Preeclampsia is the major pregnancy-induced hypertensive disorder. It modifies the expression profile of placental genes, including several serine protease inhibitors ( SERPINs ). The objective of this study was to perform a systematic expression analysis of these genes in normal and pathological placentas and to pinpoint epigenetic alterations inside their promoter regions. Expression of 18 placental SERPINs was analyzed by quantitative RT-PCR on placentas from pregnancies complicated by preeclampsia, intrauterine growth restriction, or both and was compared with normal controls. SERPINA3, A5, A8, B2, B5 , and B7 presented significant differences in expression in ≥1 pathological situation. In parallel, the methylation status of the CpG islands located in their promoter regions was studied on a sample of control and preeclamptic placentas. Ten SERPIN promoters were either totally methylated or totally unmethylated, whereas SERPINA3, A5 , and A8 presented complex methylation profiles. For SERPINA3 , the analysis was extended to 81 samples and performed by pyrosequencing. For the SERPINA3 CpG island, the average methylation level was significantly diminished in preeclampsia and growth restriction. The hypomethylated CpGs were situated at putative binding sites for developmental and stress response (hypoxia and inflammation) factors. Our results provide one of the first observations of a specific epigenetic alteration in human placental diseases and provide new potential markers for an early diagnosis.

Expression of imprinted genes is classically associated with differential methylation of specific CpG-rich DNA regions (DMRs). The H19/IGF2 locus is considered a paradigm for epigenetic regulation. In mice, as in humans, the essential H19 DMR—target of the CTCF insulator—is located between the two genes. Here, we performed a pyrosequencing-based quantitative analysis of its CpG methylation in normal human tissues. The quantitative analysis of the methylation level in the H19 DMR revealed three unexpected discrete, individual-specific methylation states. This epigenetic polymorphism was confined to the sixth CTCF binding site while a unique median-methylated profile was found at the third CTCF binding site as well as in the H19 promoter. Monoallelic expression of H19 and IGF2 was maintained independently of the methylation status at the sixth CTCF binding site and the IGF2 DMR2 displayed a median-methylated profile in all individuals and tissues analyzed. Interestingly, the methylation profile was genetically transmitted. Transgenerational inheritance of the H19 methylation profile was compatible with a simple model involving one gene with three alleles. The existence of three individual-specific epigenotypes in the H19 DMR in a non-pathological situation means it is important to reconsider the diagnostic value and functional importance of the sixth CTCF binding site.

Expression of imprinted genes is classically associated with differential methylation of specific CpG-rich DNA regions (DMRs). The H19/IGF2 locus is considered a paradigm for epigenetic regulation. In mice, as in humans, the essential H19 DMR-target of the CTCF insulator-is located between the two genes. Here, we performed a pyrosequencing-based quantitative analysis of its CpG methylation in normal human tissues. The quantitative analysis of the methylation level in the H19 DMR revealed three unexpected discrete, individual-specific methylation states. This epigenetic polymorphism was confined to the sixth CTCF binding site while a unique median-methylated profile was found at the third CTCF binding site as well as in the H19 promoter. Monoallelic expression of H19 and IGF2 was maintained independently of the methylation status at the sixth CTCF binding site and the IGF2 DMR2 displayed a median-methylated profile in all individuals and tissues analyzed. Interestingly, the methylation profile was genetically transmitted. Transgenerational inheritance of the H19 methylation profile was compatible with a simple model involving one gene with three alleles. The existence of three individual-specific epigenotypes in the H19 DMR in a non-pathological situation means it is important to reconsider the diagnostic value and functional importance of the sixth CTCF binding site.

Alpha-fetoprotein (AFP) is a major plasma protein produced during human fetal life. It is a good marker for several possible disorders affecting gestation. We previously reported that afp gene expression, which takes place mainly in yolk sac and fetal liver, also occurs in normal human placenta, specifically in early pregnancy. The aim of the present study was to determine the precise location of AFP synthesis sites within the placental villi. In situ hybridization and immunohistochemical experiments were performed on sections obtained from placentas of first-trimester and full-term pregnancies. We found that the pattern of afp gene expression was restricted to specific villous trophoblastic areas in early placentas. Both afp transcripts and AFP protein were mainly located in discontinuous regions, at junctions between two villi and at budding sites. In contrast, no AFP expression was detected in the cytotrophoblastic extravillous proliferative zone or in other placental cell types. According to the earlier studies, no AFP synthesis was detected in placental villous tissue from full-term pregnancies, using in situ hybridization and immunohistochemistry.

Villi from first-trimester human placenta were exposed to oxygen concentrations of either 2 or 20% during 3 h to construct two reciprocally subtracted libraries using the suppressive subtractive hybridization (SSH) methodology. After cloning, sequencing, and gene identification, the genes (1,071 clones corresponding to 822 different sequences) were classified according to 1) the subtracted library from which they originated and 2) within 58 groups of gene functions. We then developed a logarithm of the odds (LOD) test to identify a possible excess of genes in each group. We show that genes involved in angiogenesis are significantly overrepresented in the “hypoxic” condition (2% O 2 ), whereas apoptotic genes are overrepresented in the “normoxic” condition (20% O 2 ). Furthermore, we observed an excess of kinases relative to phosphatases and an excess of genes involved in proliferation over genes involved in cell growth in the hypoxic condition. To validate our results, we used quantitative RT-PCR to analyze the set of eight genes involved in angiogenesis on six independent placentas. Finally, we studied the distribution of gene clusters on human chromosomes to check whether their chromosomal distribution was random or not. We observed on human chromosome 11 a clear clustering of genes regulated similarly by O 2 tension, and we also discovered indications that such clustering exists on chromosomes 6 and 12.

Background: As a first step to explore the possible relationships existing between the effects of low oxygen pressure in the first trimester placenta and placental pathologies developing from mid-gestation, two subtracted libraries totaling 2304 cDNA clones were constructed. For achieving this, two reciprocal suppressive/subtractive hybridization procedures (SSH) were applied to early (11 weeks) human placental villi after incubation either in normoxic or in hypoxic conditions. The clones from both libraries (1440 hypoxia-specific and 864 normoxia-specific) were spotted on nylon macroarrays. Complex cDNAs probes prepared from placental villi (either from early pregnancy, after hypoxic or normoxic culture conditions, or near term for controls or pathological placentas) were hybridized to the membranes.Results: Three hundred and fifty nine clones presenting a hybridization signal above the background were sequenced and shown to correspond to 276 different genes. Nine of these genes are mitochondrial, while 267 are nuclear. Specific expression profiles characteristic of preeclampsia ( PE) could be identified, as well as profiles specific of intra-uterine growth retardation ( IUGR). Focusing on the chromosomal distribution of the fraction of genes that responded in at least one hybridization experiment, we could observe a highly significant chromosomal clustering of 54 genes into 8 chromosomal regions, four of which containing imprinted genes. Comparative mapping data indicate that these imprinted clusters are maintained in synteny in mice, and apparently in cattle and pigs, suggesting that the maintenance of such syntenies is requested for achieving a normal placental physiology in eutherian mammals.Conclusion: We could demonstrate that genes induced in PE were also genes highly expressed under hypoxic conditions ( P=5.10(-5)), which was not the case for isolated IUGR. Highly expressed placental genes may be in syntenies conserved interspecifically, suggesting that the maintenance of such clusters is requested for achieving a normal placental physiology in eutherian mammals.

In this paper, we applied a new theoretical model of uterine contraction to a large panel of human pregnant and nonpregnant myometrial strips, treated or not by corticotrophin-releasing hormone (CRH). This model is based on a fine analysis of the contraction curves. This analysis yielded four mathematical parameters (beta, theta, tau 1, and tau 2) related to excitability, duration of plateau phase, and time constants for relaxation describing, respectively, the different portions of the contraction cycle. This leads to specific differences in spontaneous contractile activity between pregnant and nonpregnant states. The relaxing effect of CRH in the pregnant state is presumably correlated with the origin of the strips (the lower uterine segment). Besides our observation of a specific receptor-dependent relaxing effect of CRH in both pregnant and nonpregnant myometrium, we could identify highly significant effects at given CRH concentration for beta in nonpregnant myometrium and for theta, tau 1, and tau 2 in pregnant myometrium. In addition, highly significant differences were found between pregnant and nonpregnant myometrium. Also, we discovered a strong correlation between theta and tau 1, specifically in the pregnant state. Although the biochemical signification of these results remains to be elucidated, they contribute to emphasize the complex network of CRH action at the myometrial level. Furthermore, our approach could pave the way toward a better analysis of the efficacy of the uterine contractile behavior.

Villi from first trimester human placenta were exposed to oxygen concentrations of either 2%, or 20% during 3 hours to construct two reciprocally subtracted libraries using the Suppressive Subtractive Hybridization (SSH) methodology. After cloning, sequencing and gene identification, the genes (1070 clones corresponding to 822 different sequences) were classified according to (1) the subtracted library from which they originated, and (2) within 58 groups of gene functions. We then developed a Logarithm of the Odds (LOD) test in order to identify a possible excess of genes in each group. We show that genes involved in angiogenesis are significantly over-represented in the “hypoxic” condition (2% O 2 ), while apoptotic genes are over-represented in the “normoxic” condition (20% O 2 ). Furthermore, we observed an excess of kinases relative to phosphatases and an excess of genes involved in proliferation over genes involved in cell growth in the “hypoxic” condition. To validate our results, we used quantitative RT-PCR to analyze the set of 8 genes involved in angiogenesis on 9 independent placentas. Finally, we studied the distribution of gene clusters on human chromosomes to check whether their chromosomal distribution was random or not. We observed on HSA11 a clear clustering of genes regulated similarly by O

The anti-inflammatory and utero-relaxant effects of two potent phosphodiesterase 4 (PDE4) inhibitors of the latest generation: cilomilast (one of the most advanced PDE4 inhibitors in clinical development, reportedly more selective for PDE4D) and compound A (which displays 12-fold greater selectivity toward PDE4B and/or PDE4A than toward PDE4D) were evaluated in human uterine smooth muscle. We first established that these compounds exhibit greater efficacy in inhibiting total cAMP-PDE activity in pregnant versus nonpregnant myometrium (E(max) = 78.0% +/- 3.6% and 80.3% +/- 2.2% in pregnant versus 57% +/- 4.7% and 70.5% +/- 5.9% in nonpregnant women for compound A and cilomilast, respectively; P < 0.05 for both compounds), confirming the prominent participation of PDE4 isoforms in cAMP hydrolysis in the near-term pregnant myometrium. Using pregnant myometrial explants, we have shown that both these drugs and also rolipram, the prototype PDE4 inhibitor, produce concentration-dependent inhibition of lipopolysaccharide (LPS) induced tumor necrosis factor alpha (TNFalpha) release with similar potency in each case (pD2 = 8.0 +/- 0.5, 7.9 +/- 0.2, and 7.6 +/- 0.2 for compound A, cilomilast, and rolipram, respectively). The maximum inhibition produced is 65%. Pretreatment with forskolin or 8-bromo-cAMP mimics the PDE4 inhibitor effect. Furthermore, compound A and cilomilast both produce concentration-dependent inhibition of the spontaneous contractions of myometrial strips and are more potent in pregnant than in nonpregnant myometrium (pD2 = 7.3 +/- 0.7 and 8.1 +/- 0.3 in pregnant versus 6.2 +/- 0.9 and 6.6 +/- 0.1 in nonpregnant myometrium for compound A and cilomilast, respectively; P < 0.05 for both compounds). This demonstrates that the PDE4 isoforms involved in the mechanism of contraction are different in the pregnant and nonpregnant myometrium. Our study highlights the importance of developing PDE4 inhibitors for the pharmacological management of infection-induced preterm labor.

Endothelins exhibit growth regulating properties in many cell types, and there is now considerable evidence that they play a critical pathophysiological role in human diseases such as carcinogenesis. In the choriocarcinoma cell lines JEG-3, JAR and BeWO, we demonstrate by RT-PCR that prepro endothelin (ET)-1 and prepro ET-2 mRNA were expressed, whereas prepro ET-3 was never expressed. Only ET-1 and ET-2 peptides were identified by HPLC/RIA analyses in culture media from these three choriocarcinoma cell lines. In the BeWO line, the cellular growth measured as the cell count and DNA content decreased with increasing concentrations of dimethyl sulfoxide (DMSO; range 0.5-3%). The expression of prepro ET-2 was also suppressed by DMSO, whereas no change was noticed inprepro ET-1 mRNA. All these effects were reversible when DMSO was replaced by 15% foetal calf serum. These effects of DMSO which are correlated to ET-2 down regulation in dividing BeWO cells suggest a role for this endothelin isoform in trophoblast proliferation.

The aim of this study was to identify, in cultured human cervical fibroblasts, the mechanisms by which interleukin (IL)-1beta induces the synthesis of glycosaminoglycans (GAG) and to explore the putative role of prostaglandin E(2) (PGE(2)) in this process. Exposure of the cells for 24 h to IL-1beta induced a significant (P < 0.05) dose-dependent increase in GAG synthesis. IL-1beta (1 ng/ml) induced the expression of cyclooxygenase-2 (COX-2) protein 6 h after treatment, accompanied by a 7.5-fold increase in PGE(2) production. We confirmed that NS398, a selective COX-2 inhibitor, dose-dependently blocked PGE(2) augmentation following IL-1beta treatment. AH23848, the selective EP(4) receptor antagonist, completely abolished IL-1beta-induced GAG synthesis, whereas AH6809, an EP(2) receptor antagonist, had no effect on the stimulatory effects of IL-1beta. Furthermore, we demonstrated that 6 h exposure to IL-1beta induced a notable increase in EP(4) receptor mRNA expression and a decrease in EP(1) receptor mRNA but had no effect on the expression of EP(2) and EP(3) receptor transcripts. In conclusion, these findings indicate that IL-1beta not only induced GAG synthesis by increasing COX-2 protein expression and the subsequent PGE(2) production but also enhanced the responsiveness of cervical fibroblasts to PGE(2) by selectively up-regulating EP(4) receptor mRNA expression. These results suggest that PGE(2) may regulate human cervical ripening in an autocrine/paracrine manner via EP(4) receptors.

To our knowledge, the problem of how to maintain isolated smooth cells in a "contractile" phenotypic state without deviation after subculturing has yet to be resolved. The present study characterized the in vitro contractile response of human uterine smooth muscle cell to endothelin-1, which induces contractions in isolated uterine strips. Contractile effects were qualitatively investigated using silicone rubber substrata. Endothelin-1 was able to distort and reduce the wrinkles in the silicone surface. Contractions were also quantified by measuring the resulting change in the collagen lattice area. Endothelin-1 significantly increased the contractile response in a dose-dependent manner by selectively activating endothelin A receptors. When myometrial cells were cultured within collagen lattices, a microfilament-disrupting agent, cytochalasin B, abolished contractions, and no change was observed in smooth muscle alpha-actin immunostaining. Taken together, these observations show that the uterine smooth muscle cells are contractile and respond appropriately to a potent uterotonic agent. Based on these findings, a cultured uterine smooth muscle cell model, which could be used to elucidate the mechanisms controlling uterine activity, is proposed.

The in vitro spontaneous contractions of human myometrium samples can be described using a phenomenological model involving different cell states and adjustable parameters. In patients not receiving hormone treatment, the dynamic behavior could be described using a three-state model similar to the one we have already used to explain the oscillations of intrauterine pressure during parturition. However, the shape of the spontaneous contractions of myometrium from patients on progestin treatment was different, due to a two-step relaxation regime including a latched phase which cannot be simulated using the previous model without introducing an ad hoc mechanism to account for the extra energy involved in this sustained contraction. One way to do this is to introduce an anomalous rate of ATP consumption, the biochemical reasons for which have not yet been elucidated and which cannot be mathematically simulated using our experimental data. An alternative explanation is the reduced cycling rate of actin-myosin cross-bridges known to occur during the latch-phase. Our experimental findings suggest a third possibility, namely a sol-gel transition with a specific relaxation time constant, which would maintain a significant part of the cell population in the contracted-state until the intracellular-medium returns to its normal fluid behavior.

We have previously shown that protein kinase C (PKC) zeta and/or PKC delta are necessary for endothelin-1 (ET-1)-induced human myometrial contraction at the end of pregnancy (Eude I, Paris P, Cabrol D, Ferré F, and Breuiller-Fouché M. Biol Reprod 63: 1567-1573, 2000). Here, we report that the selective inhibitor of PKC delta isoform, Rottlerin, does not prevent ET-1-induced contractions, whereas LY-294002, a phosphatidylinositol (PI) 3-kinase inhibitor, affects the contractile response. This study characterized the in vitro contractile response of cultured human pregnant myometrial cells to ET-1 known to induce in vitro contractions of intact uterine smooth muscle strips. Cultured myometrial cells incorporated into collagen lattices have the capacity to reduce the size of these lattices, referred to as lattice contraction. Neither the selective conventional PKC isoform inhibitor, Gö-6976, or rottlerin affected myometrial cell-mediated gel contraction by ET-1, whereas this effect was blocked by LY-294002. We found that treatment of myometrial cell lattices with an inhibitory peptide specific for PKC zeta or with an antisense against PKC zeta resulted in a significant loss of ET-1-induced contraction. Evidence is also presented by using confocal microscopy that ET-1 induced translocation of PKC zeta to a structure coincident with the actin-rich microfilaments of the cytoskeleton. We have shown that PKC zeta has a role in the actin organization in ET-1-stimulated cells. Accordingly, our results suggest that PKC zeta plays a role in myometrial contraction in pregnant women.

Objective: Our goal was to study the effect of amniotic fluid obtained at 16 and 39 weeks of gestation in normal human pregnancies upon prostaglandin production by human myometrial cells in culture. Study design: Amniotic fluid, sampled either at 16 weeks, during amniocentesis, or at 39 weeks, during caesarean section before labor, was fractionated by molecular-weight and then incubated with human myometrial cells in culture. We then used radioimmunoassay to measure PGE(2) and PGI(2) production. Results: The "3-30 kDa" fraction of amniotic fluid sampled at 16 weeks significantly inhibited PGE(2) and PGI(2) production by human myometrial cells. When amniotic fluid was sampled at 39 weeks, it stimulated both PGE(2) and PGI(2) production, and the "> 30 kDa" fraction increased levels of PGE(2) considerably more than of PGI(2) (420.0 +/- 88.0 ng/10(6) Cells versus 188.2 +/- 21.4 ng/10(6) cells, P < 0.001). Conclusion: Amniotic fluid contains substances whose effects in cultured myometrial cells vary according to gestational age and type of prostaglandin. These data suggest that the fetus plays a role in the regulation of myometrial activity during pregnancy. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

The role of protein kinase C (PKC)-alpha in endothelin-1 (ET-1)induced proliferation of human myometrial cells was investigated. Inhibition of conventional PKC with Go 6976 eliminated the proliferative effect of ET-1. Treatment of myometrial cells with an antisense oligonucleotide against PKC alpha efficiently reduced PKC alpha protein expression without effect on other PKC isoforms and resulted in the loss of ET-1-induced cell growth. Immunocytochemistry using an antibody against PKC alpha revealed that there was no PKC alpha immunoreactivity in the nuclei of quiescent nonconfluent untreated cells, whereas it is evenly distributed throughout the cytoplasm. Exposure of myometrial cells to ET-1 for 15 min caused the PKC alpha to shift towards the perinuclear area, and incubation for 60 min caused a shift towards the nucleus. These results reveal that PKC alpha is required for ET-1-induced human myometrial cell growth and suggest that targeting of PKC alpha by antisense nucleotides might be an important approach for the development of anticancer treatments.