α5 subunit positivity was weak in the same epithelia during the first half of the cycle but disappeared after ovulation. Stromal cell α5 positivity was present throughout the cycle but increased dramatically in decidualized endometria. We conclude that the α5 integrin subunit which disappears from the epithelium at the end of the cycle might allow migration of the epithelial cells and repair of the endometrium after menses. We also wonder if α5 positivity is part of a change in the stromal cell phenotype induced by decidualization.

T9.2 Physiological modifications of the uteroplacental vascularisation: the role of the subplacental arterio-venous shunt

Integrins are heterodimeric glycoproteins acting as membrane receptors for extracellular matrix components. The specificity of these receptors towards one particular matrix glycoprotein depends on the type of α and β subunit combination. Since integrins are involved in the migratory behaviour of cells and since cytotrophoblastic cells are constitutively invasive, we undertook to immunolocalize the α2, α5 and α6 integrin subunits in normal and hydatidiform molar trophoblast, in an implantation site as well as in decidualized and non-decidualized endometrium. α6 positivity was confined to villous cytotrophoblast and was clearly polarized towards the basement membrane. Extravillous cytotrophoblastic cells were α6-negative but became α5-positive. In contrast to normal trophoblast, villous cytotrophoblast from hydatidiform molar tissue was α5-positive. We conclude that the expression of a α5 integrin subunit on cytotropholastic cell surfaces is correlated with the appearance of an invasive phenotype. α6 and α2 integrin subunits were both localized on the surface and glandular epithelium of the endometrium and their expression was increased during the secretory phase but became low or undetectable after decidualization. In contrast, α5 subunit positivity was weak in the same epithelia during the first half of the cycle but disappeared after ovulation. Stromal cell α5 positivity was present throughout the cycle but increased dramatically in decidualized endometria. We conclude that the α5 integrin subunit which disappears from the epithelium at the end of the cycle might allow migration of the epithelial cells and repair of the endometrium after menses. We also wonder if α5 positivity is part of a change in the stromal cell phenotype induced by decidualization.

Immunocytochemical Studies of Adhesion Molecules on Mouse UNK Cells and Their Extracellular Matrix Ligands During Mouse Pregnancy

Uterine natural killer (uNK) cells are the dominant lymphocytes of pregnant mammals' uterus. Studies have identified four differentiation stage of mouse uNK cells based on Dolichos biflorus lectin cytochemistry, and their distribution showed preferential domain in the uterus through out the pregnancy. This work was done to investigate the expression of alpha5, alpha6, and beta7 integrins on uNK cells and their ligands distribution. Section of mouse uterus from sixth to seventeenth gestational days were submitted to immunocytochemistry and positive reactions for alpha5, alpha6, and beta7 integrins were found on uNK from eighth to tenth gestational days but not after twelfth gestational days. Fibronectin reactions were seemed from sixth to tenth gestational days around uNK from the myometrium and endometrium close to the myometrium. No reaction for fibronectin was seen in the decidualized and nondecidualized endometrium near the placenta. Laminin reaction was seen just in the antimesometrial side. beta7 integrin seems to be the active receptor to bind with VCAM-1 or MAdCAM-1 of endothelial cells, promoting the uNK cross through the vessels. The absence of laminin in an uNK domain suggests these cells are not dependent of laminin and alpha6 integrin for their establishment. However, fibronectin seems to support uNK migration, proliferation, differentiation, and survival in the uterus by binding with alpha5 integrin. The loss of alpha5 integrin ligation by the down regulation of fibronectin could inhibits these events and further studies are need to investigate whether unligated alpha5 can actively and initiate apoptosis, maybe in a caspase 8-dependent way that has been called integrin-mediated death.

The long-term actions of etonogestrel and levonorgestrel on decidualized and non-decidualized endometrium in a mouse model mimic some effects of progestogen-only contraceptives in women

Breakthrough bleeding (BTB), a major side effect of long-acting progestogen (p)-only contraceptives in women, is the main reason for discontinuation of their use. To understand the mechanisms of BTB, a mouse model of endometrial breakdown and repair was adapted to evaluate the effects of long-term progestogens on the endometrium. Appropriately prepared mice received either an etonogestrel (ENG)- or levonorgestrel (LNG)-releasing subdermal implant. Forty eight hours after decidualization was induced in one uterine horn the majority of tissues were highly decidualized, designated 0 day (0d). Uteri were collected subsequently at 5-day intervals (to 45d) and both decidualized and non-decidualized horns were analysed for morphological changes, leukocyte infiltration and matrix metalloproteinase expression (MMP). In decidualized horns, large blood vessels (BV) developed and disturbance of tissue integrity was observed at 5d with substantial stromal breakdown by 10d, progressing until 25d when re-epithelialization was initiated. By 45d, the tissue was restored to its pre-decidualized state but with considerable tortuosity of the luminal epithelium. Tissue remodelling was not apparent in the non-decidualized horns before 35d, when hyperproliferation of the luminal epithelium resulted in tortuosity. Changes in morphology were similar with the two progestogens, but occurred more rapidly with LNG. Apart from macrophages, few leukocytes were present in non-decidualized horns but large infiltrates of neutrophils and uterine natural killer cells (uNK) were associated with tissue breakdown in decidualized tissue, many of these cells were MMP9-positive. MMP7 was primarily associated with tissue repair. Therefore, this model mimics some of the changes observed in endometria of women using p-only contraceptives and provides an opportunity for functional studies.

Temporal and cell-specific gene expression by human endometrium after coculture with trophoblast

Objective: Our purpose was to identify temporal and stage-specific expression of endometrial genes during coculture with trophoblast cells. Study Design: Endometrial stromal cells were cultured to confluence in the presence of estradiol and progesterone. During these culture conditions the gene expression of 1 tissue specimen that secreted abundant prolactin (415 ng/mL culture medium at 21 days) was compared with a second specimen that did not. These 2 tissues were coincubated with trophoblast tissue in a specialized coculture flask. After 4 and 24 hours of culture messenger ribonucleic acid was extracted and reverse transcribed, and the complementary deoxyribonucleic acid products were amplified by polymerase chain reactions. The reverse transcriptase–polymerase chain reaction products were separated by electrophoresis, and potentially important complementary deoxyribonucleic acid fragments were reamplified, inserted into a plasmid vector, and sequenced after recovery. Sequences were submitted for Basic Local Alignment Search Tool searches of GenBank. Results: We observed up-regulation of 6 gene fragments in decidualized endometrium after 4 hours of coculture with choriocarcinoma-derived trophoblast BeWo cells, but only 1 gene fragment was up-regulated after 24 hours of exposure. Conversely, 2 fragments were down-regulated in decidualized stroma that was exposed to BeWo for 4 hours and 2 fragments were underexpressed after the 24-hour exposure. In the parallel experiment stromal cells that failed to secrete prolactin did not elicit the same regulation of expression. The nondecidualized endometrium overexpressed 1 gene fragment after 4 hours of BeWo exposure and overexpressed 4 gene fragments after exposure to BeWo for 24 hours. Underexpression of gene products also occurred with the nondecidualized endometrium, and we observed 2 fragments and 1 fragment to be underexpressed after 4 and 24 hours of BeWo exposure, respectively. To date, 3 of the candidate differential display fragments from these experiments have been cloned and sequenced. An up-regulated fragment (C6225J4EB-1) was 99% identical (167/168 sequences) to a reported nonredundant expressed sequence tags isolated from muscle, brain, ovary, testis, liver, and pregnant uterus tissues. A second up-regulated fragment (C4375J4EB-1) matched 100% identity (117/117) with a reported gene fragment in the expressed sequence tags database of GenBank that was derived from fetal heart and pregnant uterus. Additional characterization of these expressed sequence tags has not been reported. The third up-regulated fragment (C4250J24EB-2) was 100% identical (265/265) to human reduced nicotinamide adenine dinucleotide dehydrogenase III in the nonredundant gene database of GenBank. Conclusion: This report demonstrates the potential usefulness that endometrial-trophoblast coculture and differential display can offer for the molecular analysis of implantation phenomena. We have recognized both overexpression and underexpression of interesting gene fragments during the early phases of endometrial responses to paracrine regulators derived from BeWo trophoblast cells. These responses appear to be specific to the degree of endometrial transformation (decidualization) before challenge by the trophoblast and to the duration of the BeWo exposure. Sequence data identified 1 gene with an unidentified function, another gene with a known function, and a fragment not previously recognized. We submit that our model of endometrial-trophoblast coculture offers a novel tool to test cellular responses during implantation, and differential display represents a sensitive technique that can identify many of the important elements of genomic signaling during nidation. (Am J Obstet Gynecol 1999;180:806-14.)

Insulin-like Growth Factor Binding Protein Gene Expression in the Pregnant Rat Uterus and Placenta

While the insulin-like growth factor (IGF) system plays a fundamental role in regulating embryonic and placental growth, the specific contributions of the six IGF binding proteins (IGFBPs 1–6) to these processes are not well understood. We here focus on IGFBP expression in the extraembryonic environment, which both supports and constrains embryonic growth, and have usedin situhybridization to determine sites of IGFBP mRNA synthesis in the pregnant rat uterus and placenta. We find that all IGFBPs are expressed in distinct, changing patterns in the uterine endometrium, at the decidual boundary, in the decidual vasculature, and in the myometrium during pregnancy. Within the endometrium, the most prominent change is that expression of IGFBP-1 begins in some, but not all, endometrial glands prior to implantation and then expands to include all secretory epithelia shortly after implantation. During the period of rapid decidual proliferation that follows implantation, IGFBP-3, -4, and -5 transcripts are all detected in a laminar array at the boundary between the decidua and the nondecidualized endometrium. In the decidual vasculature at Day (d) 8.0, both IGFBP-3 and IGFBP-4 mRNAs are detected in dilating blood vessels, with BP-3 most prominent in the antimesometrial plexus and BP-4 primarily at the mesometrial pole. Later (d11.5), all decidual vessels express high levels of IGFBP-3 and lower levels of IGFBP-4 mRNAs. Finally, changes in expression of several IGFBPs also occur within the myometrium during pregnancy. For example, IGFBP-2 is expressed in the inner circular layer shortly after implantation, and expression increases through late gestation. In contrast, IGFBP-5 hybridization occurs over both myometrial layers before implantation, but decreases in intensity and spatial distribution as pregnancy proceeds. Finally, and most strikingly, IGFBP-6 expression, barely detectable in the d7.0 myometrium, gradually increases until it is very strongly transcribed during the placental stages. Taken together, these observations suggest multiple roles for IGFBPs in supporting implantation, regulating the extent of decidualization, modulating local levels of vascular IGFs, and regulating uterine muscular growth.

Differential Expression of Two β-Galactoside-Binding Lectins in the Reproductive Tracts of Pregnant Mice1

Two beta-galactoside-binding proteins were isolated from uteroplacental complexes of pregnant mice and identified as the S-Lac lectins galectin-1 and galectin-3. The spatiotemporal pattern of appearance of those proteins was determined by immunocytochemistry. Galectin-1 was present in all tissue compartments of the uterus except the luminal and glandular epithelium. It was found in the uteri of animals from all preimplantation stages of pregnancy, as well as in those from nonpregnant, ovariectomized, or sexually immature animals. After implantation of the embryo, cells of the decidua basalis were labeled, as were granular metrial gland cells, all trophoblastic elements of the placenta, the myometrium, and nondecidualized endometrium. By contrast, there was little evidence of galectin-3 in the uteri of nonpregnant animals or during the preimplantation stages of pregnancy. However, immunoreactive material was observed in endometrial cells of the primary decidual zone immediately after implantation and at later stages of pregnancy in the decidua basalis, metrial gland, and all trophoblastic elements of the placenta. There was no evidence of galectin-3 in the myometrium or nondecidualized endometrium. After parturition, amounts of galectin-3 in the endometrium and metrial triangle appeared to decrease as the implantation sites were resorbed. These data suggested that the function of galectin-1 is one of tissue maintenance, whereas the function of galectin-3 is related specifically to pregnancy.

Epithelial localization of insulin-like growth factor binding protein 1 in the uterus of the rat during pregnancy, deciduoma-bearing pseudopregnancy and hormone treatment.

A monospecific antibody was used to determine the immunocytochemical localization of insulin-like growth factor binding protein 1 (IGFBP-1) in the rat uterus. Immunoreactive IGFBP-1 was first detected from day 5 of pregnancy in the luminal and glandular epithelium. However, immunoreactivity was most intense from day 6 in the glandular epithelium, where it was associated with apically located granules. Immunoreactive glands were located only in non-decidualized endometrium, which was limited at the implant site to a thin basal layer by growth of the antimesometrial decidua from day 7. However, glands and associated immunoreactive IGFBP-1 were prominent in the inter-implant sites until day 9, although they were detected throughout pregnancy. Similar reactivity was detected in the glands of the basal endometrium in deciduomata-bearing animals, but these decreased in number from day 7 of pseudopregnancy. No immunoreactivity was detected during the oestrous cycle but could be induced in ovariectomized animals by sequential oestradiol and oestradiol plus progesterone treatment. The observations were consistent with IGFBP-1 representing a secretory product of the glandular epithelium and could either play a role in development of the trophoblastic component of the conceptus during the pre-placental period of anti-mesometrial implantation or in the endometrium acting as an inhibitor of local IGF-I action and in either case by transporting IGF-I from the stromal to the glandular luminal environment.

Localization of alpha 2, alpha 5 and alpha 6 integrin subunits in human endometrium, decidua and trophoblast

Integrins are heterodimeric glycoproteins acting as membrane receptors for extracellular matrix components. The specificity of these receptors towards one particular matrix glycoprotein depends on the type of α and β subunit combination. Since integrins are involved in the migratory behaviour of cells and since cytotrophoblastic cells are constitutively invasive, we undertook to immunolocalize the α2, α5 and α6 integrin subunits in normal and hydatidiform molar trophoblast, in an implantation site as well as in decidualized and non-decidualized endometrium. α6 positivity was confined to villous cytotrophoblast and was clearly polarized towards the basement membrane. Extravillous cytotrophoblastic cells were α6-negative but became α5-positive. In contrast to normal trophoblast, villous cytotrophoblast from hydatidiform molar tissue was α5-positive. We conclude that the expression of a α5 integrin subunit on cytotropholastic cell surfaces is correlated with the appearance of an invasive phenotype. α6 and α2 integrin subunits were both localized on the surface and glandular epithelium of the endometrium and their expression was increased during the secretory phase but became low or undetectable after decidualization. In contrast, α5 subunit positivity was weak in the same epithelia during the first half of the cycle but disappeared after ovulation. Stromal cell α5 positivity was present throughout the cycle but increased dramatically in decidualized endometria. We conclude that the α5 integrin subunit which disappears from the epithelium at the end of the cycle might allow migration of the epithelial cells and repair of the endometrium after menses. We also wonder if α5 positivity is part of a change in the stromal cell phenotype induced by decidualization.

Granulocyte-macrophage colony stimulating factor (GM-CSF): one of a family of epithelial cell-derived cytokines in the preimplantation uterus.

Granulocyte-macrophage colony stimulating factor (GM-CSF) is one of a number of lympho-haemapoietic cytokines, including CSF-1, interleukin-6 (IL-6) and leukaemia inhibitory factor (LIF) now known to be synthesized by epithelial cells in the murine uterus. GM-CSF synthesis is regulated primarily by the ovarian steroid hormone oestrogen, but is also subject to modulation by factors including a seminal component of seminal vesicle origin which stimulates a 20-fold increase in luminal fluid content at mating, and bacterial lipopolysaccharide (LPS) and the T-lymphocyte and natural killer (NK) cell product interferon-gamma (IFN gamma). In the non-pregnant mouse GM-CSF synthesis peaks at oestrus. Synthesis is maintained at comparable or moderately higher levels during the preimplantation period of pregnancy and in the non-decidualized endometrium during mid gestation. An embryotrophic activity is suggested by studies in vitro that indicate that GM-CSF stimulates attachment and outgrowth of blastocysts. It is postulated that GM-CSF is of major importance to the physiology of pregnancy through its role as a component of a local cytokine circuit acting to recruit and regulate function of endometrial leukocytes, and by its action as interlocutor and important effector arm in embryo-maternal interactions during gestation.

Characterization of deciduoma marker proteins in hamster uterus: detection in decidual cell cultures.

The objective of this study was first, to identify the proteins associated with decidualization of the hamster uterus by comparing the protein maps of decidualized and nondecidualized endometrium in vivo, and second, to determine whether decidual cell cultures produced these characteristic proteins. Decidualization was induced in one uterine horn, and the contralateral horn was not stimulated (control tissue). Animals were ovariectomized and a subcutaneous progesterone implant was used to maintain decidualization. Uterine proteins from nuclear and cytosol fractions were analyzed by two-dimensional electrophoresis using a highly sensitive protein staining technique. Analysis of nuclear extract and cytosol from decidualized and nondecidualized endometrium from Days 6, 7, and 8 of pseudopregnancy demonstrated the presence of 11 nuclear and five cytosolic deciduoma-associated proteins. Serum and erythrocyte proteins were identified by two-dimensional electrophoresis, and none of the 16 deciduoma-associated proteins was a serum or erythrocyte contaminant. Forty-eight-hour cultures of decidual cells harvested from Day 5 of pseudopregnancy produced all 16 of the deciduoma-associated proteins found in whole tissue in situ. Culture conditions minimized serum and erthrocyte contamination, enhancing the detection of deciduomal cell proteins. Four nuclear and two cytosolic proteins were considered deciduoma specific, i.e., they were not associated with cellular proliferation, as evidenced by their absence from cultures of rapidly dividing fetal hamster fibroblasts. Thus, these studies show that the detection of deciduomal proteins may be a useful criterion for the assessment of decidualization in vitro and in vivo.