The transcriptional repressor Blimp1/PRDM1 regulates the maternal decidual response in mice.

The transcriptional repressor Blimp1 controls cell fate decisions in the developing embryo and adult tissues. Here we describe Blimp1 expression and functional requirements within maternal uterine tissues during pregnancy. Expression is robustly up-regulated at early post-implantation stages in the primary decidual zone (PDZ) surrounding the embryo. Conditional inactivation results in defective formation of the PDZ barrier and abnormal trophectoderm invasion. RNA-Seq analysis demonstrates down-regulated expression of genes involved in cell adhesion and markers of decidualisation. In contrast, genes controlling immune responses including IFNγ are up-regulated. ChIP-Seq experiments identify candidate targets unique to the decidua as well as those shared across diverse cell types including a highly conserved peak at the Csf-1 gene promoter. Interestingly Blimp1 inactivation results in up-regulated Csf1 expression and macrophage recruitment into maternal decidual tissues. These results identify Blimp1 as a critical regulator of tissue remodelling and maternal tolerance during early stages of pregnancy.

Endometrial autophagy is essential for embryo implantation during early pregnancy.

Embryo implantation is an essential and complex process in mammalian reproduction. However, little evidence has indicated the involvement of autophagy during embryo implantation. To determine the possible role of autophagy in uterine of pregnant mice during the peri-implantation stage, we first examined the expression of autophagy-related markers ATG5 and LC3 on day 4, 5, and 6 of pregnancy (D4, D5, and D6, respectively). Compared with expression on D4, downregulation of the autophagy-related markers was observed on D5 and D6, the days after the embryo attached to the receptivity endometrium. Further examination showed that autophagy-related markers ATG5, ATG12, LC3, cathepsin B, and P62 at the implantation site were significantly decreased when comparing with the inter-implantation site. Fewer number of autophagosomes at the implantation site were also observed by transmission electron microscopy. To confirm the functional role of autophagy during embryo implantation in mice, we administered the autophagy inhibitor 3-methyladenine and chloroquine to mice. After treated with 3-methyladenine, the expression of decidual markers HOXA10 and progesterone receptor were significantly reduced. Furthermore, a reduction in implantation sites and increase in the HOXA10 and PR protein levels were observed in response to chloroquine treatment. In addition, impaired uterine decidualization and dysregulation of the PR and HOXA10 protein levels was observed after autophagy inhibited by 3-methyladenine and chloroquine in in vivo artificial decidualization mouse model. In the last, LC3 and P62 were also observed in normal human proliferative, secretory, and decidua tissues. In conclusion, endometrial autophagy may be essential for embryo implantation, and it may be associated with endometrial decidualization during early pregnancy. KEY MESSAGE: • Autophagy-related markers were significantly decreased at implantation site. • Autophagy inhibition results in abnormal decidualization. • Autophagy is essential for embryo implantation.

Tissue stiffness at the human maternal-fetal interface.

STUDY QUESTION: What is the stiffness (elastic modulus) of human nonpregnant secretory phase endometrium, first trimester decidua, and placenta? SUMMARY ANSWER: The stiffness of decidua basalis, the site of placental invasion, was an order of magnitude higher at 103 Pa compared to 102 Pa for decidua parietalis, nonpregnant endometrium and placenta. WHAT IS KNOWN ALREADY: Mechanical forces have profound effects on cell behavior, regulating both cell differentiation and migration. Despite their importance, very little is known about their effects on blastocyst implantation and trophoblast migration during placental development because of the lack of mechanical characterization at the human maternal-fetal interface. STUDY DESIGN, SIZE, DURATION: An observational study was conducted to measure the stiffness of ex vivo samples of human nonpregnant secretory endometrium (N = 5) and first trimester decidua basalis (N = 6), decidua parietalis (N = 5), and placenta (N = 5). The stiffness of the artificial extracellular matrix (ECM), Matrigel®, commonly used to study migration of extravillous trophoblast (EVT) in three dimensions and to culture endometrial and placental organoids, was also determined (N = 5). PARTICIPANTS/MATERIALS, SETTING, METHODS: Atomic force microscopy was used to perform ex vivo direct measurements to determine the stiffness of fresh tissue samples. Decidua was stained by immunohistochemistry (IHC) for HLA-G+ EVT to confirm whether samples were decidua basalis or decidua parietalis. Endometrium was stained with hematoxylin and eosin to confirm the presence of luminal epithelium. Single-cell RNA sequencing data were analyzed to determine expression of ECM transcripts by decidual and placental cells. Fibrillin 1, a protein identified by these data, was stained by IHC in decidua basalis. MAIN RESULTS AND THE ROLE OF CHANCE: We observed that decidua basalis was significantly stiffer than decidua parietalis, at 1250 and 171 Pa, respectively (P < 0.05). The stiffness of decidua parietalis was similar to nonpregnant endometrium and placental tissue (250 and 232 Pa, respectively). These findings suggest that it is the presence of invading EVT that is driving the increase in stiffness in decidua basalis. The stiffness of Matrigel® was found to be 331 Pa, significantly lower than decidua basalis (P < 0.05). LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Tissue stiffness was derived by ex vivo measurements on blocks of fresh tissue in the absence of blood flow. The nonpregnant endometrium samples were obtained from women undergoing treatment for infertility. These may not reflect the stiffness of endometrium from normal fertile women. WIDER IMPLICATIONS OF THE FINDINGS: These results provide direct measurements of tissue stiffness during the window of implantation and first trimester of human pregnancy. They serve as a basis of future studies exploring the impact of mechanics on embryo implantation and development of the placenta. The findings provide important baseline data to inform matrix stiffness requirements when developing in vitro models of trophoblast stem cell development and migration that more closely resemble the decidua in vivo. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Centre for Trophoblast Research, the Wellcome Trust (090108/Z/09/Z, 085992/Z/08/Z), the Medical Research Council (MR/P001092/1), the European Research Council (772426), an Engineering and Physical Sciences Research Council Doctoral Training Award (1354760), a UK Medical Research Council and Sackler Foundation Doctoral Training Grant (RG70550) and a Wellcome Trust Doctoral Studentship (215226/Z/19/Z).

Morphophysical dynamics of human endometrial cells during decidualization.

During decidualization, human mesenchymal-like endometrial stromal cells undergo well characterized cellular and molecular transformations in preparation for accepting a developing embryo. Modulation of cellular biophysical properties during decidualization is likely to be important in receptivity and support of the embryo in the uterus. Here we assess the biophysical properties of human endometrial stromal cells including topography, roughness, adhesiveness and stiffness in cells undergoing in vitro decidualization. A significant reduction in cell stiffness and surface roughness was observed following decidualization. These morphodynamical changes have been shown to be associated with alterations in cellular behavior and homeostasis, suggesting that localized endometrial cell biophysical properties play a role in embryo implantation and pregnancy. This cell-cell communication process is thought to restrict trophoblast invasion beyond the endometrial stroma, be essential in the establishment of pregnancy, and demonstrate the altered endometrial dynamics affecting cell-cell contact and migration regimes at this crucial interface in human reproduction.

Androgens modulate the morphological characteristics of human endometrial stromal cells decidualized in vitro.

The activated androgen receptor (AR) in decidualizing human endometrial stromal cells (HESCs) regulates genes involved in cytoskeletal organization, cell motility, and cell cycle progression. Androgens also enhance the secretion of prolactin, a widely used marker of decidualized HESCs. The purpose of the present study was to investigate the direct effects of androgens on the ultrastructural changes associated with decidual transformation of HESCs. Primary HESC cultures were established and propagated, and confluent cultures were decidualized for 6 days with 8-bromoadenosine 3',5'-cyclic monophosphate (8-br-cAMP) and progesterone (P4) in the presence or absence of dihydrotestosterone (DHT). Phase-contrast image analysis demonstrated that DHT increases the shape index of decidualizing cells, which was reversed upon cotreatment with the AR antagonist flutamide. Electron microscopy demonstrated that DHT enhances many of the ultrastructural changes induced by 8-br-cAMP and P4 in HESCs. Decidualizing cells are characterized by an abundant cytoplasm, multiple cell surface projections and, unlike undifferentiated HESCs, form 2 or more cell layers. The DHT further stimulated cytoplasmic expansion, lipid droplet formation, the production of an abundant extracellular matrix, and gap junction formation in decidualized HESCs. The present study demonstrates that androgen signaling has an impact on the morphological and ultrastructural changes associated with the decidual process. Our findings show that androgens promote the development and expansion of cytoplasmic organelles and gap junctions in decidualizing HESCs. These results suggest that androgens in early pregnancy play an important role in promoting the cellular transformation associated with decidualization.

Differences in collagen ultrastructure of human first trimester decidua basalis and parietalis: implications for trophoblastic invasion of the placental bed.

AIM: The human embryo-maternal interface in the first trimester of pregnancy is an area of extensive tissue remodeling. Because collagen is the most abundant constituent of the extracellular matrix of the placental bed, successful invasion must involve its rapid turnover. We compared the nature and distribution of collagen fibrils in decidua basalis and parietalis. METHODS: We used a direct-vision hysteroscopic technique to obtain biopsies of the decidua basalis and parietalis from 11 women undergoing pregnancy termination in the first trimester. The biopsies were subjected to light, transmission and scanning electron microscopy, and immunohistochemical studies using mouse monoclonal antibodies against cytokeratin 7 and collagen types I, III and V. RESULTS: Collagen fibrils in the stroma of decidua basalis were significantly thicker when compared to those in decidua parietalis (56.48 ± 1.37 nm vs 45.64 ± 0.85 nm; P < 0.0001 [mean ± standard error]) between 9 and 12 weeks gestation, but this difference in thickness was not observed at gestations below 9 weeks. In basalis, the fibrils appeared disrupted at most places surrounding the decidual/trophoblast cells while a uniform regular arrangement was preserved throughout most of parietalis. CONCLUSION: There are differences in the ultrastructure of collagen fibrils between basalis and parietalis, with thicker and disrupted fibrils within abundant amorphous tissue in basalis, and thinner uniform fibrils in parietalis. These differences may reflect an adaptive response by decidua or a direct consequence of the invading trophoblast cells.

Formation of atypical podosomes in extravillous trophoblasts regulates extracellular matrix degradation.

Throughout pregnancy the cytotrophoblast, the stem cell of the placenta, gives rise to the differentiated forms of trophoblasts. The two main cell lineages are the syncytiotrophoblast and the invading extravillous trophoblast. A successful pregnancy requires extravillous trophoblasts to migrate and invade through the decidua and then remodel the maternal spiral arteries. Many invasive cells use specialised cellular structures called invadopodia or podosomes in order to degrade extracellular matrix. Despite being highly invasive cells, the presence of invadapodia or podosomes has not previously been investigated in trophoblasts. In this study these structures have been identified and characterised in extravillous trophoblasts. The role of specialised invasive structures in trophoblasts in the degradation of the extracellular matrix was compared with well characterised podosomes and invadopodia in other invasive cells and the trophoblast specific structures were characterised by using a sensitive matrix degradation assay which enabled visualisation of the structures and their dynamics. We show trophoblasts form actin rich protrusive structures which have the ability to degrade the extracellular matrix during invasion. The degradation ability and dynamics of the structures closely resemble podosomes, but have unique characteristics that have not previously been described in other cell types. The composition of these structures does not conform to the classic podosome structure, with no distinct ring of plaque proteins such as paxillin or vinculin. In addition, trophoblast podosomes protrude more deeply into the extracellular matrix than established podosomes, resembling invadopodia in this regard. We also show several significant pathways such as Src kinase, MAPK kinase and PKC along with MMP-2 and 9 as key regulators of extracellular matrix degradation activity in trophoblasts, while podosome activity was regulated by the rigidity of the extracellular matrix.

Collagen fibril organization in the pregnant endometrium of decorin-deficient mice.

In the pregnant mouse endometrium, collagen fibrillogenesis is characterized by the presence of very thick collagen fibrils which are topographically located exclusively within the decidualized stroma. This dynamic biological process is in part regulated by the small leucine-rich proteoglycans decorin and biglycan. In the present study we utilized wild-type (Dcn(+/+)) and decorin-deficient (Dcn(-/-)) time-pregnant mice to investigate the evolution of non-decidualized and decidualized collagen matrix in the uterine wall of these animals. Ultrastructural and morphometric analyses revealed that the organization of collagen fibrils in the pregnant endometrium of both non-decidualized and decidualized stroma showed a great variability of shape and size, regardless of the genotype. However, the decidualized endometrium from Dcn(-/-) mice contained fibrils with larger diameter and more irregular contours as compared to the wild-type littermates. In the Dcn(-/-) animals, the proportion of thin (10-50 nm) fibrils was also higher as compared to Dcn(+/+) animals. On day 7 of pregnancy, biglycan was similarly localized in the decidualized endometrium in both genotypes. Lumican immunostaining was intense both in decidualized and non-decidualized stroma from Dcn(-/-) animals. The present results support previous findings suggesting that decorin participates in uterine collagen fibrillogenesis. In addition, we suggest that the absence of decorin disturbs the process of lateral assembly of thin fibrils, resulting in very thick collagen fibrils with irregular profiles. Our data further suggest that decorin, biglycan and lumican might play an interactive role in collagen fibrillogenesis in the mouse endometrium, a process modulated according to the stage of pregnancy.

Ultrastructural alterations in human decidua in miscarriages compared to normal pregnancy decidua.

BACKGROUND: Pregnant endometrial stroma, an immunologically privileged site in the female reproductive system, is enriched by decidual and natural killer (NK) cells. Since the cellular microenvironment in early pregnancy from the decidual tissues of normal and miscarriage cases has gained importance, with special emphasis on cell-to-cell contacts, we aimed to document the plastic structure of the cellular milieu in normal and miscarriage decidua. METHODS: Endometrial biopsies were obtained from women after legal curettage or women who had been treated by curettage after miscarriage. Samples were analysed in a light microscope (LM), a scanning electron microscope (SEM) and a transmission electron microscope (TEM). RESULTS: Decidual cells possess several polyploidic protrusions on cell membranes. NK cells were distributed among decidual cells. Decidual cells were found to develop gap junctions in the interfaces between each other. Their cytoplasms were also found to possess well-developed protein synthesising organelles. Decidual cells obtained from miscarriages showed a moderate degree of degeneration and, in between, a decreased number of junctional complexes. Mononuclear cell infiltration was found to be significantly low. CONCLUSION: We conclude that decidual cells during early pregnancy build a series of miniature cell-cell contacts to assemble a proper endometrial milieu. In contrast, in miscarriage samples, those intercellular communications seem lacking, associated with an increased number of NK cells, a phenomenon which obviously alters proper implantation and leads to the induction of embryonic disgenesis and miscarriage.

Arrangement and fine structure of collagen fibrils in the decidualized mouse endometrium.

The adaptations of the mouse uterus to pregnancy include extensive modifications of the cells and extracellular matrix of the endometrial connective tissue that surround the embryos. Around each implanted embryo this tissue redifferentiates into a transient structure called decidua, which is formed by polygonal cells joined by intercellular junctions. In the mouse, thick collagen fibrils with irregular profile appear in decidualized areas of the endometrium but not in the nondecidualized stroma and interimplantation sites. The fine organization of these thick fibrils has not yet been established. This work was addressed to understand the arrangement and fine structure of collagen fibrils of the decidua of pregnant mice during the periimplantation stage. Major modifications occurred in collagen fibrils that surrounded decidual cells: (1) the fibrils, which were arranged in parallel bundles in nonpregnant animals, became organized as baskets around decidual cells; (2) very thick collagen fibrils with very irregular profiles appeared around decidual cells. Analysis of replicas and serial sections suggests that the thick collagen fibrils form by the lateral aggregation of thinner fibrils to a central fibril resulting in very irregular profile observed in cross sections of thick fibrils. The sum of modifications of the collagen fibrils seem to represent an adaptation of the endometrium to better support the decidual cells while they hold the embryos during the beginning of their development. The deposition of thick collagen fibrils in the decidua may contribute to form a barrier that impedes leukocyte migration within the decidua, preventing immunological rejection of genetically dissimilar embryonic tissues.

Extracellular breakdown of collagen by mice decidual cells. A cytochemical and ultrastructural study

The interaction of antimesometrial decidual cells and collagen fibrils was studied by light microscopy and ultrastructural cytochemistry in fed and acutely fasted mice on days 9-11 of pregnancy. Fibrillar elements in the extracellular space consisted of collagen fibrils and filamentous aggregates (disintegrating collagen fibrils). Intracellular vacuoles exhibited typical collagen immersed in electron-translucent material (clear vacuoles) and faint cross-banded collagen immersed in electron-opaque material (dark vacuoles). Fibrillar elements showed extracellular acid phosphatase activity which was stronger in the region of mature decidua than in predecidual cells region in all animals; it was conspicuous in mature decidua of fasted animals. Intracellular acid phosphatase activity was observed in dark vacuoles and lysosomes, and was absent in clear vacuoles in all cells studied. Since acid phosphatase activity reflects the presence of lysosomal hydrolases in general, the results indicate that breakdown of extracellular collagen occurs by release of lysosomal enzymes by decidual cells and also by internalization of collagen for intracellular degradation in fed and fasted mice. Collagen breakdown may be part of the process of tissue remodeling in mature and predecidual regions, however, in mature decidua, collagen breakdown is enhanced and may therefore contribute to nutrition of the fetus, specially in acutely fasted mice.

Immunoexpression of CD30 and CD30 ligand in deciduas from spontaneous abortions.

In the present study, using immunohistochemistry, we studied the expression of CD30 and CD30-L in 35 deciduas obtained from women following elective abortion during normal physiological gestation and in 60 deciduas obtained from women after spontaneous abortion with or without signs of inflammation. The main difference was noticed in the first trimester of gestation in which was found a decrease in CD30/CD30-L-positive decidual glandular and stromal cells in a greater number of cases of spontaneous abortions with respect to cases of physiological pregnancies (70% vs 50%, p<0.05). In addition, deciduas from spontaneous abortions with inflammation and without inflammation reacted similarly. The reduced expression of CD30 and CD30-L and their cellular pattern detected in the deciduas from spontaneous abortions suggest that the CD30/CD30-L system is crucial for preventing abortions in the first trimester. Furthermore, the distinctive expression of CD30/CD30-L in deciduas from physiological pregnancies may indicate that the CD30/CD30-L system exerts its main role in the first trimester.

Endometrial glands as a source of nutrients, growth factors and cytokines during the first trimester of human pregnancy: a morphological and immunohistochemical study.

BACKGROUND: The maternal circulation to the human placenta is not fully established until 10-12 weeks of pregnancy. During the first trimester the intervillous space is filled by a clear fluid, in part derived from secretions from the endometrial glands via openings in the basal plate. The aim was to determine the activity of the glands throughout the first trimester, and to identify components of the secretions. METHODS: Samples of human decidua basalis from 5-14 weeks gestational age were examined by transmission electron microscopy and immunohistochemically. An archival collection of placenta-in-situ samples was also reviewed. RESULTS: The thickness of the endometrium beneath the implantation site reduced from approximately 5 mm at 6 weeks to 1 mm at 14 weeks of gestation. The glandular epithelium also transformed from tall columnar cells, packed with secretory organelles, to a low cuboidal layer over this period. The lumens of the glands were always filled with precipitated secretions, and communications with the intervillous space could be traced until at least 10 weeks. The glandular epithelium reacted strongly for leukaemia inhibitory factor, vascular endothelial growth factor, epidermal growth factor, transforming growth factor beta, alpha tocopherol transfer protein, MUC-1 and glycodelin, and weakly for lactoferrin. As gestation advanced uterine natural killer cells became closely approximated to the basal surface of the epithelium. These cells were also immunopositive for epidermal growth factor. CONCLUSIONS: Morphologically the endometrial glands are best developed and most active during early human pregnancy. The glands gradually regress over the first trimester, but still communicate with the intervillous space until at least 10 weeks. Hence, they could provide an important source of nutrients, growth factors and cytokines for the feto-placental unit. The endometrium may therefore play a greater role in regulating placental growth and differentiation post-implantation than previously appreciated.

Cytomorphometric analysis and surface ultrastructure of developing decidua.

A scanning electron microscopic (SEM) and morphometric analysis of the topographical changes occurring in the uterine luminal epithelial layer in association with decidual tissue (DT) formation in guinea pigs was undertaken in order to elucidate the surface ultrastructural characteristics which occur during the process of endometrial differentiation. Experimentally induced decidua formation was promoted by mechanical stimulation of the antimesometrial luminal surface during the period of maximal uterine sensitivity to stromal differentiation. DT-associated remodeling of the uterine epithelial layer was subsequently examined by light and SE microscopic analysis for apical epithelial and luminal contour alterations associated with decidua growth. Cytological changes in the luminal surface associated with DT induction included sparse microvillus growth from the apical epithelial surface, accompanied by the appearance of prominent apical membrane surface protrusions and endometrial gland openings as compared with non-DT-stimulated control samples. Decidua surface growth was characterized by a short, sparse epithelial microvillus pattern present over a highly contoured luminal uterine surface on which contoured gland openings were both numerous and prominent. These surface modifications contrasted with the flat, non-decidualized luminal surface contour which was covered by distinct, microvilli-laden, apical cell membranes, and defined by prominent intercellular membrane borders. The uterine surface at the time of maximal DT formation (i.e. growth) closely resembled that of a uterine luminal surface undergoing apoptosis and subsequent cellular reabsorption, characterized by disrupted cell surface membranes, sparse microvillus surfaces and prominent epithelial contours reflecting stromal tissue and vasculature involution. These data indicate that the alterations in the uterine luminal surface associated with DT formation are reminiscent of the endometrial changes associated with the initiation of early placentation, and may be used as a model for the analysis of the role of epithelial cell surface modifications associated with the induction and support of interstitial blastocyst implantation and early decidua formation.

Localization of alkaline phosphatase and Ca2+-ATPase in the cat placenta.

We localized alkaline phosphatase and plasma membrane calcium-ATPase (PMCA) in the cat placental syncytiotrophoblast to address their polarized distribution and their potential as markers for specific plasma membrane purification. We used enzyme- (alkaline phosphatase) and immuno- (PMCA) histochemistry and, for alkaline phosphatase, compared data to observations on the human placenta. Alkaline phosphatase activity in the cat was localized to the decidual cell membranes, to within the associated interstitial space and on the subjacent apical (maternal facing) plasma membrane of the syncytiotrophoblast. Occasional maternal capillaries were positive on their basal surface and there was focal staining within the syncytiotrophoblast. This widespread distribution is less specific than in the human placenta where alkaline phosphatase was restricted to the apical and basal plasma syncytiotrophoblast membranes, with much greater density on the apical membrane. Expression of PMCA in the cat was restricted to the basal membrane of the syncytiotrophoblast only. This specific localization of PMCA is identical to the human placenta and all other species in which its placental localization has been studied. We conclude that the plasma membranes of the cat syncytiotrophoblast show a broadly similar functional polarization to the human and that PMCA would prove a useful marker in isolation of the cat syncytiotrophoblast basal plasma membrane.

Granulated metrial gland cells in the murine uterus: localization, kinetics, and the functional role in angiogenesis during pregnancy.

Granulated metrial gland (GMG) cells are a major immune cell population in the murine pregnant uterus, and contribute to the maintenance of pregnancy by functioning as uterus-specific natural killer (NK) cells. In order to reveal their kinetics, activation, and functional roles in pregnancy, we conducted quantitative and immunohistochemical analyses in normal and immuno-modulator-treated mice. Under a light microscope, GMG cells were identified by red cytoplasmic granules in periodic-acid-Schiff (PAS)-stained sections. They progressively increased in number and size with the peak at day 12-14 of pregnancy in the decidua and metrial gland. New vessel formation was most prominent around day 8, and the total vascular area reached the peak at day 13. GMG cells were often located near the blood vessels, and expressed vascular endothelial growth factor (VEGF), suggesting their possible inducing role in angiogenesis during the development of decidua/metrial gland. While blood vessels in the non-pregnant uterus were negative for vascular cell adhesion molecule (VCAM)-1, those in the pregnant one were positive. Treatment with neutralizing antibody against VCAM-1, however, did not decrease the number of GMG cells. On the other hand, mitosis of GMG cells was frequently observed. These data suggest that the increment of GMG cells during pregnancy may largely result from local proliferation in the uterus rather than an increased influx of precursor cells. Although we attempted to induce in vivo activation of GMG cells by administration of interleukin-12 (IL-12) or alpha-galactosylceramide, a potent activator for natural killer-T (NK-T) cells, the number of GMG cells did not appreciably increase. The present study has demonstrated that GMG cells locally proliferate in the pregnant uterus, not being related to VCAM-1 expression by the uterine vasculature or systemic activation of NK cells and NK-T cells, and seem to be involved in angiogenesis in the pregnant uterus through VEGF production.

Ultrastructure of epithelial plaque formation and stromal cell transformation by post-ovulatory chorionic gonadotrophin treatment in the baboon (Papio anubis).

BACKGROUND: To understand factors controlling endometrial responses to pregnancy, we have established a model using the baboon and examined the effects of infused human chorionic gonadotrophin (HCG) on the preparation of the luminal epithelium and stromal cell differentiation for the establishment of pregnancy. METHODS: The ultrastructure of endometrium from normal day 10 post-ovulation animals, cycling females treated with either HCG or FSH (control), and a day 15 pregnant animal has been compared. RESULTS: In the control endometrium, the luminal epithelium was smooth and regular, with underlying spindle shaped stromal cells. In pregnancy, the luminal epithelium underwent a plaque reaction, while stromal cells enlarged and developed filament-rich cell processes. Infusion of HCG produced changes similar to those seen in pregnancy, with generalized plaque formation and stromal decidualization, while in the animal treated with FSH there was no response. CONCLUSIONS: This study indicates that infusion of HCG into the uterus can duplicate many of the responses of the endometrium to pregnancy, although in this case the plaque reaction involved the whole of the luminal epithelium, rather than only the implantation site as in pregnancy.

Early implantation events in the baboon (Papio anubis) with special reference to the establishment of anchoring villi.

The development of the baboon anchoring villus has been studied from day 14 to day 48 of gestation, using light and electron microscopy. At day 14, cords of trophoblast could be seen streaming into the endometrium, invading maternal vessels and forming blood-filled lacunae; by 20 days gestation some of these had differentiated into distinctive anchoring villi, with an outer covering of syncytiotrophoblast and inner cytotrophoblast cells which differed from those of floating villi in that a subpopulation detached from the syncytium to form an interconnecting network of cells within the centre of the villus. Subsequent migration of cytotrophoblast into the endometrium formed the cytotrophoblastic shell while fibrillin-like extracellular matrix biosynthesis within the body of the villus provided a firm mechanical support. At the trophoblast-decidual interface, a zone of necrosis and phagocytosis initially developed, which became less extensive with time, so that by 40 days a stable interface was evident with only residual pockets of necrosis. During this period, there was differentiation of decidual cells which by 28 days developed characteristic pedunculated cell processes, and later became surrounded by a basal lamina. The factors that control detachment of cytotrophoblast from the syncytium and the biosynthesis of the specialized, fibrillar extracellular matrix, features that are not apparent in other placental villi, require further investigation, possibly by carefully controlled in vitro experimentation.

Feto-maternal interface of human placenta inhibits angiogenesis in the chick chorioallantoic membrane (CAM) assay.

The rapidly growing chorionic villi of the human placenta characteristically show constant blood vessel growth and differentiation. In contrast, the underlying decidua reveals tissue remodeling without apparent angiogenesis. Using the chick chorioallantoic membrane (CAM) assay, we found marked inhibition of angiogenesis by the feto-maternal interface tissue derived from nine human placentas obtained minutes after delivery. Inhibition was prevented by the addition of monensin, which blocks the release of synthesized cell products, and was markedly reduced by drying or freezing the tissue before the assay. Histology, combined with statistical analysis of the constituent cell types, correlated inhibition of angiogenesis with the number of fetally-derived extravillous trophoblasts in the feto-maternal interface tissue. Electron microscopy revealed endothelial cell damage in preexisting small (but not large) CAM vessels. We conclude that decidual tissue inhibited angiogenesis by releasing a water soluble factor which was under apparent constant production by vaible trophoblast on the CAM. The extravillous trophoblast population resembles tumor cells in its migratory and invasive properties but, in contrast to tumor induced angiogenesis, it is angiostatic, perhaps to counteract angiogenic proteins leaking from the intervillous space which could be detrimental to the maternal organism if active.

Expression of vascular endothelial growth factor by granulated metrial gland cells in pregnant murine uteri.

Granulated metrial gland (GMG) cells are a characteristic uterine component belonging to a natural killer cell lineage. This study is aimed at revealing their kinetic and spatial relationship with vascular growth during pregnancy and the expression of vascular endothelial growth factor (VEGF). GMG cells and blood vessels were identified by periodic-acid-Schiff-reagent (PAS)-stained granules and positive staining for factor-VIII-related antigen, respectively. GMG cells were widely distributed in the decidua and metrial gland and showed a numerical increase with a peak at day 13 in parallel with the increase of vascular density. Preceding the maximal vascular development at day 13, microvessels with a narrow lumen representative of neovascularization prevailed at days 7-9, and the VEGF content in the decidua/metrial gland was significantly elevated at days 7-13 concurrently with mRNA expression. By immunolight microscopy combined with PAS staining, GMG cells with PAS-stained granules were positive for VEGF. Immunoelectron microscopy demonstrated that immunoreactions were diffuse in the cytoplasm but not localized in the granules. In contrast, fibroblast-like stromal cells were negative. These data indicate that GMG cells express VEGF and may play inducing roles in uterine neovascularization during pregnancy.