Cell-Free Fetal DNA, Telomeres, and the Spontaneous Onset of Parturition.

Multiple previous reports have provided compelling support for the premise that spontaneous parturition is mediated by activation of inflammation-related signaling pathways leading to increased secretion of cytokines and chemokines, the influx of neutrophils and macrophages into the pregnant uterus, increased production of uterine activation proteins (eg, connexin-43, cyclo-oxygenase-2, oxytocin receptors, etc), activation of matrix metalloproteinases, and the release of uterotonins leading to cervical ripening, membrane rupture, and myometrial contractions. The missing link has been the fetal/placental signal that triggers these proinflammatory events in the absence of microbial invasion and intrauterine infection. This article reviews the biomedical literature regarding the increase in cell-free fetal DNA (cffDNA), which is released during apoptosis in the placenta and fetal membranes at term, the ability of apoptosis modified vertebrate DNA to stimulate toll-like receptor-9 (TLR9) leading to increased release of cytokines and chemokines, and the potential "fail-safe" role for the anti-inflammatory cytokine IL-10. This article also reviews the literature supporting the key role that telomere loss plays in regard to increasing the ability of vertebrate (including placental) DNA to stimulate TLR9, and in regard to signaling the onset of apoptosis in the placenta and fetal membranes, thereby providing a biologic clock that determines the length of gestation and the timing for the onset of parturition. In summary, this literature review provides a strong rationale for future research to test the hypothesis that telomere loss and increased cffDNA levels trigger the proinflammatory events leading to the spontaneous onset of parturition in mammals: the "cffDNA/telomere hypothesis."

Expression of tumor necrosis factor-alpha and vascular endothelial growth factor in different zones of fetal membranes: a possible relation to onset of labor.

This study aimed to explore whether the altered expression of tumor necrosis factor-alpha (TNF-α), vascular endothelial growth factor (VEGF) and apoptotic changes in mid zone (MZ) and rupture zone (RZ) of fetal membranes (FM) are regulatory mechanisms associated with labor at term. Fifteen FM specimens were collected after vaginal deliveries and 13 specimens after elective caesarian section. Histological and immunohistochemical analysis were employed. Area percent of TNF-α and VEGF immunostaining and apoptotic index (AI) were evaluated using image analysis. The statistical data revealed significantly higher area % for TNF-α, VEGF immunoexpression and AI in labor compared to non-labor specimens (p < 0.0001). There was a significantly higher percentage of TNF-α immunoexpressed area in MZ compared with RZ in both groups (p < 0.0001). VEGF expression in RZ of both groups proved nearly double or triple the area % of expression relative to MZ with highly significant difference (p < 0.0001). quantitative analysis revealed near two fold increase in the AI in RZ (13.42% ± 1.2 in labor; 11.20% ± 0.96 in non-labor groups) when compared to MZ (7.20% ± 0.6 in labor; 5.08% ± 0.76 in non-labor groups) with highly significant zonal difference (p < 0.0001). Correlation analysis revealed significant correlation between apoptotic indices and area % of TNF-α (r = 0.575, p = 0.002 in non-labor; r = 0.652, p < 0.0001 in labor) and VEGF (r = 0.795, p < 0.0001 in non-labor; r = 0.668, p < 0.0001 in labor). In conclusion, Apoptosis may be regulated by TNF-α and VEGF expression in FM at labor. MZ is a step back from RZ and could participate actively in rupture of the FM during labor. TNF-α and VEGF increase with onset of labor and differentially expressed in the RZ and the MZ. These findings call for further study with tissue cultures or animal models.

The contribution of genetic and environmental factors to the duration of pregnancy.

This review describes how improvements in biometric-genetic studies of twin kinships, half-sibships, and cousinships have now demonstrated a sizeable fetal genetic and maternal genetic contribution to the spontaneous onset of labor. This is an important development because previous literature for the most part reports only an influence of the maternal genome. Current estimates of the percent of variation that is attributable to fetal genetic factors range from 11-35%; the range for the maternal genetic contribution is 13-20%. These same studies demonstrate an even larger influence of environmental sources over and above the influence of genetic sources and previously identified environmental risk factors. With these estimates in hand, a major goal for research on pregnancy duration is to identify specific allelic variation and environmental risk to account for this estimated genetic and environmental variation. A review of the current literature can serve as a guide for future research efforts.

SLIT3 is increased in supracervical human foetal membranes and in labouring myometrium and regulates pro-inflammatory mediators.

PROBLEM: Inflammation is associated with preterm birth, a worldwide healthcare issue. SLIT3 has a role in inflammation, and thus, the purpose of this study was to determine the effect of SLIT3 on labour mediators in human gestational tissues. METHOD OF STUDY: SLIT3 protein expression was performed using immunohistochemistry in foetal membranes and myometrium with no labour and after labour. Foetal membranes were also obtained from a distal site (DS) and supracervical site (overlying the cervix; SCS). SLIT3 gene silencing was achieved using siRNA in primary amnion and myometrial cells. Pro-inflammatory and pro-labour mediators were evaluated by qRT-PCR, ELISA and gelatin zymography. RESULTS: SLIT3 expression was greater in foetal membranes from the SCS compared with DS and in myometrium after term spontaneous labour onset. SLIT3 siRNA in primary amnion and myometrial cells decreased IL-1ß-induced pro-inflammatory cytokine gene expression and release (IL-6 and IL-8) and MMP-9 gene expression and release. In amnion cells, SLIT3 siRNA knockdown decreased IL-1ß-induced COX-2 expression and prostaglandin PGE2 release. There was no effect of SLIT3 siRNA on IL-1ß-induced NF-κB transcriptional activity. CONCLUSION: Our results demonstrate that SLIT3 is increased with labour, and both our amnion and our myometrial studies describe a pro-inflammatory effect of SLIT3 in these tissues.

Early onset pre-eclampsia is associated with altered DNA methylation of cortisol-signalling and steroidogenic genes in the placenta.

Placental cortisol is inactivated in normotensive pregnancies, but is frequently present in pre-eclampsia associated placentae. Since glucocorticoids are strongly associated with the programming of long-term health, we assessed DNA methylation of genes involved in cortisol signalling and bioavailability, and hormonal signalling in the placenta of normotensive and hypertensive pregnancies. Candidate genes/CpG sites were selected through analysis of Illumina Infinium HumanMethylation450 BeadChip array data on control (n = 19) and early onset pre-eclampsia (EOPET; n = 19) placental samples. DNA methylation was further quantified by bisulfite pyrosequencing in a larger cohort of control (n = 111) cases, in addition to EOPET (n = 19), late onset pre-eclampsia (LOPET; n = 18) and normotensive intrauterine growth restriction (nIUGR; n = 13) cases. DNA methylation (percentage points) was increased at CpG sites within genes encoding the glucocorticoid receptor (NR3C1 exon 1D promoter; +8.46%; P<0.01) and corticotropin releasing hormone (CRH) binding protein (CRHBP intron 3; +9.14%; P<0.05), and decreased within CRH (5' UTR; -4.30%; P = 0.11) in EOPET-associated placentae, but not in LOPET nor nIUGR cases, compared to controls. Differential DNA methylation was not observed among groups at the 11ß-hydroxysteroid dehydrogenase type 2 (HSD11B2) gene promoter. Significant hypomethylation was observed in pre-eclampsia but not nIUGR placentae for steroidogenic genes, including CYP11A1 (exon1; EOPET; -9.66%; P<0.00001, and LOPET; -5.77%; P<0.001), 3ß-hydroxy-delta-5-steroid dehydrogenase type 1 (HSD3B1 exon 2; EOPET; -12.49%; P<0.00001, and LOPET; -6.88%; P<0.001), TEA domain family member 3 (TEAD3 intron 1; EOPET; -12.56%; P<0.00001) and CYP19 (placental-specific exon 1.1 promoter; EOPET; -10.62%, P<0.0001). These data represent dysregulation of the placental epigenome in pre-eclampsia related to genes involved in maintaining the hormonal environment during pregnancy and highlights particular susceptibility in the early onset syndrome.

Nuclear factor kappa B activation occurs in the amnion prior to labour onset and modulates the expression of numerous labour associated genes.

BACKGROUND: Prior to the onset of human labour there is an increase in the synthesis of prostaglandins, cytokines and chemokines in the fetal membranes, particular the amnion. This is associated with activation of the transcription factor nuclear factor kappa B (NFκB). In this study we characterised the level of NFκB activity in amnion epithelial cells as a measure of amnion activation in samples collected from women undergoing caesarean section at 39 weeks gestation prior to the onset of labour. METHODOLOGY/PRINCIPAL FINDINGS: We found that a proportion of women exhibit low or moderate NFκB activity while other women exhibit high levels of NFκB activity (n = 12). This activation process does not appear to involve classical pathways of NFκB activation but rather is correlated with an increase in nuclear p65-Rel-B dimers. To identify the full range of genes upregulated in association with amnion activation, microarray analysis was performed on carefully characterised non-activated amnion (n = 3) samples and compared to activated samples (n = 3). A total of 919 genes were upregulated in response to amnion activation including numerous inflammatory genes such cyclooxygenase-2 (COX-2, 44-fold), interleukin 8 (IL-8, 6-fold), IL-1 receptor accessory protein (IL-1RAP, 4.5-fold), thrombospondin 1 (TSP-1, 3-fold) and, unexpectedly, oxytocin receptor (OTR, 24-fold). Ingenuity Pathway Analysis of the microarray data reveal the two main gene networks activated concurrently with amnion activation are i) cell death, cancer and morphology and ii) cell cycle, embryonic development and tissue development. CONCLUSIONS/SIGNIFICANCE: Our results indicate that assessment of amnion NFκB activation is critical for accurate sample classification and subsequent interpretation of data. Collectively, our data suggest amnion activation is largely an inflammatory event that occurs in the amnion epithelial layer as a prelude to the onset of labour.

Beta-2 adrenoceptor genotype and progress in term and late preterm active labor.

OBJECTIVE: We sought to evaluate whether beta-2 adrenoceptor (ß2AR) genotype at a functional polymorphic site encoding for amino acid residue 16 influences rate of cervical dilatation in term and late preterm active labor. STUDY DESIGN: Subjects who underwent vaginal delivery at ≥34 weeks' gestational age from May 2006 through August 2007 were identified. Each subject had provided venous blood from which DNA was extracted for ß2AR genotyping. Digital cervical examinations with paired examination times were collected from intrapartum records. Rate of cervical dilatation in active labor was determined using linear regression. Rates were compared between genotype groups. RESULTS: Among 401 subjects with satisfactory genotype and intrapartum data, overall rate of active labor was 0.76±0.01 cm/h. When labor was compared by genotype, homozygous Arg/Arg16 subjects progressed at a slower rate (0.64±0.03 cm/h) than all other pooled genotypes (0.8±0.02 cm/h, P<.001). CONCLUSION: Homozygous ß2AR genotype encoding for Arg/Arg16 was associated with slower progress in active labor.

Surfactant protein-A (SP-A) selectively inhibits prostaglandin F2alpha (PGF2alpha) production in term decidua: implications for the onset of labor.

CONTEXT: Labor is characterized by "decidual activation" with production of inflammatory mediators. Recent data suggest that surfactant protein-A (SP-A) may be critical to the onset of labor in mice. Whether this is also true in humans is unclear. OBJECTIVES: The aim was to investigate: 1) the expression of SP-A at the maternal-fetal interface; 2) the effect of SP-A on the production of inflammatory mediators by human decidua; and 3) the association between single nucleotide polymorphisms in maternal SP-A genes and spontaneous preterm birth. RESEARCH DESIGN AND METHODS: In situ expression of SP-A was investigated by immunohistochemistry and quantitative RT-PCR. Term decidual stromal cells were isolated, purified, and treated with/without SP-A (1-100 µg/ml), IL-1ß, and/or thrombin. Levels of inflammatory mediators [IL-6, IL-8, TNFα, matrix metalloproteinase-3, monocyte chemotactic protein-1, IL-1ß, PGE(2), prostaglandin F(2α) (PGF(2α))] and angiogenic factors (soluble fms-like tyrosine kinase-1, vascular endothelial growth factor) were measured in conditioned supernatant by ELISA and corrected for protein content. The effect of SP-A on eicosanoid gene expression was measured by quantitative RT-PCR. RESULTS: SP-A localized to endometrium/decidua. High-dose SP-A (100 µg/ml) inhibited PGF(2α) by term decidual stromal cells without affecting the production of other inflammatory mediators, and this effect occurred at a posttranscriptional level. Decidual SP-A expression decreased significantly with labor. Single nucleotide polymorphisms in the SP-A genes do not appear to be associated with preterm birth. CONCLUSIONS: SP-A is produced by human endometrium/decidua, where it significantly and selectively inhibits PGF(2α) production. Its expression decreases with labor. These novel observations suggest that decidual SP-A likely plays a critical role in regulating prostaglandin production within the uterus, culminating at term in decidual activation and the onset of labor.

Fetal membranes as an interface between inflammation and metabolism: increased aquaporin 9 expression in the presence of spontaneous labor at term and chorioamnionitis.

OBJECTIVE: Aquaporin 9 (AQP9) is a water channel protein characterized by its high permeability to nutrients such as lactate and glycerol, as well as urea and other small solutes. These unique properties of AQP9 suggest that this molecule may play a role in the modulation of nutrient flux through the fetal membranes in conditions associated with increased metabolic demand, such as spontaneous labor and inflammation. The objective of this study was to determine the expression of AQP9 in the chorioamniotic membranes from women with and without term labor, as well as those with preterm prelabor rupture of membranes (PPROM) with and without histologic chorioamnionitis. STUDY DESIGN: A cross-sectional study was performed which included patients in the following groups: (1) term not in labor (TNL; n = 14); (2) term, spontaneous labor (n = 14); and (3) PPROM with (n = 20) and without (n = 17) histologic chorioamnionitis. AQP9 mRNA expression in fetal membranes was quantified using quantitative real-time reverse transcription-polymerase chain reaction and analyzed with a linear model including gestational age as a covariate. RESULTS: (1) AQP9 mRNA expression was identified in all chorioamniotic membrane specimens; (2) AQP9 expression in fetal membranes was significantly higher in spontaneous term labor when compared with TNL (fold change 3.6; p = 0.01); and (3) Among patients with PPROM, the presence of histologic chorioamnionitis was associated with a higher expression of AQP9 in the chorioamniotic membranes compared with those from women without histologic chorioamnionitis (fold change 8.7; p < 0.001). CONCLUSION: Aquaporin 9 mRNA expression is higher in the fetal membranes from patients with spontaneous term labor and those with PPROM and histologic chorioamnionitis. These findings are novel, and suggest a role for aquaporin 9 in membrane-mediated transfer of nutrients to support the increased metabolic demands associated with the host immune response of the terminal pathway of parturition and histologic chorioamnionitis.

The transcriptome of cervical ripening in human pregnancy before the onset of labor at term: identification of novel molecular functions involved in this process.

OBJECTIVE: The aim of this study was to identify changes in the cervical transcriptome in the human uterine cervix as a function of ripening before the onset of labor. STUDY DESIGN: Human cervical tissue was obtained from women at term not in labor with ripe (n = 11) and unripe (n = 11) cervices and profiled using Affymetrix GeneChip HGU133Plus2.0 arrays. Gene expression was analyzed using a moderated t-test (False Discovery Rate 5%). Gene ontology and pathway analysis were performed. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used for confirmation of selected differentially expressed genes. RESULTS: (1) Ninety-one genes were differentially expressed between ripe and unripe groups. (2) Cervical ripening was associated with enrichment of specific biological processes (e.g. cell adhesion, regulation of anatomical structure), pathways and 11 molecular functions (e.g. extracelluar matrix (ECM)-structural constituent, protein binding, glycosaminoglycan binding). (3) qRT-PCR confirmed that 9 of 11 tested differentially expressed genes (determined by microarray) were upregulated in a ripe cervix (e.g. MYOCD, VCAN, THBS1, COL5A1). (4) Twenty-three additional genes related to ECM metabolism and adhesion molecules were differentially regulated (by qRT-PCR) in ripe cervices. CONCLUSION: (1) This is the first description of the changes in the human cervical transcriptome with ripening before the onset of labor. (2) Biological processes, pathways and molecular functions were identified with the use of this unbiased approach. (3) In contrast to cervical dilation after term labor, inflammation-related genes did not emerge as differentially regulated with cervical ripening. (4) Myocardin was identified as a novel gene upregulated in human cervical ripening.

Functional genomics of the pregnant uterus: from expectations to reality, a compilation of studies in the myometrium.

BACKGROUND: Studies on the human myometrium have reported on different microarrays containing different sets of genes or ESTs. However each study profiled only a small number of patients due to various constraints. More profiling information would be an addition to our knowledge base of parturition. METHODS: We compiled from five human studies, transcriptional differences between the non pregnant myometrium (NP), preterm myometrium (PTNIL), term myometrium not in labor (TNIL) and term myometrium in labor (TIL). Software modules developed by the Draghici's group at Wayne State University (Detroit, MI, USA) were used to propose a hierarchical list of several KEGG pathways most likely adjusted to changes observed in microarray experiments. RESULTS: The differential expression of 118 genes could be dispatched in 14 main KEGG pathways that were the most representative of the changes seen in NP and PTNIL, versus TNIL or TIL. Despite the potential of multiple pitfalls inherent to the use of the microarray technology, gene module analysis of the myometrial transcriptome reveals the activation of precise signaling pathways, some of which may have been under evaluated. CONCLUSION: The remodelling and maturation processes that the uterus undergoes in pregnancy appear clearly as phenomena which last during the full course of gestation. It is attested by the nature of the main signaling pathways represented, in the comparison of the PTNIL versus TNIL uterus. Comparatively, the onset of labor is a phenomenon which remains less well characterized by these methods of analysis, possibly because it is a phenomenon occurring in too short a window to have been grasped by the studies carried out up to now.

Up-regulation of the progesterone receptor (PR)-C isoform in laboring myometrium by activation of nuclear factor-kappaB may contribute to the onset of labor through inhibition of PR function.

Progesterone acting via the progesterone receptor (PR) plays a critical role in maintaining uterine quiescence during pregnancy. In the present study, we tested the hypothesis that the transactivating capability of the PR is down-regulated in the myometrium at term by a change in uterine PR isoform ratio resulting from local activation of the nuclear factor (NF)-kappaB pathway. Overexpression of the truncated PR-C isoform in human myometrial cells inhibited PR-B transactivation. Expression of PR isoforms, PR-A, PR-B, and PR-C, was characterized by immunoblotting and quantitative PCR (Q-PCR) in fundal and lower uterine segment myometrium from pregnant women in labor and not in labor and in the pregnant mouse uterus during late gestation. We observed a marked increase in levels of PR-C and transcriptionally active PR-B specifically in fundal myometrium of women in labor. In pregnant mouse uterus, levels of PR-B and PR-C also increased between 15 days post coitum and term, whereas expression of PR-A was dramatically up-regulated at 19 days post coitum. In studies of uterine tissues of mice injected intraamniotically with surfactant protein A and of human myometrial and T47D breast cancer cells in culture, up-regulation of PR isoform expression was observed in response to activation of the NF-kappaB pathway. Chromatin immunoprecipitation analysis revealed IL-1beta induced binding of NF-kappaB to the PR promoter. Collectively, these findings suggest that up-regulation of inhibitory PR isoform expression by NF-kappaB activation in both laboring human fundus and pregnant mouse uterus near term may inhibit PR transactivation and thereby lead to a loss of uterine quiescence and the onset of labor.

Expression of cyclo-oxygenase types-1 and -2 in human fetal membranes throughout pregnancy.

Human labour is associated with increased prostaglandin synthesis within the fetal membranes. We have studied the expression of the two isoforms of the central prostaglandin synthetic enzyme, cyclo-oxygenase (COX-1 and COX-2), in human fetal membranes throughout pregnancy, at mRNA, protein and activity levels. COX-1 mRNA expression was low in human amnion and chorion-decidua and did not change with gestational age. COX-2 mRNA expression in fetal membranes increased with gestational age, with significant up-regulation prior to the onset of labour and in association with labour. Protein concentrations of COX-1 did not change, whilst concentrations of COX-2 increased from the first to the third trimester. COX activity increased with gestational age and in association with labour, although prostaglandin production in fetal membranes collected after labour was reduced, suggesting reduced substrate supply. These data suggest that it is up-regulation of COX-2, rather than of COX-1, which mediates increased prostaglandin synthesis within the fetal membranes at term. Much of the increase in COX-2 expression precedes the onset of labour, suggesting that it is a cause, rather than a consequence, of labour.