Longitudinal assessment of spiral artery and intravillous arteriole blood flow and adverse pregnancy outcome.

OBJECTIVE: Superb microvascular imaging (SMI) has been shown to improve visualization of small vessels by suppressing global motions while preserving low-flow components, such as the microvessels in the placenta. We sought to determine if SMI-aided visualization of flow velocity waveforms in the spiral arteries (SA) and intravillous fetal arterioles (IVA) could predict fetal growth restriction (FGR), gestational hypertension (GH) and/or pre-eclampsia (PE). METHODS: This was a prospective longitudinal study of singleton pregnancies without fetal anomaly, receiving prenatal care in one of two medical centers over a 5-year period. Using SMI-aided color Doppler, SA and IVA flow velocity was measured at three timepoints: 11 + 0 to 14 + 0, 18 + 0 to 22 + 6 and 28 + 0 to 34 + 6 weeks of gestation. SA and IVA flow velocity waveforms were reported as resistance indices (RI). RI values were analyzed using multilevel modeling; individual regression curves were estimated and combined to obtain the reference intervals for SA-RI and IVA-RI in uncomplicated pregnancies. The primary clinical outcome was FGR and secondary outcomes were PE and GH. FGR was defined as estimated fetal weight < 10th percentile. Student's t-test was used to compare deviation from expected RI between normal and complicated pregnancies. RESULTS: Among 540 pregnancies included in the analysis, 18 (3.3%) had FGR, 31 (5.7%) PE and 61 (11.3%) GH. In uncomplicated pregnancies, the SA-RI decreased progressively with advancing gestation, whereas the IVA-RI increased with gestational age. In the third trimester, the mean SA-RI and IVA-RI values were significantly higher in the FGR group compared with pregnancies that did not develop FGR, while the mean SA-RI was significantly higher in PE compared with non-PE pregnancies. There was no significant difference in mean SA-RI or IVA-RI between pregnancies with vs those without GH at any gestational age. When all three adverse outcomes were combined, SA-RI was significantly higher in pregnancies with these outcomes when compared to uncomplicated pregnancies in the third trimester (mean ± SD, 0.29 ± 0.12 vs 0.26 ± 0.12; P = 0.02). In screening for FGR using SA-RI, the areas under the receiver-operating-characteristics curves (AUC) were 0.68, 0.73 and 0.73 in the first, second and third trimesters, respectively. The respective AUCs for IVA-RI were 0.72, 0.72 and 0.73 for each trimester. CONCLUSIONS: SA-RI and IVA-RI, measured using SMI technology, were significantly higher in pregnancies at risk for FGR in late gestation. Larger studies are needed to determine if SA and IVA flow are reliable predictors of adverse pregnancy outcome. © 2021 International Society of Ultrasound in Obstetrics and Gynecology.

Regulation of chemokine production in response to pro-inflammatory cytokines in first trimester decidual cells.

OBJECTIVE: Chemokines initiate the immune response by controlling leukocyte migration and lymphocyte development. Macrophage infiltration of the decidua has been implicated in the genesis of recurrent miscarriage and preeclampsia. Therefore, we determined whether cultured human decidual cells produce monocyte/macrophage-recruiting chemokines in response to a potent pro-inflammatory cytokine, interleukin-1beta (IL-1beta), and whether decidual cell-conditioned medium contains monocyte- and macrophage-chemoattractant activity. METHODS: Leukocyte-free first trimester decidual cells were treated for 6h with estradiol (E(2)) and medroxyprogesterone acetate (MPA) to mimic the steroidal milieu of pregnancy, or E(2) and MPA and IL-1beta (1 ng/ml) to mimic inflamed decidua. Total RNA was used for cDNA synthesis. Biotinylated cRNAs were generated and chemically fragmented for hybridization on Affymetrix HG_U133 Plus 2.0 chips followed by fluorescence labeling and optical scanning. Raw data generated from Affymetrix GCOS 1.2 (GeneChip Operating Software) were analyzed by GeneSpring 7.2 software. Subsequently microarray results were validated by real time RT-PCR and Western blotting. A functional study of monocyte migration was carried out also using conditioned media from culture. RESULTS: Five chemokines responsible for monocyte/macrophage chemoattraction and activation, including C-C motif ligand 2 (CCL2), CCL5, C-X-C motif ligand 2 (CXCL2), CXCL3 and CXCL8, were markedly elevated from 29- to 975-fold after exposure to IL-1beta in cultured first trimester decidual cells. The results of real-time RT-PCR (up-regulation from 43- to 3069-fold) and Western blotting (up-regulation from 15- to 300-fold) confirmed the microarray findings. Monocyte migration was significantly induced by the conditioned medium from IL-1beta-treated decidual cells. CONCLUSIONS: Treatment of first trimester decidual cells with IL-1beta induces secretion of monocyte/macrophage recruiting-chemokines and promotes monocyte migration. Extrapolation of these in vitro results to the milieu of implantation site suggests a mechanism whereby IL-1beta could mediate excessive macrophage infiltration of the decidua.

Regulation of synthesis of the transformation-induced protein, leukocyte plastin, by ovarian steroid hormones.

Previous studies indicated that, among normal cells, only those of hemopoietic lineages expressed the abundant leukocyte phosphoprotein, L-plastin, and that activation of the L-plastin gene frequently occurred in malignant cells of solid tumors. We discovered that the gene encoding L-plastin contains potential estrogen and progesterone response elements upstream from its promoter, suggesting that L-plastin expression is subject to ovarian steroid regulation. To determine if L-plastin synthesis is regulated by ovarian steroids (estrogens and progestins), we examined cultured uterine endometrial stromal cells (SC) which are known to be responsive to ovarian steroids in a fashion that approximates the normal endometrium. Primary SC, which synthesized estrogen receptor and progesterone receptor mRNA transcripts, dramatically elevated L-plastin transcript synthesis in response to treatment with estradiol (E2) and medroxyprogesterone acetate (MPA). Stimulation of L-plastin synthesis by E2 and MPA was also evident by examination of protein synthesis using high-resolution two-dimensional gel electrophoresis and Western blotting. By contrast, SC that were propagated through multiple culture passages exhibited a coordinate decline in L-plastin, estrogen receptor, and progesterone receptor transcript levels and L-plastin protein synthesis. No other intracellular proteins could be found that were modulated significantly by E2 and MPA, but secretory protein synthesis was profoundly affected by E2 and MPA. Like L-plastin synthesis, hormone-mediated secretory protein synthesis was lost after propagation of the SC culture and reduction of estrogen receptor and progesterone receptor transcript synthesis. Our findings suggest that L-plastin synthesis is regulated coordinately with secretory protein synthesis in endometrial SC by estrogens and progestins.

Metalloproteinase expression by control and telomerase immortalized human endometrial endothelial cells.

Vascular endothelial cells play a critical role in the maintenance of endometrial homeostasis. Indeed many pathological conditions causing abnormal endometrial bleeding including progestin only contraception, hormone replacement therapy, endometrial polyps, myomas, hyperplasia and cancer are associated with aberrant angiogenesis. Critical to the process of angiogenesis is the breakdown of the surrounding tissues by matrix metalloproteases (MMPs). In addition to the cells surrounding the endometrial endothelial cells, the endothelial cells themselves produce their own panel of MMPs. We now characterize the specific MMPs that are expressed by endothelial cells derived from human endometrium. These include MMP-1, MMP-2 and MMP-10 but not MMP-3. In addition, in order to successfully carry out consistent, homogeneous and sufficient numbers of studies we investigated the in vitro expression of the MMPs with both freshly isolated, early passaged endometrial endothelial cells (HEECs) as well as with newly telomerase immortalized HEECs (T-HEECs). The latter were karyotypically normal and expressed classic endothelial cell endpoints such as tubulogenesis on matrigel and expression of the endothelial cell markers CD-31 (PECAM), von Willebrand's factor, and the Tie-2 receptors. The levels of MMP expression as well as that of the metalloprotease inhibitors TIMP-1 and TIMP-2 were similar in parent and immortalized endothelial cells.

Regulation of tissue factor gene expression in human endometrium by transcription factors Sp1 and Sp3.

Prior studies indicate that tissue factor (TF), the primary cellular initiator of hemostasis, is persistently up-regulated in human endometrial stromal cells (HESCs) undergoing progestin-induced decidualization in vivo and in vitro. The mechanism underlying progestin enhancement of TF mRNA and protein levels in these cells involves transcriptional activation of the TF gene. Transient transfections of HESCs with the truncated TF promoters driving the luciferase reporter gene have demonstrated that the region spanning -111 to +14 bp retained differential progestin-enhancing effects. We now demonstrate that RU486 displays inhibitory effects on the progestin-induced TF promoter activity, confirming the involvement of the progesterone receptor. Since the TF minimal promoter (pTF 111 spanning -111 to +14 bp) contains three overlapping Sp1 and three Egr-1 sites, the present study determined whether Sp1 and/or Egr-1 were required for progestin-regulated TF expression. The results indicate that the three Sp1 sites are primarily responsible for both the constitutive and progestational activity of the pTF 111 promoter, whereas the Egr-1 sites have only a minor involvement in both activities. Overexpression of the Sp1 protein resulted in greater than a 6-fold induction in TF promoter activity. In contrast, no enhancement was observed when the Sp3 protein was overexpressed. The concomitant overexpression of Sp1 and Sp3 demonstrated that Sp3 completely blocked the induction of TF promoter activity by Sp1. Moreover, the addition of 10 nM mithramycin, a concentration that inhibits Sp1 binding to target DNA, blocked the progestational induction of TF mRNA expression. Immunohistochemical studies demonstrated increased Sp1 levels in perivascular stromal cells in secretory phase compared with proliferative phase endometria. In contrast, Sp3 expression was greatly decreased in stromal cells of secretory, compared with proliferative phase tissues. The levels of Egr-1 were low in both proliferative and secretory endometria. Immunocytochemistry of E2 vs. E2 + medroxyprogesterone acetate-treated HESCs demonstrated a dramatic reduction in Sp3 expression after progestin treatment, and Northern blots demonstrated progestational increases in Sp1 and reduction in Sp3 mRNA expression compared with controls. Taken together, our results demonstrate that progestin enhancement of TF gene expression in HESCs is mediated principally by Sp1. We propose that progestins regulate HESC TF gene expression in vivo by altering the ratio of Sp1 to Sp3 nuclear factors.

Chinese herbal medicine for miscarriage affects decidual micro-environment and fetal growth.

INTRODUCTION: Intrauterine growth restriction complicates 5-10% of pregnancies. This study aims to test the hypothesis that Chinese herbal formula, JLFC01, affects pregnancy and fetal development by modulating the pro-inflammatory decidual micro-environment. METHODS: Human decidua from gestational age-matched elective terminations or incomplete/missed abortion was immunostained using anti-CD68 + anti-CD86 or anti-CD163 antibodies. qRT-PCR and Luminex assay measured the effects of JLFC01 on IL-1ß- or TNF-α-induced cytokine expression in first trimester decidual cells and on an established spontaneous abortion/intrauterine growth restriction (SA/IUGR)-prone mouse placentae. The effect of JLFC01 on human endometrial endothelial cell angiogenesis was evaluated by average area, length and numbers of branching points of tube formation. Food intake, litter size, fetal weight, placental weight and resorption rate were recorded in SA/IUGR-prone mouse treated with JLFC01. qRT-PCR, Western blot and immunohistochemistry assessed the expression of mouse placental IGF-I and IGF-IR. RESULTS: In spontaneous abortion, numbers of decidual macrophages expressing CD86 and CD163 are increased and decreased, respectively. JLFC01 reduces IL-1ß- or TNF-α-induced GM-CSF, M-CSF, C-C motif ligand 2 (CCL2), interferon-γ-inducible protein-10 (IP-10), CCL5 and IL-8 production in first trimester decidual cells. JLFC01 suppresses the activity of IL-1ß- or TNF-α-treated first trimester decidual cells in enhancing macrophage-inhibited angiogenesis. In SA/IUGR-prone mice, JLFC01 increases maternal food intake, litter size, fetal and placental weight, and reduces fetal resorption rate. JLFC01 induces IGF-I and IGF-IR expression and inhibits M-CSF, CCL2, CCL5, CCL11, CCL3 and G-CSF expression in the placentae. DISCUSSION: JLFC01 improves gestation by inhibiting decidual inflammation, enhancing angiogenesis and promoting fetal growth.

Effects of estradiol on prostaglandin F2 alpha levels in primary monolayer cultures of epithelial cells from human proliferative endometrium.

Primary monolayer cultures of glandular epithelial or stromal cells isolated from human proliferative endometrium release prostaglandin F2 alpha (PGF2 alpha) into the medium. In epithelial cell cultures maintained in medium containing charcoal-stripped calf serum, estradiol (10(-8) M) increased the levels of PGF2 alpha 2.5- to 5-fold during the first 24 h and as much as 40-fold during the second and third days in culture. A similar response was seen with estradiol at a concentration of 10(-10) M. The response to estradiol was only about half as great in medium containing untreated calf serum. Preliminary measurements of PGF2 alpha levels in stromal cell cultures showed that estradiol did not increase PGF2 alpha levels in the medium and even reduced it in some preparations. The estradiol-stimulated increase in PGF2 alpha levels in the epithelial cell cultures contrasts with the lack of responsiveness of proliferative endometrium in organ culture. This finding suggests that the ability of glands to respond to estradiol is repressed in proliferative endometrium.

Lipoidal derivative of estradiol: the biosynthesis of a nonpolar estrogen metabolite.

The incubation of estradiol with human breast tumors as well as several estrogen-responsive tissues of the rat, including dimethylbenzanthracene-induced mammary tumors, leads to the formation of a nonpolar metabolite. This compound has been called a lipoidal derivative of estradiol because of its nonpolar characteristics and because it is converted back into estradiol with alkali. The properties of this metabolite are similar to those of the endogenous lipoidal derivatives of the delta 5-3 beta-hydroxysteroids, pregnenolone, 17-hydroxypregnenolone, and dehydroisoandrosterone, that were found in the adrenal. When estrone was incubated in a similar manner, a nonpolar metabolite was formed, but it was the lipoidal derivative of estradiol, not estrone. The lipoidal derivative of estradiol was not oxidized by treatment with CrO3, which showed that the lipoidal moiety is linked to the C-17 of the steroid nucleus. Unlike other estrogen metabolites, the lipoidal derivative of estradiol is found only within the tissue and cannot be detected in the incubation medium.

Differential effects of estradiol, arachidonic acid, and A23187 on prostaglandin F2 alpha output by epithelial and stromal cells of human endometrium.

Primary monolayer cultures of glandular epithelial cells and stromal cells derived from human endometrial curettings release similar amounts of prostaglandin F2 alpha (PGF2 alpha) to the incubation medium, but PGF2 alpha output is significantly increased by estradiol (E2; 10(-8) M) only in epithelial cells. The differential responsiveness to estrogens of these two cell types was further demonstrated in the presence of exogenous arachidonic acid (AA), a PG precursor that markedly elevated PGF2 alpha output by both epithelial and stromal cells. In epithelial cell cultures, the increases in PGF2 alpha output obtained with mixtures of E2 and AA were about 2.5-fold greater than the sum of the increases elicited by E2 or AA alone. In contrast, the effects of AA in stromal cell cultures were not enhanced by E2. Addition of the calcium ionophore A23187 to cultures of epithelial cells resulted in a Ca2+-dependent increase in PGF2 alpha output, which could be further augmented by E2 to levels almost twice the sum of those produced by each agonist alone. Unlike its effects on epithelial cells, A23187 did not significantly increase PGF2 alpha levels in stromal cell cultures, regardless of the addition of Ca2+ (1.5 mM) or E2 (10(-8)M) to the culture medium. These results suggest that the epithelia and not the stroma are the primary targets for the effects of E2 on PGF2 alpha output by the endometrium. The synergistic effects of E2 and AA (the substrate for PG synthase) or of E2 and A23187 (a stimulator of phospholipase-mediated release of AA from phospholipid stores) on PGF2 alpha production suggest that estrogens enhance PG production in epithelial cells by elevating PG synthase activity.

In vitro inhibition with antiestrogens of estradiol effects on prostaglandin F2 alpha production by human endometrium and endometrial epithelial cells.

It has been previously reported that neither an antiestrogen, actinomycin D, nor cycloheximide inhibited estradiol (E2)-stimulated elevations in uterine prostaglandin F2 alpha (PGF2 alpha) production in ovariectomized rats, suggesting that in contrast to other steroid-initiated events, this effect on PGF2 alpha may not involve receptor-mediated transcription-dependent actions of E2. To eliminate indirect influences, the ability of antiestrogens to affect PGF2 alpha output was reevaluated during incubations of human secretory endometrium and in cultures of epithelial cells derived from glands isolated from proliferative and secretory tissues. In these preparations, which respond to E2 with marked elevations in PGF2 alpha output, tamoxifen and its metabolite trans-4-monohydroxytamoxifen acted as virtually pure antagonists, counteracting the E2 effect while failing to influence basal PGF2 alpha output. Consistent with its effects on other estrogen-mediated end points, trans-4-monohydroxytamoxifen was at least 10 times more potent than tamoxifen, eliminating, at a 10(-6) M concentration, almost completely the stimulatory effect of 10(-8) M E2 on PGF2 alpha production by both endometrial fragments and monolayers of epithelial cells.

Expression of 11 beta-hydroxysteroid dehydrogenase during decidualization of human endometrial stromal cells.

This study evaluated the expression of the corticosteroid-metabolizing enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta HSD) during in vitro decidualization of human endometrial stromal cells. The cultured stromal cells displayed both NADP(+)-dependent (type 1) and NAD(+)-dependent (type 2) 11 beta HSD activities under basal conditions. Although the cells did not respond to estradiol (E2) added alone, catalytic levels of both isoforms were enhanced by medroxyprogesterone acetate (MPA) and further enhanced by E2 plus MPA. Type I messenger RNA (mRNA) was undetected by Northern analysis of total RNA, but was evident as a 1.5-kilobase band in polyadenylated selected RNA from E2- plus MPA-treated cultures. Use of RT-PCR to augment the sensitivity of mRNA detection revealed the presence of type I mRNA as a faint band in the MPA-treated cultures and as an intense band in the E2- plus MPA-treated cultures. Thus, type I mRNA is present as a low abundance message in the cultured stromal cells whose steady state levels parallel progestin-enhanced enzyme activity. As the expression of several progestin-regulated decidualization markers is also augmented by E2, the results of the present study reveal a correlation between enhanced 11 beta HSD expression and the decidualization reaction. Time-course measurements indicated that elevated 11 beta HSD expression is an early event in the decidualization response, which precedes E2- plus MPA-enhanced PRL production by several days. Clear dose-response effects on both type 1 and type 2 11 beta HSD activities were obtained in cells incubated with 10(-8) mol/liter E2 added together with MPA at concentrations that approximated circulating progesterone levels from the luteal phase (10(-9) mol/liter) through pregnancy (10(-7) mol/liter). Corticosteroids are thought to exert toxic and teratogenic effects on the implanting embryo and could influence trophoblast invasion by regulating extracellular matrix turnover. Therefore, the novel finding that decidualization involves marked enhancement of the corticosteroid-metabolizing capacity of stromal cells suggests a mechanism by which decidual cells could affect the health and invasiveness of implanting trophoblastic cells.

Matrix metalloproteinase and matrix metalloproteinase inhibitor expression in endometrial stromal cells during progestin-initiated decidualization and menstruation-related progestin withdrawal.

Estradiol (E) primes human endometrial stromal cells (HESCs) for the decidualizing effects of progesterone in vivo and in vitro. Matrix metalloproteinase (MMP) expression was evaluated in confluent HESCs incubated in control medium, and in medium supplemented with either E, or the synthetic progestin medroxyprogesterone acetate (P), or E + P. Measurements with a specific ELISA indicated that basal pro-MMP-1 output was unaffected by E, whereas E + P, which induces the expression of several decidualization-related markers, produced a time-dependent inhibition in HESC-secreted levels of pro-MMP-1. Consistent with progestin inhibition of MMP-1 protein expression in the HESCs, P but not E, reduced steady state levels of MMP-1 messenger RNA (mRNA) as determined by Northern analysis. By contrast, mRNA levels for MMP-2 and the MMP inhibitor TIMP-1 were not altered by either P or E. Steroid withdrawal studies indicated that after MMP-1 expression was suppressed by incubation of the HESCs with E + P, 4 days of exposure to the antiprogestin RU 486 (mifepristone) significantly up-regulated MMP-1 levels in the conditioned medium by severalfold compared with cultures maintained in E + P. The change to steroid-free control medium required a more prolonged period of withdrawal to attain up regulatory effects that were comparable with those evoked by RU 486. The ELISA measurements were validated by immunoblot analysis with a specific MMP-1 antibody, which showed corresponding changes in a band at the expected mobility of about 50 kDa. Moreover, Northern analysis revealed parallel changes in MMP-1 mRNA levels, whereas neither MMP-2 nor TIMP-1 mRNA levels were modulated by adding or withdrawing steroids. The contrast between regulated MMP-1 expression and constitutive MMP-2 expression observed in the cultured HESCs is consistent with the demonstrated presence on the MMP-1 promoter of regulatory elements such as AP-1 and PEA-3 that are absent from the MMP-2 promoter. Extrapolation of these in vitro changes in HESCs to in vivo endometrial events suggests that: 1) inhibition of MMP-1 expression by E and progesterone would stabilize the perivascular endometrial ECM to prevent local hemorrhage during endovascular invasion by the implanting trophoblast; 2) enhanced expression of MMP-1 evoked by steroid withdrawal would mediate endometrial ECM degradation leading to sloughing of the functional layer during menstruation.

Progestin regulation of plasminogen activator inhibitor type 1 in primary cultures of endometrial stromal and decidual cells.

The progestin medroxyprogesterone acetate (MPA) enhanced expression of the endothelial-type plasminogen activator inhibitor PAI-1 by stromal cells from cycling endometrium and by decidual cells from first trimester endometrium. In the cultured stromal cells, Northern analysis revealed a 4-fold increase in steady state levels of PAI-1 mRNA in response to 10(-6)-10(-8) mol/L MPA. Although the cells were refractory to 10(-8) mol/L estradiol (E2) alone, E2 plus MPA produced a further doubling of PAI-1 mRNA levels. Parallel effects on PAI-1 protein levels in the stromal cell-conditioned medium were measured by immunoassay and confirmed by immunoblot analysis. During an initial 3-day exposure, PAI-1 levels were elevated 6- and 12-fold by MPA and E2 plus MPA, respectively, compared with those in either control or E2-treated cells. In the subsequent 3 days of culture, PAI-1 levels were increased 30-fold by MPA and 70-fold by E2 plus MPA. Cultured decidual cells released significant quantities of PAI-1 under basal conditions; these levels were also elevated by MPA and increased markedly by E2 plus MPA. While PAI-2 was also detected in both stromal and decidual cell cultures, its levels were far lower than those of PAI-1 and were unaffected by exogenous steroids. Extrapolation of these in vitro results to periimplantational events in humans suggests that under progesterone regulation, decidual cell-derived PAI-1 could 1) restrain blastocyst invasion of the stroma by inhibiting trophoblast-associated urokinase-type plasminogen activator, and 2) prevent hemorrhage during trophoblast invasion of the endometrial vasculature by inhibiting fibrinolysis mediated by tissue-type plasminogen activator.

Lipocortin output by human endometrium in vitro.

Lipocortin was found to be secreted by human endometrium incubated for 1-2 days under organ culture conditions. Levels of lipocortin in the culture medium, measured by RIA, were increased by dexamethasone (10(-8)-10(-6) M) and decreased by progesterone (10(-8)-10(-6) M). Both steroids, however, decreased prostaglandin F2 alpha (PGF2 alpha) output, also estimated by RIA. These results suggest that lipocortin does not mediate the inhibition of PGF2 alpha production evoked by progesterone. Dexamethasone may inhibit PGF2 alpha production by mechanisms involved in the inhibition by progesterone in addition to those mediated by an elevation in the levels of lipocortin, a phospholipase inhibitor.

In vitro effects of ovarian steroids on prostaglandin F2 alpha output by human endometrium and endometrial epithelial cells.

Correlations of the rise in prostaglandin F2 alpha (PGF2 alpha) levels in human endometrium during the menstrual cycle with changes in plasma concentrations of ovarian steroids have suggested that progesterone (P) priming of the endometrium is necessary for the stimulation of PGF2 alpha production by estradiol (E2). However, despite the absence of significant levels of P in plasma during the follicular phase of the cycle, PGF2 alpha output by epithelial cell cultures derived from proliferative endometrium was stimulated in vitro by 10(-8) M E2 at least as well as PGF2 alpha output from secretory endometrium. In addition, basal PGF2 alpha output by proliferative endometrium under organ culture conditions was significantly greater than that by secretory endometrium. Concurrent addition of P counteracted the effects of E2 in these in vitro systems. P (10(-7) M) plus E2 (10(-8) M) resulted in PGF2 alpha output as low or lower than those of control incubations of secretory and proliferative endometrium. In the epithelial cell cultures, significant net stimulation by 10(-8) M E2 occurred in the presence of 10(-6) M P, a noteworthy finding since the rise in endometrial PGF2 alpha during the luteal phase takes place when the tissue is exposed to both E2 and P. P lowered the basal output of PGF2 alpha by endometrium in organ culture, but not by epithelial cells in monolayer cultures. E2 appears to stimulate PGF2 alpha output by increasing synthesis rather than diminishing metabolism, since exogenous [3H]PGF2 alpha was metabolized to an equivalent extent by fragments of secretory endometrium or glandular epithelial cells regardless of whether E2 was added to the incubation medium. The results from this study confirm that only secretory endometrium responds to E2 in vitro by significantly increasing PGF2 alpha output. The lack of response by proliferative endometrium, when contrasted with the marked responsiveness of epithelial cells derived from this tissue, suggests that an inhibitory influence is removed during the isolation or subsequent culture of endometrial glands.

Effects of thrombin on steroid-modulated cultured endometrial stromal cell fibrinolytic potential.

By virtue of their unique chronic expression of tissue factor, the primary initiator of hemostasis, decidualized endometrial stromal cells are capable of significant thrombin generation after vascular disruption. In addition to its potent procoagulant effects, thrombin modifies endothelial and glomerular cell fibrinolytic activity. Therefore, we evaluated whether thrombin affected the expression of endometrial stromal cell urokinase-type (uPA) and tissue-type (tPA) plasminogen activators and their primary inhibitor, type 1 plasminogen activator inhibitor (PAI-1), and whether ovarian steroids modulated putative thrombin effects. Confluent stromal cell cultures were incubated in a defined medium containing vehicle control, 10(-8) mol/L estradiol (E2), 10(-7) mol/L medroxyprogesterone acetate (MPA), or E2 plus MPA for 4 days. The medium was then collected and exchanged for medium containing the corresponding steroids with or without thrombin and the specific thrombin inhibitor, D-phenyl-alanyl-propyl-arginine-chloromethyl ketone, for an additional 24 h. The conditioned medium was then collected and analyzed for immunoreactive (ir) uPA, tPA, and PAI-1 by enzyme-linked immunosorbent assay and for PA activity by chromogenic assay, whereas Northern analysis of the cells was employed to evaluate the expression of thrombin receptor, uPA, tPA, and PAI-1 messenger ribonucleic acid (mRNA) species. The latter studies revealed that confluent cultures incubated in defined medium expressed the 3.45-kilobase thrombin receptor message. Steady state levels of thrombin receptor mRNA were unaffected by exogenous steroids. Thrombin added in the absence of exogenous steroids elevated concentrations of ir tPA, uPA, and PAI-1 compared with control cultures. Conversely, in the absence of added thrombin, MPA added alone or together with E2 inhibited levels of ir tPA and uPA while stimulating PAI-1 levels despite the lack of a response to E2 alone. Interestingly, thrombin counteracted this progestin inhibition of tPA and uPA expression and augmented the progestin-enhanced expression of PAI-1. Northern analysis revealed that steady state levels of tPA and uPA mRNA were also enhanced by thrombin in both control and steroid-containing cultures. Net PA activity reflects the balance between PA and PAI-1. In the absence of thrombin, there is virtually no detectable tPA activity and minimal uPA activity in progestin-exposed cultures. However, thrombin elicited significant increases in tPA and uPA activity in control and E2-treated cultures. Despite the molar excess of PAI-1 in MPA-treated and E2- plus MPA-treated cultures, thrombin reversed progestin inhibition of PA activity. Predictably, the addition of D-phenyl-alanyl-propyl-arginine-chloromethyl ketone, blocked the effects of thrombin on PAI-1, tPA, and uPA protein and mRNA expression and PA activity. In summary, thrombin enhances endometrial stromal cell fibrinolytic and extracellular matrix-degrading protease activity in vitro. Such processes occurring in vivo would probably play a role in menstruation and abnormal uterine bleeding.

Ovarian steroid-modulated stromelysin-1 expression in human endometrial stromal and decidual cells.

This study examined steroid-regulated expression of the metalloproteinase stromelysin-1 in primary human endometrial stromal and decidual cells. Immunoblot analysis using a specific polyclonal antibody against stromelysin-1 revealed that the progestin medroxyprogesterone acetate (MPA) produced a time-dependent reduction in a band at 50,000 mol wt. Although the cells were refractory to estradiol (E2) alone, E2 plus MPA further reduced the intensity of this stromelysin-1 zone. By 6 days of incubation, MPA inhibited levels of secreted stromelysin-1 by one third, and E2 plus MPA inhibited stromelysin-1 levels by two thirds compared with the control values. This differential responsiveness of the stromal cells to the two steroids is reported for several biochemical end points of decidualization. Northern analysis indicated pronounced inhibition of stromelysin-1 messenger ribonucleic acid (mRNA) by E2 plus MPA over a concentration range that simulated circulating progesterone levels of the luteal phase (10(-8) mol/L) through pregnancy (10(-6) mol/L). After suppression of stromelysin-1 expression in the stromal cell monolayers by E2 plus MPA, steroid withdrawal led to a several-fold enhancement of stromelysin-1 mRNA by 4 days and of the stromelysin-1 protein by 7 days. Given its actions in degrading several extracellular matrix components and activating other MMP zymogens, steroid withdrawal-enhanced stromelysin-1 activity could mediate a proteolytic cascade that promotes the rapid tissue destruction and vascular disruption associated with menstruation. Stromelysin-1 expression by cultured decidual cells isolated from first trimester endometrium was also reduced by MPA and synergistically reduced by E2 plus MPA. As activation of the 92-kilodalton gelatinase/type IV collagenase, a crucial mediator of trophoblast invasiveness, is stromelysin-1 dependent, reduced decidual stromelysin-1 production could help to limit trophoblast invasion.

Biological mechanisms underlying the clinical effects of RU 486: modulation of cultured endometrial stromal cell plasminogen activator and plasminogen activator inhibitor expression.

The abortifacient and menstrual effects of the potent antiprogestin, RU 486, are associated with both endometrial hemorrhage and extracellular matrix (ECM) degradation. Such processes reflect reduced perivascular decidual cell hemostatic and increased ECM-degrading protease activity. Therefore, we assessed the effects of RU 486 administration on the expression of immunoreactive (ir) endometrial stromal cell urokinase-type (uPA) and tissue-type (tPA) plasminogen activator and their activities as well as levels of ir type 1 plasminogen activator inhibitor (PAI-1) using a well characterized in vitro model of decidualization. Thus, confluent stromal cell cultures were exposed to vehicle control, 10(-8) mol/L estradiol (E2), 10(-7)-10(-8) mol/L medroxyprogesterone acetate (MPA), E2 plus MPA, or 10(-6)-10(-7) mol/L RU 486 alone or in combination with MPA or E2 plus MPA for 3-4 days. Compared to the vehicle control, E2 and RU 486 used alone had no effect on levels of ir PAI-1, uPA, or tPA or on PA activity in the conditioned medium. In contrast, MPA and E2 plus MPA decreased ir uPA and tPA levels and their corresponding activities, whereas MPA increased, and E2 plus MPA further increased ir PAI-1 release. These effects of progestin were blocked by a log higher concentration of RU 486. Similar results were obtained for steady state PAI-1 messenger ribonucleic acid levels. To determine if RU 486 reversed progestin-inhibited stromal cell uPA and tPA release and progestin-enhanced PAI-1 expression, confluent cultures were exposed to 10(-8) mol/L E2 plus 10(-7) mol/L MPA for 10 days, washed, and reexposed to E2 plus MPA, steroid-free medium, or RU 486 for 3-5 or 9-11 days. Compared with cultures maintained in E2 plus MPA for 3-5 days, withdrawal to a steroid-free medium failed to affect stromal cell ir PAI-1, uPA, or tPA levels. In contrast, exposure to RU 486 for 3-5 days increased ir uPA and tPA levels 5- to 8-fold (P < 0.02) while reducing PAI-1 levels by 85% (P < 0.04). By 9-11 days of treatment, steroid withdrawal and RU 486 exerted similar effects on ir PAI-1, tPA, and uPA levels. Comparable results were obtained for PAI-1, uPA, and tPA steady state messenger ribonucleic acid levels. These findings indicate that RU 486 blocks and reverses progestin-inhibited PA expression, suggesting a mechanism for RU 486-induced endometrial hemorrhage and ECM dissolution.

Transcriptional regulation of the tissue factor gene by progestins in human endometrial stromal cells.

Decidualization of estradiol (E2)-primed human endometrial cells (HESCs) by progesterone is associated with elevated levels of tissue factor (TF), the primary initiator of hemostasis. Similarly, in cultured human HESCs, the synthetic progestin, medroxyprogesterone acetate (MPA), enhances TF protein and messenger ribonucleic acid (mRNA) levels. Although ineffective alone, E2 potentiates this progestin enhancement of TF expression by HESCs. The current study examines mechanisms underlying MPA enhancement of TF mRNA expression in HESCs. In the presence of the transcription-blocking agent dichlororibofuranosylbenzimidazole, no significant differences were noted in the half-lives of TF mRNA isolated from HESCs treated with E2 alone or with E2 plus MPA. This indicates that MPA-enhanced TF mRNA levels do not reflect changes in the stability of the TF message. To test the effect of progestin on TF promoter activity and to ascertain the mechanism of promoter regulation, primary or first passaged HESCs were transfected with TF promoter constructs spanning the regions -2106 to +121 (TFp(-2106)), -278 to +121 (TFp(-278)), and -111 to +14 (TFp(-111)) bp upstream of the transcription start site. MPA was found to enhance TF transcription by 20-fold in HESCs transfected with TFp(-2106) after correcting for transfection efficiencies with a beta-galactosidase reporter plasmid. Interestingly, levels of E2- plus MPA-stimulated transcription were significantly increased using TFp(-278) compared to TFp(-2106), suggesting that the region between -2106 and -278 bp may contain an inhibitory element. In addition, rates of MPA-stimulated transcription using TFp(-111) were significantly reduced compared to values obtained using TFp(-2106) and were even further reduced compared to values obtained using TFp(-278). This suggests that regulatory elements in the -111 bp region of the TF promoter are necessary for progestin-mediated regulation of the TF gene in HESCs, but are not sufficient to account for maximal rates of TF gene transcription. Our results also demonstrated that induction of steady state TF mRNA by MPA was abolished by treating cells with E2 plus MPA in conjunction with the protein synthesis inhibitor cycloheximide. In light of the absence of a complete progesterone or estrogen response element in the published 5'-sequence of the TF promoter, our results suggest that progestin-enhanced transcription of TF mRNA in stromal cells may be mediated by an uncharacterized protein intermediate(s).

Progestin-epidermal growth factor regulation of tissue factor expression during decidualization of human endometrial stromal cells.

Perivascular decidualized human endometrial stromal cells (HESCs) are ideally positioned to prevent peri-implantational hemorrhage during endovascular trophoblast invasion by expressing tissue factor (TF), the primary cellular mediator of hemostasis. Earlier in vivo and in vitro studies have demonstrated enhanced TF expression in estradiol (E2)-primed HESCs during progestin-induced decidualization. However, the absence of estrogen or progesterone response elements from the TF gene promoter suggests that paracrine factor(s) may mediate these effects. We now demonstrate that significant elevation of TF messenger RNA and protein levels in the cultured HESCs require incubation with both epidermal growth factor (EGF) and the progestin medroxyprogesterone acetate (MPA) added, with or without E2. By contrast, no effects were elicited by adding EGF with E2, or by the separate additions of EGF, MPA, or E2 plus MPA. Our finding, that transforming growth factor-alpha, but not transforming growth factor-beta or interleukin 1-beta mimics these EGF effects, indicates that progestin-enhanced TF expression in cultured HESCs requires activation of the EGF receptor (EGFR). Western blot analysis indicated that MPA increased EGFR levels 2-to 3-fold in cultured HESCs. The current results suggest that the progestin up-regulation of TF levels in decidualized HESCs is mediated by enhanced EGFR expression.