Abstract Objectives Cellular senescence has been proposed as a pathophysiologic driver in the development of pelvic organ prolapse (POP), especially during aging. In this study, we aimed to determine if markers of cell senescence can be quantified from vaginal secretions collected from pre‐ and postmenopausal women with and without POP. Methods Vaginal swabs were collected from 81 women in four groups: premenopausal with (pre‐P) and without prolapse (pre‐NP), and postmenopausal with (post‐P) and without prolapse (post‐NP). Multiplex immunoassays (MagPix) were then used to detect and quantify the presence of 10 SASP proteins in vaginal secretions. Results The total protein concentration of vaginal secretions differed significantly among the four groups (P = 0.003) with highest mean concentrations in pre‐P [16, interquartile range (IQR) = 4.6, 38.3 μg/μL] and lowest mean concentrations in post‐P (4.4, IQR = 2.6, 7 μg/μL). The normalized concentrations of several SASP markers differed significantly among groups, with the highest concentrations being seen in the post‐P group, and the lowest concentrations being in the pre‐NP group. Using these key markers, we then constructed receiver‐operator curves to determine the relative sensitivity and specificity of these markers in predicting prolapse. Conclusions In this study, we found that SASP proteins can be detected and quantified in vaginal secretions. Several of these markers were differentially expressed among the four groups studied, with the highest normalized concentrations of SASP markers found among postmenopausal women with prolapse. Overall, the data support the theory that senescence is associated with prolapse during aging but that other factors may be important in younger women who develop POP before menopause.

Background: Penile inversion vaginoplasty is the most common gender-affirming genital surgery performed around the world. Although individual centers have published their experiences, expert consensus is generally lacking. Methods: Semistructured interviews were performed with 17 experienced gender surgeons representing a diverse mix of specialties, experience, and countries regarding their patient selection, preoperative management, vaginoplasty techniques, complication management, and postoperative protocols. Results: There is significant consistency in practices across some aspects of vaginoplasty. However, key areas of clinical heterogeneity are also present and include use of extragenital tissue for vaginal canal/apex creation, creation of the clitoral hood and inner labia minora, elevation of the neoclitoral neurovascular bundle, and perioperative hormone management. Pathway length of stay is highly variable (1–9 days). Lastly, some surgeons are moving toward continuation or partial reduction of estrogen in the perioperative period instead of cessation. Conclusions: With a broad study of surgeon practices, and encompassing most of the high-volume vaginoplasty centers in Europe and North America, we found key areas of practice variation that represent areas of priority for future research to address. Further multi-institutional and prospective studies that incorporate patient-reported outcomes are necessary to further our understanding of these procedures.

The objective of this study was to develop an in vitro model of cellular senescence using rat vaginal fibroblasts and determine the effects of treatment with senolytics.Rat vaginal tissue biopsies were collected. Primary vaginal fibroblasts were isolated and characterized by immunofluorescence. To induce cellular senescence, fibroblasts were treated with etoposide at 3, 10, and 20 mM for 24 hours, followed by treatment with the senolytics dasatinib (1 mM) and/or quercetin (20 mM). After treatment, RNA was extracted and the expression of selected genes was quantified. Immunostaining of senescence markers was also performed.Fibroblasts were confirmed by positive immunostaining for α-smooth muscle actin and vimentin, and negative immunostaining for pan-cytokeratin. Treatment with etoposide resulted in a dose-dependent increase in expression of the senescence-associated secretory phenotype markers MMP-7, MMP-9, and IL-b1 (P < 0.05) compared with controls. Immunostaining showed increased expression of γ-H2A and p21 after treatment with etoposide. Cells treated with dasatinib and quercetin after etoposide treatment had decreased expression of p21, MMP-7, MMP-9, and IL-1b compared with cells treated only with etoposide (P < 0.05).Upregulation of senescence-associated factors provided evidence that senescence can be induced in vaginal fibroblasts in vitro. Furthermore, treatment with the senolytics dasatinib and quercetin abrogated the senescence phenotype induced by etoposide in rat vaginal fibroblasts. Our findings provide a novel model for the study and development of new therapies targeting the disordered extracellular matrix associated with pelvic organ prolapse.

OBJECTIVE:The objective of this study was to develop an in vitro model of cellular senescence using rat vaginal fibroblasts and determine the effects of treatment with senolytics.METHODS:Rat vaginal tissue biopsies were collected. Primary vaginal fibroblasts were isolated and characterized by immunofluorescence. To induce cellular senescence, fibroblasts were treated with etoposide at 3, 10, and 20 mM for 24 hours, followed by treatment with the senolytics dasatinib (1 mM) and/or quercetin (20 mM). After treatment, RNA was extracted and the expression of selected genes was quantified. Immunostaining of senescence markers was also performed.RESULTS:Fibroblasts were confirmed by positive immunostaining for α-smooth muscle actin and vimentin, and negative immunostaining for pan-cytokeratin. Treatment with etoposide resulted in a dose-dependent increase in expression of the senescence-associated secretory phenotype markers MMP-7, MMP-9, and IL-b1 (P < 0.05) compared with controls. Immunostaining showed increased expression of γ-H2A and p21 after treatment with etoposide. Cells treated with dasatinib and quercetin after etoposide treatment had decreased expression of p21, MMP-7, MMP-9, and IL-1b compared with cells treated only with etoposide (P < 0.05).CONCLUSIONS:Upregulation of senescence-associated factors provided evidence that senescence can be induced in vaginal fibroblasts in vitro. Furthermore, treatment with the senolytics dasatinib and quercetin abrogated the senescence phenotype induced by etoposide in rat vaginal fibroblasts. Our findings provide a novel model for the study and development of new therapies targeting the disordered extracellular matrix associated with pelvic organ prolapse.

In adult males, spermatogonia maintain lifelong spermatozoa production for oocyte fertilization. To understand spermatogonial metabolism we compared gene profiles in rat spermatogonia to publicly available mouse, monkey, and human spermatogonial gene profiles. Interestingly, rat spermatogonia expressed metabolic control factors Foxa1, Foxa2, and Foxa3. Germline Foxa2 was enriched in Gfra1(Hi) and Gfra1(Low) undifferentiated A-single spermatogonia. Foxa2-bound loci in spermatogonial chromatin were overrepresented by conserved stemness genes ( Dusp6, Gfra1, Etv5, Rest, Nanos2, Foxp1) that intersect bioinformatically with conserved glutathione/pentose phosphate metabolism genes (Tkt, Gss, Gclc, Gclm, Gpx1, Gpx4, Fth), marking elevated spermatogonial GSH:GSSG. Cystine-uptake and intracellular conversion to cysteine typically couple glutathione biosynthesis to pentose phosphate metabolism. Rat spermatogonia, curiously, displayed poor germline stem cell viability in cystine-containing media, and, like primate spermatogonia, exhibited reduced transsulfuration pathway markers. Exogenous cysteine, cysteine-like mercaptans, somatic testis cells, and ferroptosis inhibitors counteracted the cysteine-starvation-induced spermatogonial death and stimulated spermatogonial growth factor activity in vitro.

Although it is well appreciated that ovarian stimulation protocols for in vitro fertilization (IVF) alter endometrial receptivity, the precise cellular mechanisms are not known. To gain insights into potential mechanisms by which different ovarian stimulation protocols alter the endometrium, we compared histologic and gene expression profiles of endometrium from women undergoing conventional ovarian stimulation for IVF (C-IVF) with those undergoing minimal stimulation with clomiphene citrate (MS-IVF). Sixteen women undergoing MS-IVF (n = 8) or C-IVF (n = 8) were recruited for endometrial biopsy at the time of oocyte retrieval. Endometrial glands were large, tortuous, and secretory with C-IVF but small and undifferentiated with MS-IVF. Whereas RNA sequencing did not reveal changes in estrogen receptor or its co-regulators or classic proliferation associated genes in MS-IVF, together with immunohistochemistry, Wnt signaling was disrupted in endometrium from MS-IVF cycles with significant upregulation of Wnt inhibitors. Secreted frizzled-related protein 1 (sFRP1) was increased fourfold (p < 0.01), and sFRP4 was upregulated sixfold (p < 0.01) relative to C-IVF. Further these proteins were localized to subepithelial endometrial stroma. These data indicate that MS-IVF protocols with CC do not seem to impact endometrial estrogen signaling as much as would be expected from the reported antiestrogenic properties of CC. Rather, the findings of this study highlight Wnt signaling as a major factor for endometrial development during IVF cycles.

Impaired elastogenesis and increased degradation of elastic fibers has been implicated in the pathogenesis of pelvic organ prolapse. Loss of the elastogenic organizer, fibulin-5 (FBLN5), leads to pelvic organ prolapse in mice. The objective of this study was to investigate the regulation of FBLN5 after surgical injury of the vaginal wall using the rat as a preclinical animal model. Both endogenous and recombinant FBLN5 were degraded after surgical injury. Estrogen did not alter the dramatic loss of vaginal FBLN5 in the acute phase after injury (12–48 h), but resulted in rescue of the poor recovery of FBLN5 levels in the late phase (7 d) of healing in ovariectomized animals. In contrast with estrogen, the general MMP inhibitor, actinonin, abrogated injury-induced degradation of FBLN5 significantly. Further, actinonin rescued the negative effects of injury on biomechanics, histomorphology, and elastic fibers. Control of excessive matrix degradation by local application of actinonin at the time of surgery may lead to improved elastic fiber regeneration and wound healing, thereby potentially enhancing pelvic floor recovery after reconstructive surgery for prolapse.

be susceptible to endometriosis.It has been reported that the levels of HDAC1 and HDAC2 are deregulated in endometriotic stromal cells.HDAC1 and HDAC2 are key regulators of WNT and p53 pathways.During nucleosome remodeling, the deacetylase complex physically interacts with the WNT4 chromatin in an HDAC-dependent manner, leading to suppression of the WNT4 gene and WNT4 dependent morphogenesis.Analyses of the ten other human HDAC genes are underway.

Hormonal effects on epithelial cells of the endometrium are often mediated through stromal cell receptors. Endometrium from women undergoing IVF using minimal stimulation with clomiphene citrate (CC) is characterized by marked atrophy of endometrial glands accompanied by relative increases in stromal cells (despite supraphysiologic levels of E2). Although it's commonly believed that CC exhibits its effects simply as an estrogen receptor antagonist, recent studies indicate that CC differentially affects endometiral cell compartments. A dramatic upregulation of the Wnt antagonists, secreted frizzled related proteins 1 and 4 (sFRP1,4) is seen in only stromal cells. sFRPs inhibit Wnt7a signaling pathways affecting endometrial decidualization and gland formation in endometrial cancer cell growth, yet physiologic regulation of Wnt signaling in endometrium is not well understood. To test the hypothesis that sFRPs are secreted constitutively by stroma cells but regulated in vivo by secretions from endometrial epithelial cells. Primary human endometrial stromal cells and epithelial cells (Epi, Ishikawa) were used alone or in co-culture with transwell cell culture inserts. Stromal cells (80% confluent) were pretreated for 48 h in serum free media prior to treatment with vehicle, or estradiol (E2, 3.6 nM) ± CC (20nM). Gene expression was quantified by qPCR and normalized to two housekeeping genes, GAPDH and h36B4. Endometrial tissue explants from spontaneously ovulating women (24 h after LH surge) were studied. ANOVA and Student's T test were used for statistical analyses as appropriate. In agreement with previous experiments, E2 treatment of stromal cells increased PR-B (from 1 ± 0.1 to 6.7 ± 0.4 RU, p < 0.01) and total PR (from 1.0 ± 0.11 to 5.4 ± 0.23 RU, p < 0.01). CC also increased PR gene expression in stromal cells, but did not alter expression of sFRP1 or 4. In co-culture, Epi did not alter E2-induced upregulation of PRs in stroma. However, co-culture with Epi downregulated stromal cell sFRP4 (83 ± 3%) with or without E2. The magnitude of sFRP4 suppression was dose-dependent with increasing number of Epi cells (from 104 - 105/cm2). Epi co-culture also decreased stromal sFRP1 to 34 ± 11% and expression of stromal growth factors (FGF-9 and TGF-α) significantly. Treatment of tissue explants from ovulatory women with E2 for 72 h resulted in suppression of tissue sFRP1 (from 1 ± 0.4 to 0.34 ± 0.12 RU) and sFRP4 (from 1.2 ± 0.57 to 0.33 ± 0.03 RU). CC did not have direct effects on sFRP in stroma. Rather, secretions from glandular epithelial cells suppressed stromal sFRP1 and 4 and growth factors. CC-induced inhibition of epithelial cell growth results in immature, atrophic glands that are insufficient to mediate suppression of sFRPs in the stroma. Thus, unregulated expression of stromal sFRPs suppresses Wnt signaling resulting in accentuated loss of epithelial differentiation and growth. In the absence of CC, E2 induces epithelial cell growth and suppression of stromal sFRP1 and 4 culminating in full maturation of the endometrium. Further investigation is warranted to better understand the complex dialogue between stromal and epithelial co-regulation.

Hormonal effects on epithelial cells of endometrium are often mediated through stromal cell receptors. Endometrium from women undergoing IVF using minimal stimulation (MS-IVF) with clomiphene citrate (CC) is characterized by marked atrophy of endometrial glands accompanied by relative increases in stromal cells, despite supraphysiologic levels of E2. Previously, we discovered dramatic stromal cell-specific upregulation of Wnt antagonists (secreted frizzled related proteins 1,4, SFRP) in endometrium from MS-IVF. Although sFRPs inhibit Wnt7a signaling during endometrial decidualization and gland formation in endometrial cancer, physiologic regulation of Wnt signaling in endometrium is not well understood. Our objective was to test the hypothesis that sFRPs are secreted constitutively by stroma cells but regulated in vivo by secretions from endometrial epithelial cells. Epithelial cells (Epi, Ishikawa) and primary human endometrial stromal cells were used alone or in co-culture with transwell cell culture inserts. Stromal cells were pretreated for 48 h in serum free media prior to treatment with vehicle or estradiol (E2, 3.6 nM) ± CC (20nM). Gene expression was quantified by qPCR and normalized to two housekeeping genes,GAPDHand h36B4. Endometrial tissue explants from spontaneously ovulating women (24 h after LH surge, n = 4) were studied. ANOVA and Student's T test were used for statistical analyses as appropriate. E2 treatment of stromal cells increased PR-B(from 1 ± 0.1 to 6.7 ± 0.4 RU, p < 0.01) and total PR(from 1.0 ± 0.11 to 5.4 ± 0.23 RU, p < 0.01). CC was not an ER antagonist in stromal cells also increasing PR gene expression. In contrast to in vivo results, CC did not alter expression of sFRP1or 4 in stromal cell cultures. In co-culture, Epi did not alter E2-induced upregulation of PRs in stroma. However, co-culture with Epi downregulated stromal cell sFRP4(83 ± 3%). The magnitude of sFRP4 suppression was dose-dependent with increasing number of Epi cells (from 104- 105/cm2). Epi co-culture also decreased stromal sFRP1 to 34 ± 11% and expression of stromal growth factors (FGF-9 and TGF-α) significantly. To investigate physiologic relevance, treatment of tissue explants from ovulatory women with E2 for 72 h (to induce epi growth) resulted in suppression of sFRP4(from 1.44 ± 0.3 to 0.55 ± 0.08 mRNA, p < 0.02). Although CC did not have direct effects on sFRP in stroma, secretions from glandular epithelial cells suppressed Wnt antagonists sFRP1and 4 in stroma. These results support the hypothesis that CC-induced inhibition of epithelial cell growth results in immature, atrophic glands that are insufficient to suppress Wnt inhibitors in stroma thereby accentuating loss of epithelial differentiation and growth. In the absence of CC, E2 induces epithelial cell growth and suppression of stromal sFRPs culminating in full maturation of the endometrium. Although it is believed that CC exhibits its effects simply as an ER antagonist, these studies indicate that the effects of CC are more complex and involve inhibition of Wnt signaling in cell-specific compartments.

Fibulin 5 (FBLN5) is an extracellular matrix glycoprotein that suppresses matrix metalloprotease 9 (MMP-9), angiogenesis and epithelial cell motility.Here, we investigated the regulation and function of FBLN5 in epithelial ovarian cancer (EOC).FBLN5 mRNA was down-regulated 5-fold in EOC relative to benign ovary.Not surprisingly, MMP9 mRNA and enzyme activity were increased significantly, and inversely correlated with FBLN5 gene expression.FBLN5 degradation products of 52.8 and 41.3 kDa were increased substantially in EOC.We identified two candidate proteases (serine elastase and MMP-7, but not MMP-9) that cleave FBLN5.MMP-7, but not neutrophil elastase, gene expression was increased dramatically in EOC.Recombinant FBLN5 significantly inhibited adhesion of EOC cells to both laminin and collagen I. Finally, using immunohistochemistry, we found immunoreactive FBLN5 within tumor macrophages throughout human EOC tumors.This work indicates that FBLN5 is degraded in EOC most likely by proteases enriched in macrophages of the tumor microenvironment.Proteolysis of FBLN5 serves as a mechanism to promote cell adhesion and local metastasis of ovarian cancer cells.Promotion of a stable ECM with intact FBLN5 in the tumor matrix may serve as a novel therapeutic adjunct to prevent spread of ovarian cancer.

BACKGROUND:Androgenic compounds have been implicated in induction of endometrial atrophy yet the mechanisms of androgen effects on human endometrium have not been well studied. We hypothesized that androgens may promote their endometrial effects via modulation of progesterone receptor (PR) expression.METHODS:Proliferative phase endometrial samples were collected at the time of hysterectomy. We evaluated the effect of the potent androgen 5α-dihydrotestosterone (DHT) on endometrial PR expression by treating human endometrial explants, endometrial stromal cells, and Ishikawa cells with DHT. Ishikawa cells were also treated with DHT ± the androgen receptor (AR) blocker flutamide. The PR-B, total PR messenger RNA (mRNA), and PR protein expression were assessed. Expression of cyclin D1 and D2 was checked as markers of cell proliferation.RESULTS:As expected, estradiol induced PR expression in isolated stromal cells, endometrial epithelial cells, and tissue explants. The DHT treatment also resulted in increased PR expression in endometrial explants and Ishikawa cells but not in stromal cells. Further, protein levels of both nuclear PR isoforms (PR-A and PR-B) were induced with the DHT treatment. Although flutamide treatment alone did not affect PR expression, flutamide diminished androgen-induced upregulation of PR in both endometrial explants and Ishikawa cells. Although estradiol induced both cyclin D1 and cyclin D2 mRNA, DHT did not induce these markers of cell proliferation.CONCLUSION:Androgens may mediate endometrial effects through upregulation of PR gene and protein expression. Endometrial PR upregulation by androgens is mediated, at least in part, through AR.

Although the positive effects of vaginal estrogens and the selective estrogen receptor modulator, ospemifene (OS), on the vaginal epithelium are well recognized, less is known regarding the effects of these therapies on the lower urinary tract or vaginal muscularis. Clinical evidence suggests that vaginally administered estrogen may improve overactive bladder-related symptoms. The objective of this study was to compare the effects of OS, vaginal conjugated equine estrogens (CEE), or both on the vaginal wall and lower urinary tract in a rat model of menopause. Contractile force of the bladder neck, dome, and external urethral sphincter at optimal field stimulation did not differ significantly among treatment groups. Pharmacologic responses to atropine, carbachol, and potassium chloride were similar among groups. Vaginal epithelial thickness and differentiation were differentially regulated by CEE or OS. Ospemifene altered epithelial differentiation pathways in vaginal epithelium in a unique way, and these effects were additive with local CEE. Unless contraindicated, the beneficial effects of vaginal CEE on the vaginal wall outweigh those of OS.

Although fertilization rates have increased with modern-day IVF, implantation rates have not improved to the same extent. It is believed that high estradiol (E2) levels are detrimental to endometrial receptivity. Here, we tested the hypothesis that serum from women undergoing minimal stimulation (mini-stim) and traditional gonadotropin protocols alters early differentiation of human endometrial stromal cells (HESCs) during decidualization. HESCs from women in the proliferative phase were used in vitro to test the effect of preovulatory serum on endometrial differentiation. HESCs were treated with preovulatory serum (1 d prior to hCG) from patients undergoing natural cycle, mini-stim, or traditional IVF (15% v/v) x 24 h with or without decidualization (MPA + cAMP). Charcoal-stripped fetal bovine serum (FBS, 15%) ± E2 (0.3, 2.7, or 4.5 ng/ml) or E2 + clomiphene citrate (6 ng/ml) served as controls. RNA was extracted and analyzed with qPCR. FKBP-4 and -5 (progesterone receptor chaperones and markers of early secretory endometrium) increased significantly during decidualization with 15% FBS (from 0.6 ± 0.13 to 1.0 ± 0.14 RU/GAPDH for FKBP-4; from 0.14 ± 0.04 to 1.0 ± 0.12, FKBP-5). Serum (15% v/v) from natural cycle IVF not only increased baseline levels of FKBP-4 from 0.6 to 2.03 ± 0.4, but also amplified early decidual responses from 1.0 ± 0.1 to 2.67 ± 0.15 RU/GAPDH, P < 0.05. Likewise, natural cycle serum increased baseline expression of FKBP-5 (from 0.14 ± 0.04 to 0.90 ± 0.13 RU/GAPDH) especially after decidualization (to 2.9 ± 0.09, P < 0.01). The effect of serum from mini-stim or traditional IVF was more dramatic than that of natural cycle. For example, decidualized FKBP-4 was amplified from 1 ± 0.1 (FBS) to 3.8 ± 0.08 for mini-stim and to 4.4 ± 0.3 for traditional IVF. Effects of E2 (0.3 - 4.5 ng/ml) were modest relative to preovulatory serum with only 60-80% increases in FKBP-4 and -5. Further, clomiphene citrate (6 ng/ml) did not alter these effects. Surprisingly, aquaporin 3 (AQP3, also upregulated in early secretory endometrium) was not induced within 24 h of decidualization with FBS. Expression of AQP3, however, was increased significantly with natural cycle serum from 1.0 ± 0.15 to 2.3 ± 0.33; to 2.4 ± 0.08 for mini-stim; and to 4.1 ± 0.8 for traditional IVF. Markers of early secretory endometrium are upregulated in HESCs decidualized for 24 hours. The magnitude of this change in endometrial differentiation is increased with pre-ovulatory serum from mini-stim and traditional IVF relative to natural cycle and independent of E2 or clomiphene citrate. The results suggest that accelerated endometrial development during IVF is more complex than just high E2 levels and may involve other circulating factors such as progesterone or gonadotropins.

OBJECTIVE:Reconstructive surgery for pelvic organ prolapse is plagued with high failure rates possibly due to impaired healing or regeneration of the vaginal wall. Here, we tested the hypothesis that postoperative administration of local estrogen, direct injection of mesenchymal stem cells (MSCs), or both lead to improved wound healing of the injured vagina in a menopausal rat model. METHODS:Ovariectomized rats underwent surgical injury to the posterior vaginal wall and were randomized to treatment with placebo (n = 41), estrogen cream (n = 47), direct injection of MSCs (n = 39), or both (n = 43). RESULTS:MSCs did not survive after injection and had no appreciable effects on healing of the vaginal wall. Acute postoperative administration of vaginal estrogen altered the response of the vaginal wall to injury with decreased stiffness, decreased collagen content, and decreased expression of transcripts for matrix components in the stromal compartment. Conversely, vaginal estrogen resulted in marked proliferation of the epithelial layer and increased expression of genes related to epithelial barrier function and protease inhibition. Transcripts for genes involved in chronic inflammation and adaptive immunity were also down-regulated in the estrogenized epithelium. CONCLUSIONS:Collectively, these data indicate that, in contrast to the reported positive effects of preoperative estrogen on the uninjured vagina, acute administration of postoperative vaginal estrogen has adverse effects on the early phase of healing of the stromal layer. In contrast, postoperative estrogen plays a positive role in healing of the vaginal epithelium after injury.

To evaluate the effect of myogenic stem cell-laden hydrogel scaffold on contractile function and histomorphology of the external anal sphincter (EAS) after transection without repair.

Background: Bleeding during pregnancy is a risk factor for premature rupture of the fetal membranes. Results: Thrombin causes preterm birth in mice and activates PAR-1 and TLR4 to increase MMPs and COX-2. Conclusion: Thrombin acts through multiple mechanisms to increase MMPs and PGE(2) in amnion. Significance: Thrombin plays a pivotal role in the pathogenesis of preterm labor and rupture of the membranes.Here, we investigated the effects of thrombin on matrix metalloproteinases (MMPs) and prostaglandin (PG) synthesis in fetal membranes. Thrombin activity was increased in human amnion from preterm deliveries. Treatment of mesenchymal, but not epithelial, cells with thrombin resulted in increased MMP-1 and MMP-9 mRNA and enzymatic activity. Thrombin also increased COX2 mRNA and PGE(2) in these cells. Protease-activated receptor-1 (PAR-1) was localized to amnion mesenchymal and decidual cells. PAR-1-specific inhibitors and activating peptides indicated that thrombin-induced up-regulation of MMP-9 was mediated via PAR-1. In contrast, thrombin-induced up-regulation of MMP-1 and COX-2 was mediated through Toll-like receptor-4, possibly through thrombin-induced release of soluble fetal fibronectin. In vivo, thrombin-injected pregnant mice delivered preterm. Mmp8, Mmp9, and Mmp13, and PGE(2) content was increased significantly in fetal membranes from thrombin-injected animals. These results indicate that thrombin acts through multiple mechanisms to activate MMPs and PGE2 synthesis in amnion.