Adverse birth outcome across the generations: the contribution of paternal factors.

PURPOSE: There is literature suggesting an intergenerational relationship between maternal and infant size for gestational age status and preterm birth, but much less is known about the contribution of paternal birth outcome to infant birth outcome. This study seeks to determine the association between paternal and infant small-for-gestational-age status (weight for gestational age < 10th percentile, SGA) and preterm birth (< 37 weeks gestation, PTB) in a large, diverse population-based sample in the United States. METHODS: Stratified and log-binomial multivariable regression analyses were computed on the vital records of Illinois-born infants (1989-1991) and their Illinois-born parents (born 1956-1976). RESULTS: Among non-Hispanic Whites (n = 83,218), the adjusted (controlling for maternal SGA or PTB, age, parity, education, marital status, prenatal care, and cigarette smoking) relative risk (95% confidence interval) of infant SGA and PTB for former SGA (compared to non-SGA) and preterm (compared to term) fathers equaled 1.65 (1.53, 1.77) and 1.07 (0.92, 1.24), respectively. Among African-Americans (n = 8401), the adjusted relative risk (95% confidence interval) of infant SGA and PTB for former SGA (compared to non-SGA) and preterm (compared to term) fathers equaled 1.32 (1.14, 1.52) and 1.19 (0.98, 1.45), respectively. CONCLUSION: Paternal adverse birth outcome, particularly SGA, is a modest risk factor for corresponding adverse infant outcome, independent of maternal risk status. This phenomenon appears to occur similarly among non-Hispanic White and African-American women.

Polymorphism of the Oxytocin Receptor (OXTR) Gene Affects the Circulating Oxytocin Receptor Levels in Late-Term Pregnancy in a Turkish Population.

INTRODUCTION: Postterm and late-term pregnancies still remain a serious health problem, and underlying exact mechanisms are not fully elucidated. These mechanisms are influenced by many factors. OBJECTIVE: The aim of this study was to investigate the relationship between plasma oxytocin and oxytocin receptor levels and oxytocin receptor polymorphisms in term and late-term pregnant women. METHODS: Sixty-eight singleton pregnant women with late-term pregnancy and 83 singleton pregnant women with term parturition were included in this study. A comparison was performed between pregnancies and neonates born at term (37 0/7 and 41 6/7 weeks' gestation). Plasma oxytocin, oxytocin receptor, estradiol, and progesterone levels were measured by using enzyme-linked immunosorbent assay kits. TaqMan® SNP Genotyping Assays and qPCR ProbesMaster were used to investigate the polymorphisms of rs237911, rs2228485, rs53576, and rs2254298. RESULTS: There was not any difference in gene distributions of 4 common single-nucleotide polymorphisms of oxytocin receptor of rs237911, rs2228485, rs53576, and rs2254298 between subjects in late-term and term pregnancy groups. With rs53576 of the GG genotype, serum oxytocin levels were 21.50 ± 10.69 (ng/L) in the late-term group and 62.71 ± 18.01 (ng/L) in the term group (p = 0.049). Oxytocin receptor levels in the late-term and term pregnancy groups of the GG genotype were 17.92 ± 8.15 (pg/mL) and 45.77 ± 11.66 (pg/mL), respectively (p = 0.046). CONCLUSION: Our findings suggest that the rs53576 oxytocin receptor single-nucleotide polymorphism is associated with late-term pregnancy through acting by direct modulation of oxytocin and oxytocin receptor levels.

Differential placental methylation in preeclampsia, preterm and term pregnancies.

INTRODUCTION: Preeclampsia (PE) is one of the leading causes of maternal mortality and morbidity worldwide. Recently, the role of epigenetic modifications in preeclampsia has been a focus of research. This study was to identified genes or pathways that may be associated with PE, and discuss whether the changes in the methylation level of these genes is related to the pathogenesis of PE. METHODS: The methylation levels of placental tissues between PE (n = 4), preterm birth (PB, n = 4) and term birth (TB, n = 4) were detected by Illumina Infinium HumanMethylation850 K BeadChip. Pyrosequencing and qRT-PCR were used to validated the methylation and expression levels of the genes with the most significant differences. RESULTS: The global methylation levels of placenta tissues in PE and PB were both higher compared to TB. After eliminated the effect of gestational age, there were 808 gene probes differentially methylated in PE compared to PB. We found 137 genes with 130 genes hypermethylated and 7 genes hypomethylated. CMIP, BLCAP and MICA genes were with the most significant differential methylation. The expression level of CMIP and BLCAP were both negatively correlated to the methylation levels, while the expression level of MICA was not related to its methylation levels. CONCLUSION: The methylation levels in placenta tissues were associated with gestational ages. We indicated the expression levels of the significantly methylated genes were negatively correlated with the methylation levels, further functional researches were still needed to find out whether they are associated with the onset of preeclampsia.

Reduced Expression of Hydrogen Sulfide-Generating Enzymes Down-Regulates 15-Hydroxyprostaglandin Dehydrogenase in Chorion during Term and Preterm Labor.

Chorionic NAD-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) plays a pivotal role in controlling the amount of prostaglandins in the uterus and has been implicated in the process of labor. Prior studies identified hydrogen sulfide-generating enzymes cystathionine-ß-synthetase (CBS) and cystathionine-γ-lyase (CSE) in fetal membranes. We investigated whether hydrogen sulfide is involved in the regulation of PGDH expression in the chorion during labor. The chorionic tissues were obtained from pregnant women at preterm in labor and at term in labor or not in labor at term. Levels of CSE and CBS and hydrogen sulfide production rate were down-regulated in term in labor and preterm in labor groups compared with not in labor at term group. The CBS level correlated to PGDH expression in the chorion. Hydrogen sulfide donor NaHS and precursor l-cysteine dose-dependently stimulated PGDH expression and activity in cultured chorionic trophoblasts. The effect of l-cysteine was blocked by CBS inhibitor and CBS siRNA but not by CSE inhibitor and CSE siRNA. Hydrogen sulfide treatment suppressed miR-26b and miR-199a expression in chorionic trophoblasts. miR-26b and miR-199a mimics blocked hydrogen sulfide upregulation of PGDH expression. Our results indicate that hydrogen sulfide plays pivotal roles in maintenance of PGDH expression in the chorion during human pregnancy. Reduced expression of hydrogen sulfide-generating enzymes contributes to an increased amount of prostaglandins in the uterus during labor.

A20, an essential component of the ubiquitin-editing protein complex, is a negative regulator of inflammation in human myometrium and foetal membranes.

STUDY QUESTION: Does A20 regulate mediators involved in the terminal processes of human labour in primary myometrial and amnion cells? SUMMARY ANSWER: A20 is a nuclear factor-kappa B (NF-κB) responsive gene that acts as a negative regulator of NF-κB-induced expression of pro-labour mediators. WHAT IS KNOWN ALREADY: Inflammation is commonly implicated in spontaneous preterm birth and the processes involved in rupture of foetal membranes and uterine contractions. In myometrium and foetal membranes, the pro-inflammatory transcription factor NF-κB regulates the transcription of pro-labour mediators in response to inflammatory stimuli. In non-gestational tissues, A20 is widely recognised as an anti-inflammatory protein that inhibits inflammation-induced NF-κB signalling. STUDY DESIGN, SIZE, DURATION: Primary human amnion and myometrial cells were used to determine the effect of pro-inflammatory mediators on A20 expression and the effect of A20 siRNA on the expression and secretion of pro-labour mediators. The expression of A20 was assessed in myometrium and foetal membranes from non-labouring and labouring women at preterm and or term (n = 8 or nine samples per group). PARTICIPANTS/MATERIALS, SETTING, METHODS: The effects of pro-inflammatory mediators and of A20 siRNA in cell cultures were determined by quantitative RT-PCR (qRT-PCR), western blots, immunoassays, gelatin zymography and luciferase assays. A20 expression in tissue samples was assessed by qRT-PCR. Statistical significance was ascribed to a P value < 0.05. MAIN RESULTS AND THE ROLE OF CHANCE: In primary cells isolated from myometrium and or amnion, the pro-inflammatory cytokines IL1B and TNF, the bacterial products flagellin and fsl-1, and the viral double stranded RNA analogue poly(I:C) significantly increased A20 mRNA expression via NF-κB. A20 siRNA studies in primary myometrial and amnion cells demonstrated an augmentation of inflammation-induced expression and or secretion of pro-inflammatory cytokines (IL1A, IL6), chemokines (CXCL1, CXCL8, CCL2), adhesion molecules (ICAM1, VCAM1), contraction-associated proteins (PTGS2, PTGFR, PGF2α) and the extracellular matrix degrading enzyme MMP9, as well as NF-κB activation. Inhibition of NF-κB activity significant attenuated inflammation-induced expression of pro-labour mediators in A20 siRNA transfected cells. Finally, A20 mRNA expression was decreased in myometrium and foetal membranes with labour, and in foetal membranes with chorioamnionitis. LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: The conclusions of this study are solely reliant on the data from in vitro experiments using cells isolated from myometrium and amnion. WIDER IMPLICATIONS OF THE FINDINGS: The results of this study raise the possibility that targeting A20 may be a therapeutic approach to reduce inflammation associated with spontaneous preterm birth. STUDY FUNDING AND COMPETING INTEREST(S): Associate Professor Martha Lappas is supported by a Career Development Fellowship from the National Health and Medical Research Council (NHMRC; grant no. 1047025). Funding for this study was provided by the NHMRC (grant no. 1058786), Norman Beischer Medical Research Foundation and the Mercy Research Foundation. There are no competing interests.

Folate Transporters in Placentas from Preterm Newborns and Their Relation to Cord Blood Folate and Vitamin B12 Levels.

Folate deficiency during pregnancy has been related to low birth weight, preterm (PT) birth and other health risks in the offspring; however, it is unknown whether prematurity is related to low folate transport through the placenta due to altered expression of specific folate transporters. We determined placental expression (mRNA and protein concentrations by RT-qPCR and WB respectively) of specific folate transporters: RFC, PCFT/HCP1 and FOLR1 in chorionic (fetal) and basal (maternal) plates of placentas of PT pregnancies (PT, 32-36 weeks, n = 51). Term placentas were used as controls (T, 37-41 weeks, n = 47). Folates and vitamin B12 levels were measured by electrochemiluminescence in umbilical cord blood of newborns. FOLR1 mRNA expression was lower and protein concentration higher in PT placentas (both plates) relative to the control group (p <0.05). In addition, gestational age was positively correlated with mRNA expression (Rho = 0.7), and negatively with protein concentration (Rho = -0.7 for chorionic and -0.43 for basal plate). PCFT/HCP1 mRNA was lower in PT placentas, without changes in protein levels. RFC did not differ in PT placentas compared to controls. PT newborns presented higher cord blood folate level (p = 0.049) along with lower vitamin B12 concentration compared to controls (p = 0.037).In conclusion, placental FOLR1 mRNA was positively associated with gestational age. Conversely, FOLR1 protein concentrations along with folate/vitamin B12 ratio in cord blood were negatively associated with gestational age. Placental FOLR1 is likely the main placental folate transporter to the fetus in newborns.

Fetal DNA methylation of autism spectrum disorders candidate genes: association with spontaneous preterm birth.

OBJECTIVE: Autism spectrum disorder (ASD) is associated with preterm birth (PTB), although the reason underlying this relationship is still unclear. Our objective was to examine DNA methylation patterns of 4 ASD candidate genes in human fetal membranes from spontaneous PTB and uncomplicated term birth. STUDY DESIGN: A literature search for genes that have been implicated in ASD yielded 14 candidate genes (OXTR, SHANK3, BCL2, RORA, EN2, RELN, MECP2, AUTS2, NLGN3, NRXN1, SLC6A4, UBE3A, GABA, AFF2) that were epigenetically modified in relation to ASD. DNA methylation in fetal leukocyte DNA in 4 of these genes (OXTR, SHANK3, BCL2, and RORA) was associated with PTB in a previous study. This study evaluated DNA methylation, transcription (reverse transcription polymerase chain reaction), and translation patterns (immunostaining and Western blot) in fetal membrane from term labor (n = 14), term not in labor (TNIL; n = 29), and spontaneous preterm birth (PTB; n = 27). Statistical analysis was performed with analysis of variance; a probability value of < .05 was significant. RESULTS: Higher methylation of the OXTR promoter was seen in fetal membranes from PTB, compared with term labor or TNIL. No other gene showed any methylation differences among groups. Expression of OXTR was not different among groups, but the 70 kDa OXTR protein was seen only in PTB, and immunostaining was more intense in PTB amniocytes than term labor or TNIL. CONCLUSION: Among the 4 genes that were studied, fetal membranes from PTB demonstrate differences in OXTR methylation and regulation and expression, which suggest that epigenetic alteration of this gene in fetal membrane may likely be indicating an in utero programing of this gene and serve as a surrogate in a subset of PTB. The usefulness of OXTR hypermethylation as a surrogate for a link to ASD should be further evaluated in longitudinal and in vitro studies.

Small-for-gestational age and preterm birth across generations: a population-based study of Illinois births.

Small for gestational age (weight for gestational age <10th percentile, SGA) and preterm birth (<37 weeks, PTB) are the major determinants of infant mortality rates and racial disparities therein. To determine the generational inheritance patterns of SGA and PTB among non-Hispanic Whites and African-Americans. Stratified and multivariable binominal regression analyses were performed on an Illinois transgenerational dataset of White and African-American infants (1989-1991) and their mothers (1956-1976) with appended US census income information. Former SGA White mothers (N = 8,993) had a twofold greater infant SGA frequency than former non-SGA White mothers (N = 101,312); 14.4 versus 6.9 %, RR = 2.1 (2.0-2.2). Former SGA African American (N = 4,861) mothers had a SGA birth frequency of 25.7 % compared to 16.1 % for former non-SGA mothers (N = 28,090); RR = 1.5 (1.5-1.6). The adjusted (controlling for maternal age, education, marital status, parity, prenatal care usage, cigarette smoking, and hypertension) RR (95 % CI) of infant SGA for former SGA (compared to non-SGA) White and African-American mothers equaled 2.0 (1.9-2.1 and 1.5 (1.5-1.6), respectively. The adjusted RR (95 % CI) of infant preterm birth for former preterm (compared to term) White and African-American mothers were 1.1 (1.0-1.2). The findings were minimally changed among mothers with a lifelong residence in impoverished or affluent neighborhoods. In both races, approximately 8 % of SGA births were attributable to maternal SGA. There is a transgenerational association of SGA but not preterm birth among non-Hispanic Whites and African-Americans. In both races, a similar proportion of SGA births are attributable to maternal SGA.

Apelin is decreased with human preterm and term labor and regulates prolabor mediators in human primary amnion cells.

A critical role of proinflammatory mediators including cytokines, prostaglandins, and extracellular matrix remodeling enzymes in the processes of human labor and delivery, at term and preterm, has been demonstrated. In nongestational tissues, apelin plays an important role in a number of physiologic processes, including the regulation of inflammation. However, the role and regulation of apelin and the apelin receptor (APJ) in human gestational tissues are not known. The aims of this study were to determine the effect of (i) preterm and term labor on apelin and APJ expression in human gestational tissues and (ii) apelin small interfering RNA (siRNA) knockdown in human primary amnion cells on prolabor mediators. Human placenta and fetal membranes were collected from term nonlaboring women and women after spontaneous labor and delivery. Preterm and term spontaneous labor were associated with significantly lower apelin expression in fetal membranes. On the other hand, there was no effect of labor on APJ expression and no effect of term labor on placental apelin or APJ expression. Transfection of primary amnion cells with apelin siRNA was associated with significantly increased interleukin (IL)-1ß-induced IL-6 and IL-8 release and cyclooxygenase-2 messenger RNA (mRNA) expression and resultant prostaglandin E2 and prostaglandin F2α release. There was no effect of apelin siRNA on matrix metalloproteinase (MMP)-9 mRNA expression and pro MMP-9 release. In summary, human labor downregulates apelin expression in human fetal membranes. Furthermore, a role of apelin in the regulation of proinflammatory and prolabor mediators in human fetal membranes is supported by our studies.

Gene expression profile of endotoxin-stimulated leukocytes of the term new born: control of cytokine gene expression by interleukin-10.

INTRODUCTION: Increasing evidence now supports the association between the fetal inflammatory response syndrome (FIRS) with the pathogenesis of preterm labor, intraventricular hemorrhage and bronchopulmonary dysplasia. Polymorphonuclear leukocyte (PMNs) and mononuclear cell (MONOs) infiltration of the placenta is associated with these disorders. The aim of this study was to reveal cell-specific differences in gene expression and cytokine release in response to endotoxin that would elucidate inflammatory control mechanisms in the newly born. METHODS: PMNs and MONOs were separately isolated from the same cord blood sample. A genome-wide microarray screened for gene expression and related pathways at 4 h of LPS stimulation (n = 5). RT-qPCR and ELISA were performed for selected cytokines at 4 h and 18 h of LPS stimulation. RESULTS: Compared to PMNs, MONOs had a greater diversity and more robust gene expression that included pro-inflammatory (PI) cytokines, chemokines and growth factors at 4 h. Only MONOs had genes changing expression (all up regulated including interleukin-10) that were clustered in the JAK/STAT pathway. Pre-incubation with IL-10 antibody, for LPS-stimulated MONOs, led to up regulated PI and IL-10 gene expression and release of PI cytokines after 4 h. DISCUSSION: The present study suggests a dominant role of MONO gene expression in control of the fetal inflammatory response syndrome at 4 hrs of LPS stimulation. LPS-stimulated MONOs but not PMNs of the newborn have the ability to inhibit PI cytokine gene expression by latent IL-10 release.

Differential expression of calcium transport channels in placenta primary cells and tissues derived from preeclamptic placenta.

Preeclampsia is a pregnancy-specific disease characterized by hypertension, proteinuria, and oxidative stress in the placenta. During the last trimester of gestation, calcium (Ca(2+)) transport from mother to fetus increases dramatically in response to the increased demand for Ca(2+) caused by bone mineralization in the fetus. Ca(2+) supplementation can significantly reduce the incidence and severity of preeclampsia or delay its onset. Ca(2+) transport channels (CTCs) include transient receptor potential vanilloid 6 (TRPV6), plasma membrane Ca(2+) ATPase (PMCA1), and Na(+)/Ca(2+) exchangers (NCKX3 or NCX1). We hypothesized that trans-placental Ca(2+) exchange in preeclamptic trophoblasts may be compensated for successful fetal bone mineralization. The roles of cell membrane channels (TRPV6, PMCA1, NCKX3 and NCX1) were examined in placental primary cells and in normotensive and preeclamptic placentas. The biomarker gene for preeclampsia, soluble fms-like tyrosine kinase-1 (sFLT1) or marker for oxygen-sensitive gene, hypoxia-sensitive inducible factor 1α (HIF-1α), were up-regulated in the preeclamptic placentas and hypoxic cells. The detection of sFLT1 and HIF-1α genes demonstrated that our experimental conditions were suitable to verify a preeclamptic condition. In women experiencing preterm labor, CTC expressions was found to be increased in the fetal and maternal regions of the preeclamptic placenta compared to the observed in normotensive placenta. During term labor, TRPV6 and PMCA1 were highly expressed in the fetal and maternal sections of preeclamptic placenta, while the expression of NCKX3 and NCX1 was reduced. In addition, the expression of CTCs was altered in hypoxia-stressed placental cells. Taken together, our findings demonstrated that the expression of CTCs was regulated by hypoxia stress in placenta tissues and cells, suggesting that our experimental in vitro hypoxic conditions were similar to those of preeclampsia. Furthermore, impaired Ca(2+) metabolism found in preeclamptic syncytiotrophoblasts was resulted from hypoxic stress, which may induce expression of Ca(2+) transport proteins in the placenta to maintain the balance between maternal and fetal Ca(2+) demand during pregnancy.

TMED2/p24ß1 is expressed in all gestational stages of human placentas and in choriocarcinoma cell lines.

Members of the transmembrane emp24 domain (Tmed)/p24 family of proteins are required for transport of proteins between the endoplasmic reticulum and the Golgi. One member of this family, Tmed2/p24ß1, is expressed during placental development in mice and its expression is required for normal development of the labyrinth layer. Although TMED2 is conserved in humans, little is known about its expression and function in human placenta. We examined TMED2 expression in human placenta between 5.5 and 40 weeks of gestation and showed that TMED2 is expressed in syncytiotrophoblast, cytotrophoblast, and stromal cells. We also found high levels of TMED2 expression in BeWo but not in JEG-3 choriocarcinoma cell line. We used the BeWo cell line to determine TMED2 subcellular localization in placental cells and show its co-localization with the endoplasmic reticulum Golgi intermediate compartment. Our findings show conservation of TMED2 expression in human placenta and suggest that this protein may also play a role during placental development in humans.

The effects of labor on differential gene expression in parturient women, placentas, and fetuses at term pregnancy.

Labor and its associated pain are thought to have unique impacts on parturient women. The goal of this study was to investigate the effects of labor and associated pain on differential gene expression profiles in the maternal, fetal, and placental compartments. We used microarrays to analyze maternal blood (MB), fetal cord blood (CB), and placental tissue samples in pregnant women after term vaginal deliveries (laboring group) and in term pregnant women after scheduled Ceasarean sections (nonlaboring group). The upregulated genes in the MB of the laboring group are involved in cytokine and nuclear factor-kappa B signaling pathways, regulation of the networks of toll-like receptor 4, and suppressor of cytokine signaling 3. Upregulated genes in the CB of the laboring group are involved in responding to stress and stimuli by regulating the network genes of the T-cell receptor beta locus and the FK506 binding protein 8. Differentially expressed genes in the placenta of the laboring group are involved in nitric oxide transport, gas transport, response to hydrostatic pressure, oxygen transport, acute phase responses, and the tumor necrosis factor-mediated signaling pathway, which are important during the transient hypoxemia and hypoperfusion that occur in the placenta during uterine contractions. Interestingly, few of the genes exhibited simultaneous changes in all three compartments, indicating that different pathways and complex interactions may be involved in human labor. In conclusion, human labor and its associated pain elicit unique gene regulatory changes in MB, placenta, and CB.

Interleukin-6 is an essential determinant of on-time parturition in the mouse.

IL-6 abundance in amniotic fluid and uterine tissues increases in late gestation or with infection-associated preterm labor. A role in regulation of labor onset is suggested by observations that IL-6 increases expression of genes controlling prostaglandin synthesis and signaling in isolated uterine cells, but whether IL-6 is essential for normal parturition is unknown. To evaluate the physiological role of IL-6 in parturition in mice, we investigated the effect of Il6 null mutation on the timing of parturition and expression of genes associated with uterine activation. Il6 null mutant mice delivered 24 h later than wild-type mice, although circulating progesterone fell similarly in both genotypes during the prepartal period. Il6 null mutant mice were also refractory to low doses of lipopolysaccharide sufficient to induce preterm delivery in wild-type mice. The characteristic late-gestation elevation in uterine expression of Oxtr mRNA encoding oxytocin receptor, and peripartal increases in Ptgfr and Ptgs2 mRNAs regulating prostaglandin synthesis and signaling were delayed by 24 h in Il6 null mutant mice. Conversely, Ptger4 mRNA encoding the prostaglandin E receptor-4 was abnormally elevated in late-gestation in Il6 null mutant mice. Administration of recombinant IL-6 from d 11.5 postcoitum until term restored the normal timing of delivery and normalized Ptger4 mRNA expression in late gestation. We conclude that IL-6 has a key role in controlling the progression of events culminating in parturition and that it acts downstream of luteolysis in the uterus to regulate genes involved in the prostaglandin-mediated uterine activation cascade.

Progestin and thrombin regulate tissue factor expression in human term decidual cells.

CONTEXT: Perivascular cell membrane-bound tissue factor (TF) initiates hemostasis via thrombin generation. The identity and potential regulation of TF-expressing cells at the human maternal-fetal interface that confers hemostatic protection during normal and preterm delivery is unclear. OBJECTIVES: The objective of the study were to identify TF-expressing cells at the maternal-fetal interface in term and preterm decidual sections by immunohistochemistry and evaluate progestin, thrombin, TNF-alpha, and IL-1beta effects on TF expression by cultured human term decidual cells (DCs). INTERVENTIONS AND MAIN OUTCOME MEASURES: Serial placental sections were immunostained for TF. Leukocyte-free term DC monolayers were incubated with 10(-8) M estradiol (E2) or E2 plus 10(-7) M medroxyprogestrone acetate (MPA) +/- thrombin or TNF-alpha or IL-1beta. ELISA and Western blotting assessed TF in cell lysates. Quantitative real-time RT-PCR measured TF mRNA levels. RESULTS: Immunolocalized TF in DC membranes in preterm and term placental sections displayed higher Histologic Scores than villous mesenchymal cells (P < 0.05). TF was undetected in interstitial or extravillous trophoblasts. Compared with DCs incubated with E2, MPA and 2.5 U/ml thrombin each doubled TF levels (P < 0.05) and E2 + MPA + thrombin further doubled TF levels (P < 0.05), whereas TNF-alpha and IL-1beta were ineffective. Western blotting confirmed the ELISA results. Quantitative RT-PCR revealed corresponding changes in TF mRNA levels. CONCLUSIONS: In human term placental sections, DC-expressed TF exceeds that of other cell types at the maternal-fetal interface and is localized at the cell membranes in which it can bind to factor VII and meet the hemostatic demands of labor and delivery via thrombin formation. Unlike the general concept that TF is constitutive in cells that highly express it, MPA and thrombin significantly enhanced TF expression in term DC monolayers.

Placenta-specific methylation of the vitamin D 24-hydroxylase gene: implications for feedback autoregulation of active vitamin D levels at the fetomaternal interface.

Plasma concentrations of biologically active vitamin D (1,25-(OH)(2)D) are tightly controlled via feedback regulation of renal 1alpha-hydroxylase (CYP27B1; positive) and 24-hydroxylase (CYP24A1; catabolic) enzymes. In pregnancy, this regulation is uncoupled, and 1,25-(OH)(2)D levels are significantly elevated, suggesting a role in pregnancy progression. Epigenetic regulation of CYP27B1 and CYP24A1 has previously been described in cell and animal models, and despite emerging evidence for a critical role of epigenetics in placentation generally, little is known about the regulation of enzymes modulating vitamin D homeostasis at the fetomaternal interface. In this study, we investigated the methylation status of genes regulating vitamin D bioavailability and activity in the placenta. No methylation of the VDR (vitamin D receptor) and CYP27B1 genes was found in any placental tissues. In contrast, the CYP24A1 gene is methylated in human placenta, purified cytotrophoblasts, and primary and cultured chorionic villus sampling tissue. No methylation was detected in any somatic human tissue tested. Methylation was also evident in marmoset and mouse placental tissue. All three genes were hypermethylated in choriocarcinoma cell lines, highlighting the role of vitamin D deregulation in this cancer. Gene expression analysis confirmed a reduced capacity for CYP24A1 induction with promoter methylation in primary cells and in vitro reporter analysis demonstrated that promoter methylation directly down-regulates basal promoter activity and abolishes vitamin D-mediated feedback activation. This study strongly suggests that epigenetic decoupling of vitamin D feedback catabolism plays an important role in maximizing active vitamin D bioavailability at the fetomaternal interface.

Expression of secreted frizzled-related protein 4 in the primate placenta.

Secreted frizzled-related protein 4 (sFRP4) blocks the Wnt signalling pathway by competitively binding Wnt ligands (frizzled receptors). This pathway is important during development and oncogenesis. It is, however, complex with a large number of interacting proteins, isoforms and receptors. The Wnt signalling pathway has a role in human placental development and implantation, particularly in the trophoblast. Humans and macaque monkeys exhibit a similar remodelling of the decidual spiral arteries. The expression of sFRP4 in human and macaque placentas at different gestational ages have been examined with immunohistochemistry, in-situ hybridization, real-time polymerase chain reaction, and western blotting. This study demonstrates that sFRP4 is expressed predominantly in the villous syncytiotrophoblast and the invasive intermediate cytotrophoblast, and in the amnion. These observational studies suggest that sFRP4 has a role in placental development and implantation, and may be an important factor in the development of the decidual fibrinoid zone, and in trophoblast apoptosis and a band of apoptosis in the underlying decidua deep into the trophoblast.

Signature pathways identified from gene expression profiles in the human uterine cervix before and after spontaneous term parturition.

OBJECTIVE: This study aimed to discover "signature pathways" that characterize biologic processes, based on genes differentially expressed in the uterine cervix before and after spontaneous labor. STUDY DESIGN: The cervical transcriptome was characterized previously from biopsy specimens taken before and after term labor. Pathway analysis was used to study the differentially expressed genes, based on 2 gene-to-pathway annotation databases (Kyoto Encyclopedia of Genes and Genomes [Kanehisa Laboratories, Kyoto University, Kyoto, Japan] and Metacore software [GeneGo, Inc, St. Joseph, MI]). Overrepresented and highly impacted pathways and connectivity nodes were identified. RESULTS: Fifty-two pathways in the Metacore database were enriched significantly in differentially expressed genes. Three of the top 5 pathways were known to be involved in cervical remodeling. Two novel pathways were plasmin signaling and plasminogen activator urokinase signaling. The same analysis with the Kyoto Encyclopedia of Genes and Genomes database identified 4 significant pathways that the impact analysis confirmed. Multiple nodes that provide connectivity within the plasmin and plasminogen activator urokinase signaling pathways were identified. CONCLUSION: Three strategies for pathway analysis were consistent in their identification of novel, unexpected, and expected pathways, which suggests that this approach is both valid and effective for the elucidation of biologic mechanisms that are involved in cervical dilation and remodeling.

Interleukins-1, -4, -6, -10, tumor necrosis factor, transforming growth factor-beta, FAS, and mannose-binding protein C gene polymorphisms in Australian women: Risk of preterm birth.

OBJECTIVE: The purpose of this study was to examine the relationship between preterm birth and 22 single nucleotide polymorphisms in genes that encode cytokines and mediators of apoptosis and host defense. STUDY DESIGN: Two hundred two white women with a spontaneous preterm birth of <35 weeks of gestation were compared with 185 white women with term births. Genotyping was performed with polymerase chain reaction and sequence specific primers. Multivariable analyses included demographic and genetic variables. RESULTS: Alcohol (multivariable odds ratio, 2.3; P = .001] and substance use (multivariable odds ratio, 3.7; P = .01) were associated with preterm birth at <35 weeks of gestation. Smoking (multivariable odds ratio, 2.3; P = .03), haplotypes IL10 -1082A/-819T/-592A (multivariable odds ratio, 2.1; P = .04), tumor necrosis factor ( TNF )+488A/-238G/-308G (multivariable odds ratio, 2.4; P = .04), and IL4 -509C/C (multivariable odds ratio, 3.4; P = .02), and the presence of MBL2 codon 54Asp (multivariable odds ratio, 2.3; P = .02) were associated independently with preterm birth at <29 weeks of gestation. Homozygosity for IL10 -1082G/-819C/-592C haplotype (multivariable odds ratio, 1.9; P = .02) was more common in women with preterm premature rupture of membranes. CONCLUSION: Polymorphisms in immunoregulatory genes may influence susceptibility to preterm birth or premature rupture of membranes.