Genes upregulated in the amnion at labour are bivalently marked by activating and repressive histone modifications.

Inflammatory genes are expressed increasingly in the foetal membranes at late gestation triggering birth. Here we have examined whether epigenetic histone modifications contribute to the upregulation of proinflammatory genes in the amnion in late pregnancy and at labour. Amnion samples were collected from early pregnancy, at term in the absence of labour and after spontaneous birth. The expression of the labour-associated proinflammatory genes PTGS2, BMP2 and NAMPT was determined by reverse transcription-coupled quantitative real-time PCR (qRT-PCR). Chromatin immunoprecipitation (ChIP) and sequential double ChIP were performed to determine the levels and co-occurrence of activating histone-3, lysine-4 trimethylation (H3K4me3) and repressive histone-3, lysine-27 trimethylation (H3K27me3) at the gene promoters. H3K4 methyltransferase, H3K27me3 demethylase and H3K27 methyltransferase expression was determined by qRT-PCR and immunofluorescence confocal microscopy. PTGS2, BMP2 and NAMPT expression was upregulated robustly between early pregnancy and term (P < 0.05). The promoters were marked bivalently by both the H3K4me3 and H3K27me3 modifications. Bivalence was reduced at term by the decrease of the H3K27me3-modified fraction of promoter copies marked by H3K4me3 indicating epigenetic activation. Messenger RNAs encoding the H3K4-specific methyl transferases MLL1,-2,-3,-4, SETD1A,-B and the H3K27me3-specific demethylases KDM6A,-B were expressed increasingly while the H3K27 methyl transferase EZH2 was expressed decreasingly at term. Histone modifying enzyme proteins were detected in amnion epithelial and mesenchymal cells. These results with prototypical proinflammatory genes suggest that nucleosomes at labour-promoting genes are marked bivalently in the amnion, which is shifted towards monovalent H3K4me3 modification at term when the genes are upregulated. Bivalent epigenetic regulation by histone modifying enzymes may control the timing of labour.

Inflammatory and steroid receptor gene methylation in the human amnion and decidua.

Correct timing of parturition requires inflammatory gene activation in the gestational tissues at term and repression during pregnancy. Promoter methylation at CpG dinucleotides represses gene activity; therefore, we examined the possibility that DNA methylation is involved in the regulation of labour-associated genes in human pregnancy. Amnion and decidua were collected at 11-17 weeks of gestation and at term following elective Caesarean delivery or spontaneous labour. Methylation of the inflammatory genes PTGS2, BMP2, NAMPT and CXCL2 was analysed using the Methyl-Profiler PCR System and bisulphite sequencing. Methylation of the glucocorticoid, progesterone and oestrogen receptor genes, involved in the hormonal regulation of gestational tissue function, and the expression of the DNA methyltransferases DNMT1, -3A and -3B were also determined. Variable proportions of inflammatory and steroid receptor gene copies, to a maximum of 50.9%, were densely methylated in both tissues consistent with repression. Densely methylated copy proportions were significantly different between genes showing no relationship with varying expression during pregnancy, between tissues and in individuals. Methylated copy proportions of all genes in amnion and most genes in decidua were highly correlated in individuals. DNMT1 and -3A were expressed in both tissues with significantly higher levels in the amnion at 11-17 weeks than at term. We conclude that the unmethylated portion of gene copies is responsible for the full range of regulated expression in the amnion and decidua during normal pregnancy. Dense methylation of individually variable gene copy proportions happens in the first trimester amnion influenced by sequence context and affected strongly by individual circumstances.

Molecular evidence of a (pro)renin/ (pro)renin receptor system in human intrauterine tissues in pregnancy and its association with PGHS-2.

Prorenin stimulates decidual prostaglandin (PG) production in vitro, the (pro)renin receptor ((P)RR) may mediate this action. The role of prorenin in amnion PG synthesis has not been examined, despite this being the key site of PG synthesis. To determine if (P)RR, prorenin and PGHS-2 are co-localized in gestational tissues and if expression is altered by labour, term amnion, chorion, decidua and placenta were collected during elective caesarean section or after spontaneous labour. Prorenin, (P)RR and PGHS-2 mRNA abundance was determined by real-time RT-PCR. (P)RR protein was examined by immunohistochemistry. The effect of recombinant human (rh) prorenin on PGHS-2 mRNA abundance in amnion explants was determined. Prorenin and (P)RR mRNA were highest in decidua and placenta, respectively. Decidual prorenin, (P)RR and placental (P)RR mRNA abundance decreased with labour. (P)RR protein was present in all gestational tissues. After labour, decidual prorenin was positively correlated with amnion PGHS-2 mRNA and rh-prorenin significantly increased PGHS-2 mRNA abundance in amnion explants. We conclude that the decidua is the principal source of prorenin and is downregulated with labour. All gestational tissues are targets for prorenin. Decidual prorenin may be involved in the labour-associated increase in amnion PGHS-2 abundance via the (P)RR.

Optimization of a solid phase extraction procedure for prostaglandin E2, F2 alpha and their tissue metabolites.

The primary prostaglandins PGE(2) and PGF(2 alpha) are metabolized in tissues by a series of enzymatic and non-enzymatic reactions. To measure metabolic rates and individual reaction rates it is necessary to extract the parent prostaglandins and metabolites before the separation and quantification of each compound is achieved. Here we have established and optimized a solid phase extraction (SPE) procedure to recover PGE(2), PGF(2 alpha) and their six enzymatic and non-enzymatic tissue metabolites from aqueous solutions including urine, plasma and tissue homogenate. We have used octadecyl-bonded silica gel as the stationary phase and methanol-water mixtures as binary mobile phases. The volumes and concentrations of the washing and elution solutions were optimized individually for each PG. Recoveries of all PG standards were quantitative except for PGEM, which was recovered at 80% efficiency. Biological matrix components interfered with the extraction in a PG- and matrix-specific fashion. Inclusion of 1% formic acid in the loading mixture raised recoveries from urine, plasma and tissue homogenate to >or=90%. This SPE method is the first that has been optimized by systematic elution studies for PGE(2), PGF(2 alpha) and the complement of their tissue metabolites. The procedure is simple, robust and can serve as an effective pre-purification step before downstream separation and quantification of each tissue metabolite of PGE(2) and PGF(2 alpha) from complex biological matrices.

Mechanisms regulating prostaglandin H2 synthase-2 mRNA level in the amnion and chorion during pregnancy.

Increasing prostaglandin H(2) synthase (PGHS)-2 expression in the fetal membranes is implicated in the production of prostaglandins (PGs) that stimulate labour. We have determined the activity of the PGHS-2 gene in the amnion and chorion throughout gestation and defined the contribution of transcriptional and post-transcriptional mechanisms to the increase of PGHS-2 mRNA levels. We also measured PGHS-1 mRNA abundance to assess the participation of the two isoenzymes in fetal membrane PG-production during pregnancy. Amnion and chorion were collected from non-labouring women at 10-19 weeks (early), at 28-36 weeks (preterm) and at term (37-41 weeks). We determined PGHS-1 and -2 mRNA abundance and assessed PGHS-2 gene activity by measuring PGHS-2 heterogeneous nuclear RNA levels using real-time RT-PCR. PGHS-2 gene activity and mRNA levels were up-regulated in both tissues with advancing gestation. Path analysis demonstrated that the PGHS-2 mRNA up-regulation involved both transcriptional and post-transcriptional components. PGHS-2 mRNA abundance increased 9-11 fold between the early (10-19 weeks) and preterm (28-36 weeks) groups and remained high at term. The underlying mechanism was predominantly transcriptional in the amnion and post-transcriptional in the chorion. PGHS-1 mRNA expression precipitously decreased between early gestation and term. Thus, PGHS-2 mRNA abundance is up-regulated well in advance of term and is not a trigger for labour. There is a switch in PGHS mRNA expression during pregnancy with PGHS-1 dominating in the early period and PGHS-2 dominating at term.

Prostaglandin H2 synthase-1 and -2 expression in guinea pig gestational tissues during late pregnancy and parturition.

Increased intrauterine prostaglandin (PG) production is crucial for the initiation of parturition. To investigate the mechanisms controlling intrauterine PG synthesis, we examined the expression of the key PG biosynthetic isoenzymes, PG-H2 synthase (PTGS)-1 and -2, in the amnion, visceral yolk sac (VYS), placenta and myo-endometrium of pregnant guinea pigs. This animal model was chosen because the hormonal milieu of pregnancy and the role of PGs in the hormonal control of parturition are similar to those in the human. PTGS1 mRNA abundance, measured by real-time RT-PCR, increased in the amnion and the placenta during the last third of gestation. During labour, PTGS1 mRNA levels decreased precipitously in all four tissues. PTGS1 protein abundance, assessed by immunoblotting, increased to high levels in the amnion and the placenta by the end of pregnancy and remained high during labour. PTGS2 mRNA expression was higher in the placenta than in the other tissues, but did not change before and during labour. PTGS2 protein expression decreased in the placenta and remained low in the other tissues during labour. Immunohistochemistry showed pervasive PTGS1 protein expression in the amnion and strong expression in the parietal yolk sac membrane (PYS) covering the placenta. PTGS2 was expressed in the PYS and the endometrium. The PTGS inhibitor piroxicam, administered in doses that inhibited PTGS1 but not PTGS2, significantly prolonged gestation. These data suggest that PGs generated by intrauterine PTGS1 are involved in the timing of birth in guinea pigs. The induction of PTGS1 in the amnion and the PYS is a critical event leading to labour in guinea pigs and models analogous changes in the human gestational tissues before labour.

Regulation of 15-hydroxyprostaglandin dehydrogenase (PGDH) gene activity, messenger ribonucleic acid processing, and protein abundance in the human chorion in late gestation and labor.

The prostaglandin (PG)-inactivating enzyme 15-hydroxyprostaglandin dehydrogenase (PGDH) is highly expressed in the chorion leave. To assess the involvement of PGDH in the regulation of intrauterine PG levels, we have determined the mechanisms that control chorionic PGDH expression in women at term and preterm labor. PGDH gene activity decreased at term and during normal labor. PGDH mRNA abundance also decreased at term due to changing splice variant distribution. Gene activity predicted PGDH mRNA abundance preterm and after normal labor, but not at term before labor. PGDH mRNA decayed rapidly in cultured tissues and was stabilized by transcriptional arrest. PGDH protein levels varied without being significantly different between the patient groups. PGDH mRNA levels predicted PGDH protein levels at term, but not preterm and after labor. PGDH gene activity, mRNA variant, and immunoreactive protein levels were not different between the preterm labor and preterm not in labor groups. Thus, PGDH mRNA is transiently down-regulated before term labor by a posttranscriptional mechanism(s). Protein turnover controls PGDH protein abundance at preterm and after normal labor. At term, PGDH protein levels become dependent on the rapidly turning over PGDH mRNA. This may allow rapid changes in PGDH protein abundance and uterotonic PG concentrations promoting labor.

The control of prostaglandin endoperoxide H-Synthase-2 expression in the human chorion laeve at term.

OBJECTIVE: Prostaglandin endoperoxide H synthase-2 (PGHS-2), the key enzyme of prostaglandin biosynthesis in gestational tissues, is expressed in the chorion laeve at term. We have determined the mechanisms that control the level of PGHS-2 mRNA in the chorion membrane in order to assess the significance of chorion-derived prostaglandins in term labor. METHODS: Chorion membranes were collected after elective cesarean delivery (CD, n = 21) and after spontaneous labor (SL, n = 20) at term. The PGHS-2 gene transcription rate was measured by transcriptional run-on, and PGHS-2 mRNA and heterogeneous RNA (hnRNA) abundance was determined by quantitative real-time reverse transcriptase polymerase chain reaction. PGHS-2 mRNA stability, PGHS-2 hnRNA processing rate, and the short-term dynamics of the two RNA species were characterized in 0-24-hour-long tissue incubations. RESULTS: The transcriptional activity of the PGHS-2 gene predicted (P <.02) the abundance of PGHS-2 mRNA and hnRNA in individual tissues. PGHS-2 gene activity and hnRNA processing rate were not different in the CD and SL groups. PGHS-2 mRNA was constitutively stable before and after labor, and its abundance spontaneously increased sixfold in tissues incubated for 24 hours. At the same time, PGHS-2 gene activity decreased by 80% within 2 hours and rebounded to 60% of its initial level by 24 hours. CONCLUSIONS: PGHS-2 mRNA is highly stable, and its abundance is transcriptionally controlled in the chorion laeve at term. Labor is not associated with changing PGHS-2 gene activity. Endogenous factors drive PGHS-2 gene transcription in the chorion, and the stable PGHS-2 mRNA accumulates in the tissue at term. This accumulation has little or no impact on the timing of labor.

The in vivo control of prostaglandin H synthase-2 messenger ribonucleic acid expression in the human amnion at parturition.

Prostaglandin H synthase-2 (PGHS-2) activity and mRNA rise in the human amnion at late gestation, contributing to the increase in intrauterine PG production crucial for labor and delivery. In the present investigation we have determined the mechanism that controls amniotic PGHS-2 mRNA levels in vivo at term parturition. Amnion membranes were collected after elective cesarean section (n = 20), and after spontaneous labor (n = 20). PGHS-2 relative gene transcription rates were determined by transcriptional run-on, and PGHS-2 mRNA and heterogeneous nuclear RNA (hnRNA) relative abundance were measured by quantitative real-time RT-PCR. The PGHS-2 mRNA degradation rate was determined by incubating amnion in the presence of the transcription inhibitor 5,6-dichlorobenzimidazole riboside. The dynamics of PGHS-2 hnRNA and mRNA abundance were characterized in 0- to 24-h tissue incubations. The PGHS-2 relative gene transcription rate was a significant (P < 0.05) predictor of PGHS-2 hnRNA and mRNA abundance, and PGHS-2 hnRNA was also a predictor (P < 0.01) of PGHS-2 mRNA levels both before and after labor. Interestingly, even though PGHS-2 gene activity remained unchanged, PGHS-2 mRNA abundance increased with labor and displayed constitutive stability before and after labor. PGHS-2 mRNA levels spontaneously increased by 400% (P < 0.01) upon incubation for 24 h, whereas the transcription rate dropped by 95% during the first 2 h, then rebounded significantly between 6-24 h. Thus, PGHS-2 mRNA abundance is transcriptionally controlled in term amnion. Labor does not increase PGHS-2 gene activity or mRNA stability. The PGHS-2 gene is probably induced before labor by a factor(s) originating in the amnion membrane, and the resulting stable mRNA accumulates progressively in the tissue throughout labor and delivery.