Samples Of Myometrium Research Articles

The expression of estrogen receptor isoforms alpha, beta and insulin-like growth factor-I in uterine leiomyoma

Background. A prospective study focusing on leiomyoma was conducted to investigate the expression of estrogen receptor-α (ERα), estrogen receptor-β (ERβ) and insulin-like growth factor-I (IGF-I) to elucidate their roles in the pathogenesis of leiomyoma.Methods. Twenty-one leiomyomal and six paired myometrial tissues were included in the study. The levels of ERα, ERβ and IGF-I mRNAs in leiomyoma and adjacent myometrium were analyzed by quantitative reverse transcription-polymerase chain reaction.Results. All tissues showed a higher ERα expression level. The correlation between ERα mRNA and IGF-I mRNA levels was significantly positive (r = 0.527, p = 0.014). The correlation between ERβ mRNA and IGF-I mRNA levels was also positive, but not statistically significant. In six paired leiomyoma and myometrium samples, the levels of ERα and ERβ mRNA exhibited no difference, and IGF-I mRNA levels were elevated in leiomyoma compared with paired myometrium (p < 0.05).Conclusions. The expression of IGF-I mRNA increases in leiomyoma and ERα mRNA is positively correlated with IGF-I mRNA, which implies that estrogen upregulates the gene encoding IGF-I through ERα in leiomyoma; ERβ may also be involved in the IGF-I signaling pathway in leiomyoma.

The Use of p16 in Enhancing the Histologic Classification of Uterine Smooth Muscle Tumors

Uterine smooth muscle tumors can usually be divided histologically into leiomyoma (L) and leiomyosarcoma (LMS). Occasionally, the histologic features are indeterminate and classified as smooth muscle tumor of uncertain malignant potential (STUMP). Recent gene expression studies have found p16 overexpressed in LMS when compared with normal myometrium. This study evaluated the protein expression of p16 by immunohistochemistry in LMS, L, and normal myometrium. Additionally, 8 tumors originally classified as STUMP were evaluated for p16 expression and correlated to their clinical outcome. A tissue microarray was constructed and composed of 15 LMS, 8 STUMPs, 22 L, and 10 samples of normal myometrium. p16 expression was correlated with clinical outcome and histologic features. Twelve of the 15 LMS strongly and diffusely expressed p16, 3 of the L had focal p16 staining, and none of the normal myometria were p16 positive. Three of the tumors originally classified as STUMP developed metastatic disease and 2 of these tumors had strong, diffuse p16 positivity. Histologically, these 2 cases were characterized by coagulative tumor cell necrosis and only mild cytologic atypia. p16 is preferentially expressed in LMS with only rare L showing positivity. Histologically, tumors with coagulative tumor cell necrosis alone were clinically LMS. In those cases in which the type of necrosis is uncertain (coagulative tumor cell vs. hyalinized), the addition of p16 may aid in discerning a subset of STUMP that should be classified as LMS.

Reduced expression of 14-3-3 gamma in uterine leiomyoma as identified by proteomics

To use proteomic techniques, including two-dimensional electrophoresis (2DE), Western blot, and mass spectrometry, to screen and identify proteins that were expressed differently in patients with uterine leiomyoma versus normal myometrium. First, identify proteins important for leiomyogenesis by comparing protein expression in leiomyoma and normal myometrium. Second, examine the expression level of selected proteins on a larger panel of leiomyomas. Academic research laboratory. Consenting premenopausal patients scheduled to undergo hysterectomy for symptomatic uterine leiomyomas. Paired samples of leiomyoma and adjacent myometrium were obtained and submitted to 2DE, mass spectrometry, Western blot, reverse transcription polymerase chain reaction (RT-PCR), and immunohistochemistry. Protein expression. A comparison of protein patterns revealed 37 protein spots significantly changed, with 10 protein spots with concordantly increased and 27 protein spots with concordantly decreased intensity in leiomyoma compared with normal myometrium. Ten of these proteins were identified by tandem mass spectrometry. Interestingly, one of the proteins, which exhibited a marked down-regulation in leiomyoma tissues, was the recently identified highly conserved cellular protein 14-3-3-gamma. The reduction of the 14-3-3 gamma in leiomyoma tissues was confirmed by comparative immunoblotting (n = 25) and RT-PCR (n = 25) as well as by immunohistochemistry (n = 35) of individual-matched neoplastic and normal myometrium tissue specimens. The consistency of 14-3-3 gamma reduction in uterine leiomyoma suggests that 14-3-3 protein may play a role in the origin or growth of leiomyoma.

Stiffness matters: mechanical properties of human leiomyomas and their extracellular matrix composition

Uterine leiomyomas produce an extracellular matrix (ECM) that is abnormal in its volume, composition, and structure. ECM is largely composed of collagen, proteogylcans, and glycosaminoglycans. Alterations in ECM content can influence tissue stiffness and modify mechanical signals on the cells leading to fibrosis, tumor growth, increased cell proliferation and reduced apoptosis. Since mechanotransduction has not been characterized in leiomyoma, we sought to quantify tissue mechanical properties and ECM composition in matched leiomyoma and myometrial samples. Analysis of IRB-approved, surgically-obtained leiomyoma and myometrial samples. We measured the mechanical response of paired samples of myometrium and leiomyoma samples subjected to 10% compressive strain, and calculated their respective Young's moduli (slope of the stress-strain curve at equilibrium). Total glycosaminogylcan (GAG) content was assayed using the Blyscan method. Hydroxyproline assays were conducted to assess mature, crosslinked collagen content. The average Young's moduli, a measure of tissue stiffness, for leiomyoma and myometrium were 18.4 kPa ± 2.88 and 4.9 kPa ± 1.3 respectively (P<0.05). Total sulfated GAG content per wet weight of tissue in leiomyoma and myometrium was 1.27 ug/ug ± 0.08 of tissue and 1.02 ug/ug ± 0.23, respectively. The hydroxyproline content in leiomyoma and myometrium was 8.75 ug/ug ± 1.61 of tissue and 7.29 ug/ug ± 1.32 respectively. The overall water content was 79.4% ± 0.83% in leiomyoma and 84.1% ± 0.37% in myometrium (P<0.01). Leiomyoma had a stiffness 3 fold greater than the myometrial samples from the same uterus. The increased stiffness was accompanied by a decrease in water content, a moderate increase in total sulfated gylcosaminoglycan content, and a slight increase in hydroxyproline content. The altered structure-function relationships reported in this study for leiomyomas support the hypothesis that differences in the ECM mechanical properties and composition may contribute to the an altered state of cell growth and proliferation in leiomyoma.

The focal adhesion protein Hic-5 is highly expressed in the rat myometrium during late pregnancy and labour and co-localizes with FAK

BackgroundMyometrial growth and remodeling of the cytoskeleton and focal adhesions during late pregnancy may be critical aspects of myometrial activation and thus labour. Yet our understanding of these aspects is inhibited by the paucity of information concerning the components of focal adhesions in the myometrium. The focal adhesion protein hydrogen peroxide-inducible clone-5 (Hic-5) has recently been found in mononuclear smooth muscle but was not examined in the myometrium during pregnancy. Thus, the goal of this study was to characterize Hic-5 mRNA and protein expression in the rat myometrium during pregnancy and labour.MethodsRat myometrium samples were obtained from non-pregnant animals, pregnant animals on days (d) 6, 12, 15, 17, 19, 21, 22, 23 (active labour) and 1 day postpartum (PP). In addition, myometrium samples were collected from rats within a progesterone-delayed labour paradigm. Hic-5 mRNA expression was analyzed by Northern blot analysis while Hic-5 protein expression was examined by immunoblot and immunofluorescence analysis.ResultsHic-5 mRNA expression on d15, d19 and d21 was found to be significantly elevated compared to d6 and d12 of pregnancy and expression on d23 was significantly elevated over d6 (p < 0.05). Immunofluorescence analysis demonstrated that detection of Hic-5 protein in the circular muscle layer appeared to increase from d17 onwards, except PP, and Hic-5 was detectable in the cell cytoplasm and more continuously associated with myometrial cell membranes. In the longitudinal muscle layer Hic-5 was readily detectable by d15 and thereafter and primarily associated at myometrial cell membranes. Co-immunofluorescence analysis of potential Hic-5 and focal adhesion kinase (FAK) association in situ demonstrated a limited level of co-localization on d19, d23 and PP in the circular muscle layer while in the longitudinal muscle layer Hic-5 and FAK were readily co-localized at myometrial cell membranes.ConclusionHic-5 is highly expressed in the rat myometrium during late pregnancy and labour and co-localizes with FAK in situ. Our results are consistent with a potential role for Hic-5 in focal adhesion remodeling in the rat myometrium during late pregnancy.

Increased expression of latent TGF-ß binding protein-1 and fibrillin-1 in human uterine leiomyomata

We compared latent TGF-ss binding protein-1 (LTBP-1) and fibrillin-1 (FBN-1) expression in leiomyomata and myometrium, correlated with leiomyomata size. We studied in vivo and in vitro effects of ovarian steroids using matched leiomyomata and myometrium samples from both phases of the menstrual cycle. Leiomyomata were divided into small (<or=2 cm), medium (3-5 cm) and large (>or=6 cm) groups. We validated LTBP-1 and FBN-1 expression using QPCR, western blot and immunohistochemistry. LTBP-1 and FBN-1 mRNA and protein expressions were higher in the medium-sized group compared with myometrium in the proliferative phase (P = 0.01; P = 0.01). FBN-1 mRNA expression was higher in the secretory phase (P = 0.01). LTBP-1 mRNA and protein expression was higher in the medium group compared with the small and large groups in the proliferative phase (P = 0.04; P = 0.04). No differences between groups were seen in FBN-1 expression in either phase. 17Beta-estradiol (E2) increased mRNA and protein expression of LTBP-1 and FBN-1 in cultured leiomyoma smooth muscle cells (LSMC) (P < 0.05). No change in FBN-1 and LTBP-1 expression was observed when cells were treated with E2 plus progesterone. Estrogen may be involved in LTBP-1 and FBN-1 expression in leiomyomata. Extracellular matrix metabolism may be different in medium-sized leiomyoma.

The effects of the selective progesterone receptor modulator asoprisnil on the morphology of uterine tissues after 3 months treatment in patients with symptomatic uterine leiomyomata

Asoprisnil is a selective progesterone receptor modulator with mixed progesterone agonist/antagonist activity which controls uterine bleeding via an endometrial effect. This study examined full-thickness endometrial, leiomyoma and myometrial morphology in hysterectomy specimens from patients with uterine leiomyomata, after treatment with asoprisnil for 3 months. In this double-blind, randomized, placebo-controlled study, 33 subjects with uterine leiomyomata were randomized to receive asoprisnil 10, 25 mg or placebo for an average of 95 days prior to hysterectomy. Samples of endometrium, myometrium and leiomyoma tissue were subjected to systematic morphological assessment with quantification of mitotic activity. In patients treated with 10 or 25 mg asoprisnil, a unique pattern called 'non-physiologic secretory effect' was evident in endometrium, recognizable through partially developed secretory glandular appearances and stromal changes. Endometrial thickness was decreased, and there were low levels of mitotic activity in endometrial glands and stroma. Unusual thick-walled muscular arterioles and prominent aggregations of thin-walled vessels were present in endometrial stroma, but not in myometrium or non-endometrial vascular beds. Mitotic activity was decreased in leiomyomata. Asoprisnil induces unique morphological changes and is associated with low levels of glandular and stromal proliferation in endometrium, and in leiomyomata. These changes are likely to contribute to the amenorrhoea experienced after exposure to the medication.

Expression profile of several genes in human myometrium and uterine leiomyoma

To screen several genes that are differentially expressed in uterine leiomyoma and matched unaffected myometrium by using microarray-based hybridization and real-time polymerase chain reaction analyses. Screen by arrays for < or =2,400 known genes in leiomyoma and control myometrium. University clinical research laboratory. Four patients with leiomyoma scheduled for surgery during the proliferative phase. Four paired samples of leiomyoma and adjacent myometrium were obtained from patients undergoing hysterectomy. Fold-changes in expression of leiomyoma and matched myometrium (L/M). A comparison of expression patterns revealed 73 genes significantly up- or down-regulated in each paired tissue sample, of which 30 genes showed increased expression (mean L/M of >2) and 43 showed decreased expression (mean L/M of <0.5) in leiomyoma compared with normal myometrium. When considering only growth factors, pleiotropin (PTN) was expressed 3.5-fold more in leiomyomas compared with in myometrium. No other growth factors were similarly affected. In addition, real-time polymerase chain reaction analysis correlated well with microarray data. Data obtained from the present study suggest that several genes are selectively overexpressed in leiomyomas compared with in myometrium. Increased expression of growth factor PTN may represent a promising target for therapy.

Myometrial prostaglandin E2 synthetic enzyme mRNA expression: spatial and temporal variations with pregnancy and labour

We have investigated the hypothesis that the expression of the enzymes involved in PGE(2) synthesis in the human uterus is co-ordinated. We have studied (i) the mRNA expression of the enzymes involved in PGE(2) synthesis [phospholipases (cPLA(2) and sPLA(2)), prostaglandin H synthase (PGHS)-2 and PG E synthases (PGES-1 and -2)] and their relationship to the expression of inflammatory cytokines in samples of myometrium obtained from pregnant women undergoing caesarean section (LSCS) either before or after the onset of labour at or before term; and (ii) the effect of IL-1beta, IL-6, TNF-alpha, PGE(2) and stretch on PGE(2) enzyme mRNA expression. We found that cPLA(2), sPLA(2) and PGHS-2 mRNA expression were greater in labour samples; cPLA(2), sPLA(2), PGHS-2, PGES-1 and -2 mRNA expression were greater in lower- than upper-segment samples; and there was no effect of gestational age. PGHS-2 mRNA levels correlated with those of PGES-1, cPLA(2), IL-1beta and IL-8; PGES-1 mRNA levels correlated with those of IL-1beta, IL-8 and cPLA(2). In primary cultures of uterine myocytes, cPLA(2) mRNA expression was increased by IL-1beta and IL-6; PGHS-2 mRNA expression was increased by IL-1beta, PGE(2) and stretch; and PGES-1 mRNA expression was increased by IL-1beta only. These data show that labour is associated with increased expression of the enzymes involved in PGE(2) synthesis and their expression is greater in the lower uterine segment. The presence of associations between the levels of PGE(2) enzyme mRNA expression and the effects of IL-1beta suggest that their expression is co-ordinated and that IL-1beta is the responsible factor.

Ethnic distribution of estrogen receptor-α polymorphism is associated with a higher prevalence of uterine leiomyomas in black Americans

To investigate whether polymorphisms in the estrogen receptor alpha (ERalpha) gene are associated with an increased risk of uterine leiomyomas (ULMs). Genomic DNA was isolated from normal myometrium samples collected at the time of the hysterectomy. Volunteers in an academic research environment. One hundred ninety-eight women with surgically confirmed ULMs and 229 matched controls with nonfibroid uteri. Hysterectomy samples were collected from volunteers. The two PvuII and XbaI intronic polymorphisms in the ERalpha gene using polymerase chain reaction and restriction fragment length polymorphism. The ERalpha PP genotype was associated with a significantly increased risk of ULM in black and white women, but not in Hispanic women. Women with the ERalpha PP genotype were 6.42 times (confidence limits 2.04-20.16) more likely to have ULMs than other genotypes. The ERalpha PP genotype was also significantly associated with larger tumor burden (>400 g). The overall prevalence of the PP genotype was significantly higher in black women (35%) than white (13%) or Hispanic (16%) women. Myometrial cell lines expressing the PP genotype exhibited enhanced proliferative response to estrogen in vitro compared with their pp counterparts. The ERalpha PP genotype is a genetic risk factor for ULM development among surgically treated women. The higher prevalence of this genotype in blacks might explain the increased occurrence of this tumor among black women.

Expression of Transient Receptor Channel Proteins in Human Fundal Myometrium in Pregnancy

Cation channels comprised of transient receptor potential (TrpC) proteins may play a role in signal-regulated calcium entry and calcium homeostasis in myometrium. The objective of this study was to determine the relative abundance of specific TrpC mRNAs expressed in human myometrium and determine if TrpC mRNA and protein concentrations differ in fundal myometrium before and after the onset of labor. A quantitative real-time polymerase chain reaction (Q-RT-PCR) procedure was developed for determining the concentration of TrpC mRNA expression in immortalized and primary human myometrial cells and myometrial fundus tissues from patients before and after the onset of labor. The corresponding TrpC proteins were detected by Western blot analysis and immunohistochemistry. hTrpC1, 3, 4, 5, 6, and 7 mRNAs were expressed in two lines of immortalized human myometrial cells and in primary human myocytes. In all of these cells, hTrpC1 and hTrpC4 mRNAs were the most abundant, followed by hTrpC6. A similar distribution was observed in fundal myometrium samples from patients before and after the onset of labor. hTrpC4 mRNA was significantly lower after the onset of labor; there were no significant changes in the concentrations of other TrpC mRNAs. Immunohistochemistry identified hTrpC1, 3, 4, and 6 proteins in myometrial smooth muscle cells. Western blot analysis of myometrial membranes demonstrated no statistically significant changes in hTrpC1, 3, 4, and 6 proteins between samples collected before and after the onset of labor. We have demonstrated that hTrpC1 and hTrpC4 are the most abundant TrpC mRNAs in human myometrium, with TrpC6 being the next most abundant. There was no increase in TrpC mRNA or protein in fundal myometrium with the onset of labor. Nonetheless, these isoforms may play significant roles in signal regulated calcium entry in human myometrium.

The effects of dexmedetomidine on spontaneous contractions of isolated gravid rat myometrium

The direct effects of dexmedetomidine on isolated gravid rat myometrium were investigated in this in vitro study; such effects may have clinical repercussions in the administration of anesthesia to obstetric patients. Samples of myometrium were taken from 12 gravid rats. Myometrial strips were dissected microscopically and mounted on the myograph at a resting tension of 1 g in bath that contained Krebs solution. After spontaneous contractions of the myometrium had been steadily established, increasing concentrations of dexmedetomidine were added to baths via micropipette, and the effects of these additions were recorded via myograph. Dexmedetomidine in vitro caused a significant increase in the amplitude, frequency, and area under the curve of myometrial contractions in a dose-dependent manner. Results of this study demonstrate that dexmedetomidine increases spontaneous contractions in rat myometrium; however, further investigation is needed to clarify the usefulness of dexmedetomidine in the administration of obstetric anesthesia.

Efficiency of different biopsy methods of adenomyosis diagnosis, comparative analisis

Introduction.Usage of low invasive methods in adenomyosis diagnosis helps to diagnose adenomyosis definitely by histological examination of myometrium samples.

7q deletion mapping and expression profiling in uterine fibroids

Uterine fibroids are some of the most common tumours of females, but relatively little is known about their molecular basis. Several studies have suggested that deletions on chromosome 7q could have a role in fibroid formation. We analysed 165 sporadic uterine fibroids to define a small 3.2 megabase (Mb) commonly deleted region on 7q22.3-q31.1, flanked by clones AC005070 and AC007567. We also used oligonucleotide microarrays to compare the expression profiles of 10 samples of normal myometrium and 15 fibroids, nine of which displayed 7q-deletions. Activating transcription factor 3, patched homolog (Drosophila), homeo box A5, death-associated protein kinase 1, and retinoic acid receptor responder 3 were downregulated, and excision repair crosscomplementing 3, transcription factor AP-2 gamma and protein kinase C beta 1 were upregulated in fibroids. New pathways were discovered related to fibroid formation. The presence or absence of 7q-deletions did not dramatically affect the global expression pattern of the tumours; changes, however, were observed in genes related to vesicular transport and nucleic acid binding.

Insulin receptor tyrosine kinase activity and substrate 1 (IRS-1) expression in human myometrium and leiomyoma

Background Uterine leiomyomas are the commonest tumors of the genital tract. Growth factors seem to be implicated in the development of leiomyoma. Objective To determine the insulin receptor (IR) tyrosine kinase activity – phosphorylation of exogenous substrate poly(Glu 4: Tyr 1) – and insulin receptor substrate 1 expression in normal myometrium and leiomyoma. Study design The study group consisted of 12 women with leiomyoma undergoing routine hysterectomy. Samples of leiomyoma and adjacent normal myometrium were obtained at the time of operation. Plasma membrane fractions were prepared and samples were incubated with and without insulin and incubated with exogenous substrate poly(Glu 4: Tyr 1). IRS-1 expression was studied in the whole lysate via Western blotting using specific antibodies. Data were analyzed using Student's t-test. Results The phosphorylation of the exogenous substrate poly(Glu 4: Tyr 1) in myometrium (1.566 ± 0.177) and in leiomyoma (1.98 ± 0.612) were similar ( P = 0.774). The IRS-1 levels in myometrium (0.190 ± 0.022) and in leiomyoma (0.226 ± 0.022) were not different ( P = 0.184). Conclusions There was no difference in IR tyrosine kinase activity (phosphorylation of exogenous substrate) and IRS-1 expression between normal myometrium and leiomyomata. Other steps in the insulin signaling cascade require further study to investigate the role of insulin receptor in leiomyomata.

Gene expression studies provide clues to the pathogenesis of uterine leiomyoma: new evidence and a systematic review

Uterine leiomyomas are extremely common and a major cause of pelvic pain, bleeding, infertility, and the leading indication for hysterectomy. Familial and epidemiological studies provide compelling evidence that genetic alterations play an important role in leiomyoma development. Using Affymetrix U133A GeneChip we analysed expression profiles of 22,283 genes in paired samples of leiomyoma and adjacent normal myometrium. We compared our results with previously published data on gene expression in uterine leiomyoma and identified the overlapping gene alterations. We detected 80 genes with average differences of > or = 2-fold and false discovery rates of < 5% (14 overexpressed and 66 underexpressed). A comparative analysis including eight previous gene expression studies revealed eight prominent genes (ADH1, ATF3, CRABP2, CYR61, DPT, GRIA2, IGF2, MEST) identified by at least five different studies, eleven genes (ALDH1, CD24, CTGF, DCX, DUSP1, FOS, GAGEC1, IGFBP6, PTGDS, PTGER3, TYMS) reported by four studies, twelve genes (ABCA, ANXA1, APM2, CCL21, CDKN1A, CRMP1, EMP1, ESR1, FY, MAP3K5, TGFBR2, TIMP3) identified by three studies, and 40 genes reported by two different studies. Review of gene expression data revealed concordant changes in genes regulating retinoid synthesis, IGF metabolism, TGF-beta signaling and extracellular matrix formation. Gene expression studies provide clues to the relevant pathways of leiomyoma development.

The AG1478 tyrosine kinase inhibitor is an effective suppressor of leiomyoma cell growth.

Uterine leiomyomas are the most common benign smooth muscle cell tumours in women. Formation of leiomyomas, still not completely understood, is viewed as a multistep process, with involvement of ovarian steroid hormones, cytokines and growth factors. Our study aimed to identify tyrosine kinase inhibitors as potential 'signal transduction therapeutics' for leiomyomas, underlying the effect of ovarian steroidal hormones. The selective epidermal growth factor (EGF) receptor blocker AG1478 was evaluated as a potential target, since EGF has been shown to mediate estrogen action and to play a crucial role in regulating leiomyoma growth. Paired cultures of leiomyoma and normal myometrium samples were established and the suppressive effect of AG1478 on the cells prior and subsequent to steroidal hormone treatment was examined: cell proliferation, recovery after treatment, cell cycle analysis and immunochemical analysis of relevant proteins. Leiomyoma cell growth is effectively blocked by AG1478 and is unaffected by the presence of physiological concentrations of progesterone and estradiol. AG1478 (10 microM) completely suppressed proliferation and the cells did not recover after cessation of treatment. The growth-arresting properties of AG1478, unaffected by ovarian steroidal hormones, identify it as a potential lead agent for the non-surgical management of uterine leiomyomas.

Urokinase plasminogen activator (uPA) and its receptor (uPAR) in gestational tissues: Measurements and clinical implications

Background: Urokinase plasminogen activator (uPA) and urokinase plasminogen activator receptor (uPAR) are central molecules for uPA/uPAR/plasmin-dependent proteolysis, which is thought to play a significant role in the development of pregnancy, as well as its many complications. Objective: To measure the levels of uPA and uPAR in the placenta and myometrium, as well as in the foetal membranes and amniotic fluid. Study design: The study group consisted of 35 women with normal course of pregnancy, but with complications arising during delivery, which led to Caesarean section. Samples of placenta, myometrium, foetal membranes, amniotic fluid and blood were obtained at the time of operation. Tissue extracts were prepared. Measurements were made by the ELISA method. Results: uPA and uPAR concentration in gestational tissues, including amniotic fluid, is 100–200 times higher than in plasma. Among tissues, the highest uPA level was found in placenta (1.32±0.48 ng/mg of protein), and the highest uPAR level in foetal membranes (3.33±1.20 ng/mg of protein). Conclusions: uPA and uPAR are present in all gestational tissues, in some in relatively high concentrations. Our results support the modern clinical hypothesis that fibrinolytic system can participate in mechanisms of such obstetric complications as pre-term pre-mature rupture of foetal membranes and placental abruption.

Human myometrial adaptation to pregnancy: cDNA microarray gene expression profiling of myometrium from non-pregnant and pregnant women.

The human uterus undergoes profound physiological tissue remodelling during pregnancy. In the myometrium, altered gene expression must underlie these extensive molecular and structural changes. The purpose of this study was to compare expression profiles of pregnant and non-pregnant myometrium, in order to identify genes that participate in this process. mRNA from 14 non-pregnant and four pregnant human myometrial samples were analysed using a human UniGEM V microarray with 7075 cDNA elements. A total of 602 transcripts from the microarray were up-regulated >/=2.0-fold in pregnant myometrium, with 37 transcripts up-regulated >/=4.0-fold. In contrast, eight transcripts were down-regulated >/=2.0-fold in pregnancy. To ensure accurate representation of differential gene expression, Northern blot analyses using total RNA from 16 samples of non-pregnant and pregnant myometrium were used to examine mRNA levels for four of the genes that were differentially expressed by microarray analysis, namely plasminogen activator inhibitor type 1 (PAI-1), milk fat globule-EGF factor 8 protein (MFGE8), secreted frizzled-related protein 4 (sFRP4) and estrogen receptor alpha (ERalpha). On the microarray these transcripts were up-regulated 7.5-fold for PAI-1 and 4.9-fold for MFGE8 in pregnant myometrium, and down-regulated 3.7-fold for sFRP4 and 2.9-fold for ERalpha in pregnancy. Northern blot analyses confirmed these changes. Our findings suggest that microarray technology is a useful tool for examining global changes in gene expression that occur as the myometrium differentiates from non-pregnant to pregnant status. Defining these changes provides new insight into the structural and functional adaptations of human myometrium during pregnancy.

Modulation of RhoA-Rho kinase-mediated Ca2+ sensitization of rabbit myometrium during pregnancy - role of Rnd3.

During pregnancy, the uterus undergoes major functional and structural remodelling. It is well known that during the major part of pregnancy, the myometrium normally remains relatively quiescent but is able to generate powerful contractions at the time of parturition. However, the intracellular molecular events regulating myometrial contractility during pregnancy still remain poorly understood. We applied differential gene expression screening using cDNA array technology to probe myometrium samples from non-pregnant and mid-pregnant (15 days) rabbits. Among the differentially expressed genes, the farnesylated small G-protein of the Rho family, Rnd3, was found to be upregulated (3.6-fold) at mid-pregnancy. Upregulation of Rnd3 was confirmed at the protein level by a 3.4-fold increase in Rnd3 expression in mid-pregnant myometrium. Measurements of contractile properties of beta-escin permeabilized smooth muscle strips revealed that the upregulation of Rnd3 correlated with an inhibition of RhoA-Rho kinase-mediated Ca2+ sensitization at mid-pregnancy. Treatment of muscle strips from mid-pregnant myometrium with the farnesyl-transferase inhibitor manumycin A (10 muM) led to the recovery of RhoA-Rho kinase-dependent Ca2+ sensitization. At late pregnancy (31 days), upregulation of RhoA and Rho kinase expression was associated with an increase in Ca2+ sensitivity of contractile proteins that was inhibited by the Rho kinase inhibitor Y-27632 (10 muM). These data thus demonstrate the time-dependent regulation of the RhoA-Rho kinase-mediated Ca2+ sensitization during the course of pregnancy. The depression of this mechanism at mid-pregnancy followed by its constitutive activation near term is associated with a co-ordinated modulation of Rnd3, RhoA and Rho kinase expression. The RhoA-Rho kinase signalling pathway and its regulators might thus represent potential targets for the development of new treatments for pre-term labour.