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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 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.

Normal term and spontaneous preterm births (PTB) are documented to be associated with oxidative stress (OS), and imbalances in the redox system (balance between pro- and antioxidant) have been reported in the maternal-fetal intrauterine compartments. The exact mechanism of labor initiation either at term or preterm by OS is still unclear, and this lack of understanding can partially be blamed for failure of antioxidant supplementation trials in PTB prevention. Based on recent findings from our laboratory, we postulate heterogeneity in host OS response. The physiologic (at term) and pathophysiologic (preterm) pathways of labor are not mediated by OS alone but by OS-induced damage to intrauterine tissues, especially fetal membranes of the placenta. OS damage affects all major cellular elements in the fetal cells, and this damage promotes fetal cell senescence (aging). The aging of the fetal cells are predominated by p38 mitogen activated kinase (p38MAPK) pathways. Senescing cells generate biomolecular signals that are uterotonic, triggering labor process. The aging of fetal cells is normal at term. However, aging is premature in PTB, especially in those PTBs complicated by preterm premature rupture of the membranes (pPROM), where elements of redox imbalances and OS damage are more dominant. We postulate that fetal cell senescence signals generated by OS damage are likely triggers for labor. This review highlights the mechanisms involved in senescence development at term and preterm by OS damage and provides insight into novel fetal signals of labor initiation pathways.

Pregnancy and childbirth represent a critical time period when a woman can be reached through a variety of mechanisms with interventions aimed at reducing her risk of a preterm birth and improving her health and the health of her unborn baby. These mechanisms include the range of services delivered during antenatal care for all pregnant women and women at high risk of preterm birth, services provided to manage preterm labour, and workplace, professional and other supportive policies that promote safe motherhood and universal access to care before, during and after pregnancy. The aim of this paper is to present the latest information about available interventions that can be delivered during pregnancy to reduce preterm birth rates and improve the health outcomes of the premature baby, and to identify data gaps. The paper also focuses on promising avenues of research on the pregnancy period that will contribute to a better understanding of the causes of preterm birth and ability to design interventions at the policy, health care system and community levels. At minimum, countries need to ensure equitable access to comprehensive antenatal care, quality childbirth services and emergency obstetric care. Antenatal care services should include screening for and management of women at high risk of preterm birth, screening for and treatment of infections, and nutritional support and counselling. Health workers need to be trained and equipped to provide effective and timely clinical management of women in preterm labour to improve the survival chances of the preterm baby. Implementation strategies must be developed to increase the uptake by providers of proven interventions such as antenatal corticosteroids and to reduce harmful practices such as non-medically indicated inductions of labour and caesarean births before 39 weeks of gestation. Behavioural and community-based interventions that can lead to reductions in smoking and violence against women need to be implemented in conjunction with antenatal care models that promote women's empowerment as a strategy for reducing preterm delivery. The global community needs to support more discovery research on normal and abnormal pregnancies to facilitate the development of preventive interventions for universal application. As new evidence is generated, resources need to be allocated to its translation into new and better screening and diagnostic tools, and other interventions aimed at saving maternal and newborn lives that can be brought to scale in all countries. Declaration. This article is part of a supplement jointly funded by Save the Children's Saving Newborn Lives programme through a grant from The Bill & Melinda Gates Foundation and March of Dimes Foundation and published in collaboration with the Partnership for Maternal, Newborn and Child Health and the World Health Organization (WHO). The original article was published in PDF format in the WHO Report Born Too Soon: the global action report on preterm birth (ISBN 978 92 4 150343 30), which involved collaboration from more than 50 organizations. The article has been reformatted for journal publication and has undergone peer review according to Reproductive Health's standard process for supplements and may feature some variations in content when compared to the original report. This co-publication makes the article available to the community in a full-text format. © 2013 Requejo et al.; licensee BioMed Central Ltd.

To examine the effect of interleukin-10 on production and regulation of gelatinases by amniochorion in an in vitro model of infection.We placed amniochorionic membranes collected from eight women who had elective repeat cesareans at term in an organ explant culture system. After 48 hours in culture, the membranes were stimulated with lipopolysaccharide (50 ng/mL), and some were costimulated with interleukin-10 (500 ng/mL). Tissue and media samples were collected after 24-hour stimulation. Quantitative polymerase chain reactions and enzyme-linked immunosorbent assays were used to evaluate matrix metalloproteinase 2 and matrix metalloproteinase 9 messenger RNA and proteins, respectively.Lipopolysaccharide stimulation induced 55.14 transcripts of matrix metalloproteinase 9, compared with 0.83 in control tissues (P.001). Costimulation with interleukin-10 and lipopolysaccharide significantly reduced matrix metalloproteinase 9 messenger RNA levels to 10 transcripts (P.001). Lipopolysaccharide stimulation produced 29.25 ng/mL of immunoreactive matrix metalloproteinase 9, which was reduced to 6.3 ng/mL (P(adj) =.016) after costimulation with interleukin-10. Although not significant, matrix metalloproteinase 2 messenger RNA levels were higher in lipopolysaccharide-stimulated tissues (4.37 x 10(6) transcripts) compared with control (2.8 x 10(5) transcripts; P(adj) =.08), with a significant decrease in matrix metalloproteinase 2 messenger RNA levels in interleukin-10- costimulated tissues (2.9 x 10(6); P(adj) =.007). Interleukin-10 costimulation resulted in a significant decrease in matrix metalloproteinase 2 protein production (203.1 [lipopolysaccharide] and 149.75 [with interleukin-10]; P(adj).001).Interleukin-10 eliminated lipopolysaccharide induction of matrix metalloproteinase 2 and 9 in amniochorion.

Objective: Lipopolysaccharide and tumor necrosis factor α levels are both elevated in the amniotic fluid of women during infection-associated preterm labor and premature rupture of fetal membranes. Our laboratory has shown that apoptosis is associated with premature rupture of fetal membranes but is not associated with preterm labor. The exact pathway that leads to apoptosis-mediated premature rupture of fetal membranes is still unclear. Because infection and increased inflammatory cytokine response are associated with the majority of cases of premature rupture of fetal membranes, we examined the roles of bacterial lipopolysaccharide and tumor necrosis factor α in inducing the proapoptotic caspase pathway in fetal membranes. Study Design: Amniochorionic membranes collected from women undergoing elective repeat cesarean delivery at term were placed in an organ explant system. At the end of a 48-hour incubation period, membranes were stimulated with lipopolysaccharide (50 ng/mL) and recombinant tumor necrosis factor (50 ng/mL). Total ribonucleic acid extracted from these samples was subjected to reverse transcription and two separate sets of multiple polymerase chain reaction. One set studied the expression of Fas, Fas ligand, caspase 8, Fas-associated death domain, and tumor necrosis factor receptor-associated death domain genes and the second set studied the expression of caspase 2, 4, 6, 7, and 10. Caspase 2, 3, and 9 expression was also studied by reverse transcriptase–polymerase chain reaction. Results: Multiple polymerase chain reactions and reverse transcriptase–polymerase chain reactions documented the induction of Fas and caspase 2, 3, 7, 8, and 9 genes in amniochorion after lipopolysaccharide and tumor necrosis factor stimulation compared with the nonstimulated controls. Neither lipopolysaccharide nor tumor necrosis factor induced Fas ligand expression in human fetal membranes. Caspase 3, 4, and 6, Fas-associated death domain, and tumor necrosis factor receptor–associated death domain expressions were constitutive in all the tissues tested; however, tumor necrosis factor receptor–associated death domain expression appeared stronger in tumor necrosis factor–stimulated tissues. Conclusion: The presence of the signal docking proteins tumor necrosis factor receptor–associated death domain and Fas-associated death domain and the induction of caspase cascade initiators (caspase 2, 8, and 10) and effector caspases (caspase 3, 6, 7, and 9) by lipopolysaccharide and tumor necrosis factor suggest that tumor necrosis factor–tumor necrosis factor receptor–mediated apoptosis may occur in the human fetal membrane. (Am J Obstet Gynecol 2001;184:1392-8.)

Objective: On a clinical level, the etiologies associated with premature rupture of the membranes and preterm labor are virtually identical, though these conditions end in distinctly different events. This study was designed to determine differences between preterm labor and preterm premature rupture of membranes by using molecular markers of extracellular matrix degradation and apoptosis. Study Design: Amniochorion and amniotic fluid samples were collected from gestational age–matched groups of women undergoing cesarean delivery before term. Samples were collected from 2 groups of women, women with premature rupture of membranes and women with preterm labor with no rupture of membranes. Changes in the expression pattern of messenger ribonucleic acid for matrix metalloproteinases (MMP), tissue inhibitor of metalloproteinases (TIMP), and pro-apoptotic (p53 and Bax) and anti-apoptotic (Bcl-2) proteins were identified by quantitative polymerase chain reaction. Enzyme-linked immunosorbent assay was used to determine the levels of these proteins in the amniotic fluid. Multiplex polymerase chain reaction was performed to study the expression of Fas-Fas ligand–associated pro-apoptotic genes. Unpaired nonparametric, 2-tailed Mann-Whitney U test was used to determine statistical significance of quantitative polymerase chain reaction and enzyme-linked immunosorbent assay ( P Results: Quantitative polymerase chain reaction results demonstrated an increased mRNA expression for MMP2, MMP9, and MT1-MMP and a decreased expression for TIMP2 in prematurely ruptured membranes compared with preterm labor membranes. Enzyme-linked immunosorbent assay documented increases in the amniotic fluid concentrations of immunoreactive and bioactive MMP2 and MMP9 and immunoreactive MMP3 and a decreased TIMP2 concentration in fluids obtained from the premature rupture of membranes group compared with the preterm labor group. The pro-apoptotic genes p53 and bax were up-regulated in premature rupture of membranes when compared with preterm labor. Anti-apoptotic gene ( Bcl-2 ) expression was increased in preterm labor membranes compared with prematurely ruptured membranes. Interleukin-18 (a pro-apoptotic cytokine) was increased in the amniotic fluid during premature rupture of membranes compared with preterm labor. Prematurely ruptured membranes also demonstrated fragmented deoxyribonucleic acid and expression of Fas and caspase 8 (apoptosis initiator), which were all absent in preterm labor membranes. Conclusions: We have begun to delineate 2 divergent molecular pathways for premature rupture of membranes and preterm labor. Most likely, this is the beginning of the identification of differences that will become evident with the use of molecular biology. (Am J Obstet Gynecol 184:1399-406.)

Problem: IL-18 is a novel cytokine, which promotes inflammation and apoptosis. This study examines its expression pattern, site of production, and levels in the amniotic fluid (AF) during pregnancy complications such as preterm labor and preterm premature rupture of membranes (pPROM). The ability of IL-18 to induce the Fas–Fas ligand (FasL)/caspase-mediated apoptotic pathway is also studied.

Objective: To estimate the effect of lipopolysaccharide on gelatinases and tissue inhibitors of matrix metalloproteinase 2 (gelatinase inhibitor) balance in human fetal membranes. Methods: Amniochorionic membranes in organ explant were stimulated with 1000 ng/mL lipopolysaccharide for 24 hours after a 48-hour preincubation period. Quantitative competitive polymerase chain reaction (PCR) was done to quantitate messenger RNAs for gelatinase A and B (matrix metalloproteinase 2 and 9) and tissue inhibitor of metalloproteinase 2. Protein levels were assayed by enzyme-linked immunosorbant assay. The molar ratio between gelatinases and tissue inhibitor of metalloproteinase 2 was calculated. Statistical evaluation was done by Mann-Whitney U test. Results: Lipopolysaccharide stimulation produced 3.6 × 106 and 366 transcripts of gelatinase A and B, respectively, compared with only 5.9 × 104 (P = .009) and three transcripts (P = .006), respectively, in the controls. Lipopolysaccharide stimulation released 210 ng/mL compared with 7 ng/mL of gelatinase A and B proteins compared with 120 (P = .01) and 4.6 ng/mL (P = .3) in controls, respectively. Control amniochorion produced 5.7 × 105 transcripts of tissue inhibitor of metalloproteinase 2, whereas lipopolysaccharide stimulation produced 4.1 × 105 transcripts (P = .69). Lipopolysaccharide reduced the release of this inhibitor from 114 ng/mL to 68 ng/mL (P = .007). The molar ratio between gelatinases and tissue inhibitor of metalloproteinase 2 increased from a balanced ratio of 1:1 to 3.1:1 after 1000 ng/mL of lipopolysaccharide. Conclusion: Lipopolysaccharide increased the expression and release of gelatinases and decreased its inhibitor, which shifted the balance in favor of gelatinase activity leading to membrane degradation that predisposes to premature rupture of membranes.

Objective : To determine the expression and site of production of stromelysins in fetal membranes and to measure stromelysin 1 levels in amniotic fluid and amniochorion culture media. Methods : Amniochorionic membranes were cultured from organ explant. Membranes were stimulated with lipopolysaccharide for 24 hours after a 48-hour preincubation period. Membranes were also collected from women after vaginal deliveries. RNA samples from those tissues were subjected to reverse transcriptase—polymerase chain reaction using primers specific for stromelysin 1, stromelysin 2, stromelysin 3, and matrilysin. In situ hybridization and immunohisto-chemistry were used to localize stromelysin mRNA and peptide. Levels of stromelysin 1 in culture media and amniotic fluid collected from women with preterm premature rupture of membranes (PROM) and at term with intact membranes were compared using enzyme-linked immunosorbant assay. Results : Amniochorion in culture and from laboring and nonlaboring women expressed all three stromelysins. In situ hybridization showed stromelysin mRNA in amnion, chorion, and extracellular matrix. Immunohistochemical analysis localized stromelysin 1 protein to those same regions. Amniotic fluid levels of stromelysin 1 were higher in preterm PROM amniotic fluids (median 3.2 ng/mL) compared with term deliveries with intact membranes (median 1.3 ng/mL) ( P = .02). Lipopolysaccharide stimulation in culture increased the release of stromelysin 1 from fetal membranes compared with control (median 70.35 versus 15.8 ng/mL, respectively, P = .05). Conclusion : Human fetal membranes are a source of stromelysins 1, 2, and 3. Increased stromelysin 1 during preterm PROM and in vitro after lipopolysaccharide stimulation suggests a possible effect of that matrix metalloproteinase in PROM.

Specifically, pPROM is directly antecedent to 35-40% of spontaneous PTB and its etiology includes several epidemio- logic, behavioral, environmental, and genetic factors. The interactions of risk factors and overlapping biomolecular pathways make pPROM a complex syndrome. Lack of under- standing of the risk factors and risk-induced pathophysiology for pPROM has hampered our ability to develop diagnostic markers for predicting high-risk pregnancies for pPROM or appropriate intervention strategies to reduce its risk. Human fetal membranes are comprised of a single layer of amnionepitheliallayer thatlinestheinteriorofamnioticcavity and multilayered chorion trophoblast layers connected to maternal decidua. 2 The 2 layers are attached through an extracellular matrix (ECM) region, rich in various types of collagens providing the tensile strength and structural integrity to the membranes. Type IV collagen-rich basement membrane connects the 2 layers to the ECM. Proteolysis, specifically directed to dismantle the structural integrity of the ECM, weakens the membranes, and causes immunological, me- chanical, and functional disruption of the layers resulting in pPROM. 3 In essence, pPROM is a disease of the fetal mem- branes 4 and currently there are no strategies to reduce or manage the risks to minimize its impact on maternal-fetal health. Researchers are engaged in addressing 3 major questions related to pPROM: (1) what are the initiators of proteolytic activity resulting in pPROM? (2) what are the effectors (biochemicals) of proteolysis, membrane weakening, and rupture and can these biochemicals serve as markers to pre- dict risk prior to rupture? and (3) if diagnosed early, how do we reduce the risk of pPROM prior to its occurrence and minimize the impact on maternal-fetal well-being? Careful characterization of the pPROM phenotypes is required to better understand causality and pathophysiology.

PROBLEM: Matrix metalloproteinases play a critical role in fetal membrane extracellular matrix (ECM) homeostasis. Remodeling of the ECM during normal placental development is a balanced activity between various matrix metalloproteinases and their tissue-specific counter- regulatory proteins (tissue inhibitors of matrix metalloproteinases [TIMPs]). We have reported the presence of TIMP-1 and TIMP-2 in placental membranes in culture. In this study we have investigated the membrane expression of TIMP-1 and TIMP-2 during labor and nonlabor conditions and also the presence of two novel TIMP family members (TIMP-3 and TIMP-4). METHOD OF STUDY: Amniochorionic membranes collected from women undergoing Cesarean section and were cultured in an organ explant system. Membranes were also collected from laboring women after vaginal delivery. Samples were subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) using primers specific for TIMP-1, TIMP-2, TIMP-3, and TIMP-4. Localization of TIMP mRNAs was accomplished by in situ hybridization, and peptides were localized by immunocytochemistry. RESULTS: RT-PCR data demonstrated the expression of all the TIMPs in tissues from laboring and nonlaboring women as well as in cultured membranes. TIMP-4 expression was seen in RT-PCR, however, only a faint band was visible in all the tissues tested. In situ hybridization localized the TIMP mRNAs to the amnion, chorion, and to scattered cells in the connective tissue. CONCLUSION: Human fetal membrane cells (amniochorion and decidua) express mRNA for all the TIMPs studied so far.

OBJECTIVE: Amniochorion is a source of interleukin-8 during infection and inflammation. In this study we investigate the role of 2 immunoinhibitory cytokines, transforming growth factor and interleukin-10, in regulating interleukin-8 production from human fetal membranes and define their mechanism of regulation. STUDY DESIGN: Amniochorion was placed in an organ explant system for 72 hours. Tissues were stimulated with lipopolysaccharide (50 ng/mL), lipopolysaccharide plus transforming growth factor-β (50/50, 50/100), transforming growth factor-β (50 and 100 ng/mL), lipopolysaccharide plus interleukin-10 (50/50 and 50/100), and interleukin-10 (50 and 100 ng/mL) in culture. Tissue and media samples were frozen until quantitation of interleukin-8 messenger ribonucleic acid and protein. Quantitation of messenger ribonucleic acid was performed by quantitative competitive polymerase chain reaction and protein by enzyme-linked immunoassay, respectively. RESULTS: Lipopolysaccharide-stimulated tissues produced approximately 6 × 10 6 molecules per microliter of interleukin-8 messenger ribonucleic acid compared with 6 × 10 3 molecules per microliter in controls. Transforming growth factor-β alone and lipopolysaccharide plus transforming growth factor-β stimulation produced 6 × 10 5 and 6 × 10 4 molecules of interleukin-8 messenger ribonucleic acid per microliter, respectively. Tissues stimulated with lipopolysaccharide plus 50 ng/mL interleukin-10 produced approximately 600 molecules per microliter of interleukin-8 messenger ribonucleic acid, whereas no amplifiable messenger ribonucleic acid was detected in tissues treated with lipopolysaccharide plus 100 ng/mL interleukin-10. Tissues treated with interleukin-10 alone produced 6 × 10 3 molecules of messenger ribonucleic acid, similar to control levels. Enzyme-linked immunosorbent assay data showed similar levels of interleukin-8 peptide release from lipopolysaccharide and lipopolysaccharide plus transforming growth factor-β–treated fetal membranes. A dose-dependent decrease in interleukin-8 peptide release was seen in tissues treated with lipopolysaccharide plus interleukin-10, whereas stimulation with transforming growth factor or interleukin-10 alone resulted in interleukin-8 peptide release similar to that of control levels. CONCLUSION: Transforming growth factor-β seems to have no effect on interleukin-8 protein production in the presence of an infectious agent; however, a drop in messenger ribonucleic acid levels was observed. Interleukin-10 in the presence of lipopolysaccharide showed down-regulation of interleukin-8 messenger ribonucleic acid expression and peptide production. These data suggest that fetal membrane interleukin-8 production can be controlled by interleukin-10 during an infectious process. (Am J Obstet Gynecol 1998;179:794-9.)

Objective Group-B Streptococcus has been associated with preterm labor and other pregnancy related complications. This study was performed to evaluate the effect of peptidoglycan polysaccharide (PGPS) derived from a beta hemolytic Streptococcal cell wall on amniochorion cytokine production and to compare PGPS effects with lipopolysaccharide (LPS), which is the Gram negative counterpart of PGPS. Study design Amniochorionic membranes collected from women not in labor, and undergoing elective repeat C-section were placed in an organ explant system. Membranes were stimulated separately with 50 ng/ml of small (100p), large (10s) fractions of PGPS or LPS respectively immediately after collection and after a stabilization period of 48 hrs. Media samples were collected at 3, 6, 9, 12 and 24 hrs for protein analysis after each stimulation. Media samples were analyzed by ELISA for IL-6 and IL-8. Results Both forms of PGPS and LPS stimulated IL-6 and IL-8 production by human fetal membranes. Of note is that LPS stimulated IL-6 to a greater degree than IL-8, while PGPS stimulated IL-8 to a greater degree than IL-6. No statistical difference was seen in the levels of either one of these cytokines for the larger or smaller fragments of PGPS. Time course studies documented a 3-hour lag phase when tissues are stimulated directly after collection which was absent when tissues are stimulated after a 48-hour stabilization period. Conclusion Both PGPS and LPS stimulate cytokine production differently from fetal membranes. This supports the theory that different bacteria may affect the host in contrasting ways which may lead to a distinct host response, i.e. PROM vs. PTL.

Preterm birth (PTB) is a clinically important and heterogeneous syndrome leading to neonatal compromise. This chapter proposes that oxidative stress (OS) and the complex biology of OS may specifically contribute significantly to pathways implicated in PTB, including inflammation, apoptosis, autophagy, senescence, and altered collagen metabolism. This chapter further suggests that the apparent conflict in the association between exposure to OS and PTB seen in epidemiologic studies on the one hand and the apparent lack of effect in antioxidant therapy in clinical trials to improve clinical outcome on the other stems from the need for a more complete understanding of the underlying biology of ROS.

OBJECTIVE: This study was designed to detect the regulatory effect of the immunoinhibitory cytokines interleukin-10 and transforming growth factor-β on the amniochorion production of tumor necrosis factor-α. STUDY DESIGN: Amniochorionic membranes were collected from women undergoing elective repeat cesarean section with no history of infection. Membranes were placed in organ explant culture for 48 hours and then stimulated with lipopolysaccharide (50 ng/ml), lipopolysaccharide plus interleukin-10 (50/50, 50/100 ng/ml), interleukin-10 (50 and 100 ng/ml), lipopolysaccharide plus transforming growth factor-β (50/50 and 50/100 ng/ml), and transforming growth factor-β (50 and 100 ng/ml). At the end of a 24-hour stimulation tissue samples were frozen for ribonucleic acid analysis and media samples were frozen for enzyme-linked immunosorbent assay. Quantitation of the messenger ribonucleic acid was accomplished by quantitative competitive polymerase chain reaction, and tumor necrosis factor-α protein was assayed by use of enzyme-linked immunosorbent assay. RESULTS: Lipopolysaccharide stimulation of fetal membranes produced approximately 60,000 molecules of tumor necrosis factor-α messenger ribonucleic acid, whereas control tissue produced none. Lipopolysaccharide plus interleukin-10 stimulation resulted in a dose-dependent decrease in tumor necrosis factor-α messenger ribonucleic production (transcriptional regulation) to 6000 (50/50) and 600 (50/100) molecules. Enzyme-linked immunosorbent assay performed on media samples from these experiments demonstrated a dose-dependent reduction in tumor necrosis factor-α peptide release. Stimulation of membranes with lipopolysaccharide plus transforming growth factor-β had minimal effects on tumor necrosis factor-α messenger ribonucleic acid and protein production compared with lipopolysaccharide-treated samples. Membranes stimulated with interleukin-10 alone showed no effect on messenger ribonucleic acid or protein levels and remained similar to the levels seen in control tissues. In the absence of lipopolysaccharide, transforming growth factor-β treatment produced a dramatic decrease in tumor necrosis factor-α peptide levels without affecting messenger ribonucleic acid levels. CONCLUSION: In the presence of a stimulatory agent, interleukin-10 down-regulates tumor necrosis factor-α release from cultured human amniochorionic membranes. Transforming growth factor-β seems to have some stimulatory effect on transcription, and no effect on translation was seen with concurrent lipopolysaccharide stimulation. However, down-regulation of tumor necrosis factor-α peptide by transforming growth factor was seen in fetal membranes when not overridden by an inflammatory stimulant. This study suggests that interleukin-10 and transforming growth factor-β can regulate tumor necrosis factor-α release from amniochorion under different conditions and by a different mechanism. (Am J Obstet Gynecol 1997;177:803-9.)

Introduction Nicotinamide adenine dinucleotide phosphate oxidases (NOX 1–5) are enzymes that generate cellular reactive oxygen species (ROS) besides mitochondria and might be important ROS sources associated with pregnancy complications, particularly preterm premature rupture of membranes (pPROM), that has been related to ROS. Objective To characterize NOX enzymes expression in human fetal membranes. Methods Differential expression and localization of NOX isoforms in human fetal membranes collected from women with uncomplicated pregnancies at term, preterm birth (PTB) or pPROM and in vitro in normal term membranes maintained in an organ explant system stimulated with water-soluble cigarette smoke extract (wsCSE) were documented by real time PCR and immunohistochemistry. Results Fetal membranes from term deliveries, PTB and pPROM expressed NOX 2, 3 and 4 mRNAs whereas NOX 1 and 5 were not detected. NOX 2 expression was 2.3-fold higher in PTB than pPROM ( p = 0.005) whereas NOX 3 was 2.2-fold higher in pPROM compared to PTB ( p = 0.04). NOX 2 and 3 expressions at term mimicked pPROM and PTB, respectively. No difference in NOX 4 expression was observed among the studied groups. NOX 2, 3 and 4 were localized to both amniotic and chorionic cells. Expression of NOX 2, 3 and 4 were not significant in wsCSE-stimulated membranes compared to untreated controls. Discussion/conclusions NOX enzymes are present in the fetal membranes and are differentially expressed in PTB and pPROM. Absence of any changes in NOXs expression after wsCSE stimulation suggests ROS generation in the membranes does not always correlate with NOX expression.

The objective of this study was to assess the role of biomarker interactions as predictors of spontaneous preterm birth (PTB) using multifactor dimensionality reduction (MDR) analysis. With MDR, a nonparametric, unsupervised, model-free approach, we tested for biomarker interactions within maternal-fetal compartments in 2 racial groups: African Americans (AA) and Caucasians (C).A total of 36 biomarkers from maternal plasma (MP), cord plasma (CP), and amniotic fluid (AF) were analyzed from 191 patients. The MDR combined attribute selection, construction, and classification to detect biomarker interactions that were assessed for generality and significance using 10× cross-validation and permutation testing. Selected significant interactive models were replicated with additional samples.The interactive model containing interleukin (IL)-2, angiopoietin 2 (ANGPT-2), and IL-6 receptor was significant in AA MP. In AA CP, the IL-8 and tumor necrosis factor (TNF) receptor 1 model was significant. In AA AF, the ANGPT-2 and macrophage inflammatory protein 1 alpha model was significant. Replication of the AA MP model using 54 additional AA MP samples confirmed predictability of these biomarkers. In C AF, interaction was observed between ANGPT-2, monocyte chemotactic protein 3, and TNF-α, but no other interactions were significant in C.Using MDR, we identified biomarker interactions that are predictors of PTB even in the absence of a main effect with a single biomarker.

Objective To develop classification models of demographic/clinical factors and biomarker data from spontaneous preterm birth in African Americans and Caucasians. Design Secondary analysis of biomarker data using multivariate adaptive regression splines (MARS), a supervised machine learning algorithm method. Setting Analysis of data on 36 biomarkers from 191 women was reduced by MARS to develop predictive models for preterm birth in African Americans and Caucasians. Samples Maternal plasma, cord plasma collected at admission for preterm or term labor and amniotic fluid at delivery. Methods Data were partitioned into training and testing sets. Variable importance, a relative indicator (0–100%) and area under the receiver operating characteristic curve (AUC) characterized results. Results Multivariate adaptive regression splines generated models for combined and racially stratified biomarker data. Clinical and demographic data did not contribute to the model. Racial stratification of data produced distinct models in all three compartments. In African Americans maternal plasma samples IL-1RA, TNF-α, angiopoietin 2, TNFRI, IL-5, MIP1α, IL-1β and TGF-α modeled preterm birth (AUC train: 0.98, AUC test: 0.86). In Caucasians TNFR1, ICAM-1 and IL-1RA contributed to the model (AUC train: 0.84, AUC test: 0.68). African Americans cord plasma samples produced IL-12P70, IL-8 (AUC train: 0.82, AUC test: 0.66). Cord plasma in Caucasians modeled IGFII, PDGFBB, TGF-β1, IL-12P70, and TIMP1 (AUC train: 0.99, AUC test: 0.82). Amniotic fluid in African Americans modeled FasL, TNFRII, RANTES, KGF, IGFI (AUC train: 0.95, AUC test: 0.89) and in Caucasians, TNF-α, MCP3, TGF-β3, TNFR1 and angiopoietin 2 (AUC train: 0.94 AUC test: 0.79). Conclusions Multivariate adaptive regression splines models multiple biomarkers associated with preterm birth and demonstrated racial disparity.

Spontaneous preterm birth (PTB

Pregnancy complications are hard to diagnose and treat due to complex etiologies and pathophysiologies. Adding to this difficulty is the observation that the distribution of risk is not even across populations of different geographical ancestry. Pregnancy complications are disproportionately high in African American populations. This chapter provides an overview of various factors that can contribute to higher risk and likely racial disparity.

Preterm birth remains the major cause of neonatal mortality and morbidity, mediated largely by an inflammatory process. The sirtuin (SIRT) family of cellular regulators has been implicated as key inhibitors of inflammation. We have previously reported a role for SIRT1, SIRT2, and SIRT6 in regulating inflammation-induced prolabor mediators. In this study, we determined the effect of term labor and pro-inflammatory cytokines on SIRT3, SIRT4, SIRT5, and SIRT7 expression in human myometrium. Functional studies were also used to investigate the effect of small interfering RNA (siRNA) knockdown of SIRTs in regulating inflammation-induced prolabor mediators. Western blot analysis and qRT-PCR were used to determine SIRT3, SIRT4, SIRT5, and SIRT7 mRNA and protein expression in human myometrium. Small interfering RNA knockdown of SIRT3 in myometrial primary cells determined its role in response to inflammatory stimuli IL1B and TNF. SIRT3 mRNA and protein expression levels were significantly lower in term laboring myometrium compared with term nonlaboring myometrium. There was no effect of labor on SIRT4, SIRT5 or SIRT7 protein expression. The pro-inflammatory cytokines IL1B and TNF significantly decreased levels of SIRT3 mRNA and protein expression. SIRT3 knockdown by siRNA significantly augmented IL1B- and TNF-stimulated IL6, CXCL8, and CCL2 mRNA expression and release; PTGS2 mRNA expression and subsequent PGF2alpha release; the mRNA expression and secretion of the adhesion molecule ICAM1 and the extracellular matrix remodeling enzyme MMP9; and nuclear factor kappa B1 (NFkappaB1) transcriptional activity. In human myometrium, SIRT3 expression decreases with term labor and regulates the mediators involved in the terminal effector pathways of human labor and delivery through the NFkappaB1 pathway.

OBJECTIVE: This study was designed to investigate the expression of inflammatory cytokines (interleukin-1β and interleukin-6) by fetal membranes in response to infection in vivo and to endotoxin in organ culture. Study design: Amniochorionic membranes were collected from infected and uninfected women and analyzed for cytokine messenger ribonucleic acid and protein. Normal membranes were cultured and exposed to endotoxin. Messenger ribonucleic acid expression was analyzed by reverse transcriptase-polymerase chain reaction. Cellular localization of messenger ribonucleic acid and protein was determined by in situ hybridization and immunocytochemical evaluation, respectively. RESULSTS: Messenger ribonucleic acid for interleukin-1β appeared to be increased in infected or endotoxin-stimulated amniochorionic membranes, whereas interleukin-6 messenger ribonucleic acid was only observed in infected membranes or after endotoxin stimulation. Interleukin-1β messenger ribonucleic acid was localized exclusively to chorionic cells, whereas protein was obsreved in both chorion and amnion. Interleukin-6 messenger ribonucleic acid and protein were produced in both amniotic and chorionic cells. CONCLUSION: Amniochorionic membranes are a site of inflammatory cytokine production. These findings may have significance in preterm labor or premature rupture of membranes.

Problem Intra-amniotic pathogens and by-products activate innate immune responses encompassing multitudes of signaling molecules and pathways that can result in spontaneous preterm birth (PTB). This study investigates fetal membrane response to bacterial stimulation using a bioinformatics approach. Method of study Dysregulated biomarker (IL1-β, IL-2, IL-8, IL-10, and TNF-α) data from fetal membranes at term stimulated with Ureaplasma urealyticum, Ureaplasma parvum, Mycoplasma hominis, E. coli, Group B Streptococci, Polyporhans gingivalis, or Gardnerella vaginalis with 50% (v/v) amniotic fluid (AF) were analyzed by Ingenuity Pathway Analysis. Results In racially stratified analysis, networks representing late-stage immune inflammation were seen in African-Americans in AF absence. Inflammation was dominant in AF presence as well. In Caucasians, late-stage immune response was dominant with AF, but not in its absence. Conclusions Fetal membrane biofunctions in response to bacteria reflect early- and late-stage innate immune defenses that vary based on the presence of AF and subject race.

Objective: To determine amniotic fluid myeloperoxidase concentration in women with preterm prelabor rupture of the membranes with microbial invasion of the amniotic cavity and histological chorioamnionitis. Methods: One hundred eightyone women with singleton pregnancies with a gestational age between 24+0 and 36+6 weeks were included in this study. Amniocenteses were performed, and myeloperoxidase concentration in the amniotic fluid was determined using ELISA. Result: Women with microbial invasion of the amniotic cavity had higher median myeloperoxidase concentration than women without this condition (149.2 ng/mL vs. 54.6 ng/mL; p = 0.0006). Women with the presence of histological chorioamnionitis had higher median myeloperoxidase concentration than women without histological chorioamnionitis (103.7 ng/mL vs. 50.0 ng/ mL; p = 0.0001). The presence of both microbial invasion of the amniotic cavity and histological chorioamnionitis was associated with higher median myeloperoxidase concentration (456.0 ng/ mL vs. 52.9 ng/mL; p < 0.0001). The results remained significant after adjusting for gestational age. Conclusions: Increased amniotic fluid myeloperoxidase in microbial invasion of the amniotic cavity and histological chorioamnionitis confirm a role of myeloperoxidase in preterm prelabor rupture of the membranes pathophysiology.

Infection during pregnancy triggers inflammation, which can increase myometrial contractions and the risk of premature labor and delivery. In this study, we assessed the effects of vitamin D, an anti-inflammatory ligand on cytokines, chemokines, toll-like receptors, and contractile-associated proteins on immortalized human myometrial smooth muscle (UtSM) cells stimulated with lipopolysaccharide (LPS), a bacterial endotoxin, or interleukin (IL)-1β and measured Toll-like receptor (TLR)-10 expression in pregnant myometrial tissues. A superarray analysis revealed downregulation of the chemokines monocyte chemoattractant protein (MCP)-1, Chemokine (C-X-C motif) ligand (CXCL)-10, CXCL-11, and chemokine (C-X3-C motif) ligand (CX3CL)-1; the proinflammatory cytokines IL-13 and tumor necrosis factor (TNF)-α; the TLR-4 and -5 and triggering receptor expressed on myeloid cells (TREM)-2 and upregulation of the anti-inflammatory cytokine IL-10, as well as Toll interacting protein (TOLLIP) and TREM-1 in vitamin D-treated UtSM cells. In the presence of LPS, vitamin D caused dose-dependent decreases in the messenger RNA expression of MCP-1, IL-1β, IL-13, TNF-α, TLR-4, and TLR-5, the contractile-associated proteins connexin 43, the oxytocin receptor, and the prostaglandin receptor but caused increases in IL-10 and TLR-10 in UtSM cells. The TLR-10 expression was higher in human myometrial tissue obtained from women at term not in labor compared to labor. Vitamin D also attenuated IL-1β-induced MCP-1, IL-6, connexin 43, cyclooxygenase (COX)-2, and prostaglandin receptor expression. Western analysis showed that vitamin D decreased MCP-1, TLR-4, and connexin 43 in the presence of LPS and decreased connexin 43 in the presence of IL-1β. Our results suggest that vitamin D can potentially decrease infection-induced increases in cytokines and contractile-associated proteins in the myometrium.

Spontaneous preterm birth (PTB) and preterm prelabor rupture of membranes (pPROM) occur more frequently in African-American women than in other racial groups. This may be due to an enhanced inflammatory response to pathogens associated with the condition. It is also possible that amniotic fluid (AF) has different immunomodulatory properties in African-American women that increase their risk of PTB and pPROM. To test this, we cultured fetal membranes from European-American and African-American women with sterile medium (control), Escherichia coli , Gardnerella vaginalis , Group B streptococci (GBS), Polyporphorans gingivalis , Mycoplasma hominis , Ureaplasma urealyticum or Ureaplasma parvum in the presence and absence of 50% autologous AF. Cytokine concentrations were quantified in the conditioned medium. All bacterial species increased IL-8 production. IL-1β and TNF-α production were stimulated by LPS, E. coli , and G. vaginalis compared with control, but responses to Group B streptococci and P. gingivalis were limited to IL-1β and TNF-α respectively. Genital mycoplasmas stimulated TNF-α and IL-10 but had no effect on IL-1β production. African-Americans had twice the IL-1β response to E. coli as European-Americans ( P =0.031). Conversely, European-Americans produced more IL-8 in response to LPS than African-Americans ( P =0.026). AF had both pro- and anti-inflammatory properties that varied between races and pathogens. These results suggest that the host response to fetal membrane infections is complex and not generalizable. Interventions to prevent PTB and pPROM may need to be customized based on a patient's race, type of bacterial infection and factors in her AF.

Aims: To document racial disparity in an immune modulator, hyaluronan (HA), an antimicrobial, histone H2B (H2B), and an antioxidant, superoxide dismutase (SOD) in amniotic fluid (AF) from African-American (AA) and Caucasian (C) subjects with spontaneous preterm birth (PTB). Methods: In a case (PTB) control (term) study, AF samples were analyzed for HA, H2B and SOD by ELISA. Differences in analyte concentrations between races were documented and a secondary analysis based on histologic chorioamnionitis (HC) was also performed. Results: No differences in the median HA, H2B and SOD were seen between cases and controls. AA cases had lower HA but higher H2B and SOD than controls. Analyte concentrations were not different between C cases and controls. AA samples at term had lower H2B and SOD compared to C samples at term. Cases with HC showed higher H2B and SOD. Conclusion: We report ethnic disparity in AF antimicrobial, immune mediator and antioxidant factors. Dysregulated AF production of HA, H2B and SOD was associated with PTB in AA, not in C, suggesting an overwhelming inflammatory response in AA PTB, whereas inflammation is likely a secondary phenomenon in C PTB.

Problem Preterm premature rupture of fetal membranes (pPROM) occurs in 30–40% of spontaneous preterm births (PTB) and is associated with intra-amniotic infection and inflammation. The membranes may sense and respond to microbes via Toll-like receptors (TLRs); however, little is known about their expression and regulation in this tissue. The objective of this study was to evaluate the expression of TLRs 1–10 in fetal membranes after exposure to pathogens associated with intra-amniotic infection and PTB. Method of study Normal human term fetal membrane explants were exposed to various bacteria. After 24 hrs, RNA was extracted and quantitative RT-PCR performed for TLRs1–10. Results Treatment of fetal membranes with Mycoplasma hominis increased expression of TLR4, TLR6, and TLR8 mRNA. Ureaplasma parvum upregulated TLR8 mRNA, and Porphyromonas gingivalis significantly increased fetal membrane TLR7 expression. In contrast, treatment with Gram-negative Escherichia coli (and its cell wall component lipopolysaccharide) downregulated TLR10 mRNA. No effect was detected for Ureaplasma urealyticum, Gardnerella vaginalis, or Group B Streptococcus. Conclusion These findings demonstrate that different types of bacteria have distinct effects on fetal membrane TLR expression patterns. Moreover, these findings highlight the disparity of fetal membrane responses to infection and thus suggest heterogeneity in the mechanisms by which infection-associated pregnancy complications, such as pPROM and PTB, arise.

Despite the increasing speculation that oxidative stress and abnormal energy metabolism may play a role in Autism Spectrum Disorders (ASD), and the observation that patients with mitochondrial defects have symptoms consistent with ASD, there are no comprehensive published studies examining the role of mitochondrial variation in autism. Therefore, we have sought to comprehensively examine the role of mitochondrial DNA (mtDNA) variation with regard to ASD risk, employing a multi-phase approach. In phase 1 of our experiment, we examined 132 mtDNA single-nucleotide polymorphisms (SNPs) genotyped as part of our genome-wide association studies of ASD. In phase 2 we genotyped the major European mitochondrial haplogroup-defining variants within an expanded set of autism probands and controls. Finally in phase 3, we resequenced the entire mtDNA in a subset of our Caucasian samples (∼400 proband-father pairs). In each phase we tested whether mitochondrial variation showed evidence of association to ASD. Despite a thorough interrogation of mtDNA variation, we found no evidence to suggest a major role for mtDNA variation in ASD susceptibility. Accordingly, while there may be attractive biological hints suggesting the role of mitochondria in ASD our data indicate that mtDNA variation is not a major contributing factor to the development of ASD.

Micronutrient deficiencies are hypothesized to play a role in spontaneous preterm birth (PTB;37 weeks of gestation) and possibly the racial disparity in rates of PTB between black and white women. Yet relatively few studies have addressed the role of micronutrient deficiencies in spontaneous PTB among black and white women in the United States. The purpose of this study was to investigate whether 25-hydroxy vitamin D (25-OH-D), folate, and omega-6/omega-3 fatty acid status are associated with spontaneous PTB among black and white women in the United States.Biospecimens and medical record data for this study were derived from a subsample of the 1547 women enrolled into the Nashville Birth Cohort during 2003-2006. We randomly selected 80 nulliparous and primiparous women for whom stored plasma samples from the delivery admission were available and analyzed the stored plasma for 25-OH-D, folate, and total omega-6/omega-3 fatty acids. We used multivariate logistic regression to assess the odds of spontaneous PTB among women with 25-OH-D20 ng/mL, folate5 ug/L, and omega-6/omega-315.An omega-6/omega-3 ratio15 was significantly associated with spontaneous PTB for white (adjusted odds ratio [aOR] 4.25, 95% confidence interval [CI] 1.25-14.49) but not black women (aOR 1.90, 95% CI: 0.69-5.40), whereas no significant relationships were observed for folate and 25-OH-D status and PTB for black or white women.Maternal plasma total omega-6/omega-3 fatty acid ratio15 at delivery was significantly associated with spontaneous PTB for white, but not black, women.

Objective The objective of the study was to determine the effect of statins on lipopolysaccharide (LPS)-induced inflammatory response in a mouse model of preterm birth (PTB). Study Design Day 15 CD1 mice were randomly allocated to intraperitoneal LPS injection (100 μg) or control. Mice in the LPS group were pretreated, 16 and 2 hours prior, with pravastatin (10 μg/g), simvastatin (10 μg/g), or vehicle control. Animals were sacrificed 6 hours after LPS. Cytokine messenger ribonucleic acid (mRNA) expression in the uterus and cervix, and concentrations in the maternal serum and amniotic fluid (AF) were determined. Results Pravastatin reduced interleukin (IL)-1β and IL-6 mRNA expression in the uterus and cervix, respectively, and serum IL-1β and granulocyte-macrophage colony-stimulating factor (GM-CSF) concentrations. Simvastatin reduced IL-1β and IL-6 mRNA expressions in the uterus, IL-6 and tumor necrosis factor alpha (TNF-α) in the cervix, and IL-1β, IL-2, IL-12p70, IL-13, TNF-α, GM-CSF, and interferon-γ concentrations in the serum and IL-6 in AF. Conclusion Statins reduce the LPS-induced inflammatory responses in a mouse model of PTB.

Catechol-O-methyltransferase (COMT) activity has been reported to be higher in African Americans (AA) than Caucasians (Cau). COMT converts 2- and 4-hydroxy (OH) estrogens to 2- and 4-methoxyestrogens, respectively, and can increase estrogenic milieu locally in tissues. To assess whether the increased incidence of preterm birth (PTB) among AA women is associated with single-nucleotide polymorphism (SNP) in the COMT gene, we examined variations in maternal and fetal COMT genes and their association with pregnancy outcomes (term vs preterm pregnancies) using 4 functional SNPs: rs4633, rs4680, rs4818, and rs6269 in both AA and Cau. We analyzed samples from 267 AA women (191 term and 76 preterm pregnancies) and 339 Cau (194 term and 145 preterm pregnancies) in this study. The results showed a significant difference (P < .05) in allele and genotype frequencies between term and preterm AA and Cau women in 3 SNPs in both maternal and fetal DNA. The analysis revealed that in AA fetal COMT genes, SNP rs4818 is associated with PTB at the allele (C; P < .001), genotype (C/C; P < .01), and 2- (P < .03) and 3 (P < .04)-window haplotype levels. Multidimensionality reduction analysis also showed a significant (P < .01) association between rs4818 and PTB. In conclusion, our study demonstrated that a synonymous polymorphism, rs4818 in the fetal COMT gene, is associated with PTB in AA.

PROBLEM: Preterm labor and PROM are major complications of pregnancy. We have reported the possible role of amniochorionic membrane as it relates to the production of cytokines and the early onset of labor. Amniochorion is capable of responding to an infectious process with the production of IL-6 and IL-1β. Here we examine the expression of TNF-α and TNFR in amniochorion. METHOD: Amniochorionic membranes were collected and maintained in an organ explant system. Samples were frozen and/or fixed for RT-PCR, in situ hybridization, and immunocytochemistry. RESULTS: RT-PCR demonstrated mRNA for TNF-α and in situ hybridization localized mRNA in chorion and amnion. Immunocytochemistry demonstrated TNF-α peptide in amnion but not in chorion. Immunocytochemical localization of TNFR indicates presence of that peptide in both amnion and chorion. CONCLUSIONS: We conclude that the fetal membranes are sources of TNF-α and TNFR, supporting our previous work indicating that fetal membranes are active participants in the response to intraamniotic infection.

Objective. To investigate associations among serum 25-hydroxy-vitamin D (25-OH-D), folate, omega-6/omega-3 fatty acid ratio and bacterial vaginosis (BV) during pregnancy.Methods. Biospecimens and data were derived from a random sample (N=160) of women from the Nashville Birth Cohort. We compared mean plasma nutrient concentrations for women with and without BV during pregnancy (based on Nugent score ≥7) and assessed the odds of BV for those with 25-OH-D 15.Results. The mean plasma 25-OH-D was significantly lower among women with BV during pregnancy (18.00±8.14 ng/mL versus24.34±11.97 ng/mL,P=0.044). The adjusted odds of BV were significantly increased among pregnant women with 25-OH-D Conclusion. Vitamin D and folate deficiencies were strongly associated with BV (Nugent score ≥7) during pregnancy.

Understanding spontaneous preterm birth ([PTB] < 37 weeks) is difficult due to heterogeneities associated with multitudes of risk factors and pathophysiological pathways. Several biomarkers are routinely used clinically for predicting preterm labor; however, these factors are either nonspecific or detected too late. Systematic review of literature on PTB biomarkers in the last 40 years to map out the existing knowledge and gaps in understanding PTB biomarkers. Search strategies: Five electronic databases were searched for human studies on PTB biomarkers published in any language between 1965 and 2008. Selection criteria: The phenotype of interest for final data extraction was exclusively spontaneous PTB with no rupture of membranes. Data extraction included (a) general characteristics of the study (clinical setting, period, and study design), (b) study/participant characteristics (inclusion and exclusion criteria, race/ethnicity, number of participants, gestational age at sampling, (c) characteristics of the biomarker (type, rationale for its selection, type of biological sample, and assay used, and (d) concentration of biomarkers in cases and controls. Data collection and analysis: The search yielded 7255 citations and data were extracted from 217 articles which met our inclusion and exclusion criteria. A total of 116 different biomarkers were reported and these were assayed 578 times in the 217 included studies. Over two thirds of the 217 studies were performed on North American or European populations. No reliable biomarkers emerged as a risk predictor of PTB. Identifying similar studies on biomarkers for the prediction of PTB was a very challenging task due heterogeneities in study design, sampling issues (types, timing and processing), assay methods, and analyses. Major areas of concern identified in this review include poor phenotype definition, nonideal study designs and poor rationale for biomarker selection and assays and population stratification issues.

To give an overview of the literature for evidence of nutrient deficiencies as contributors to the disparity in preterm birth (PTB) between African-American and Caucasian women.Structured literature survey.We searched MEDLINE to identify observational and experimental studies that evaluated the relation between nutrient intake and/or supplementation and PTB. For nutrients for which studies supported an association, we searched MEDLINE for studies of the prevalence of deficiency in the USA by race.Summarized findings on nutrients for which there is both evidence of a role in PTB and variability in the prevalence of deficiency by race.Nutrient deficiencies for which there are varying levels of evidence for an association with PTB and a greater burden among African-American compared with Caucasian women include deficiencies of iron, folic acid, zinc, vitamin D, calcium and magnesium, and imbalance of ω-3 and ω-6 polyunsaturated fatty acids. There are inadequate high-quality studies that investigate the role of nutrient deficiencies in PTB, their potential interaction with other risks, the proportion of excess risk for which they account, and whether supplementation can reduce the risk of, and racial disparities in, PTB in US populations.Deficiencies of several nutrients have varying levels of evidence of association with PTB and are of greater burden among African-American compared with Caucasian women. Although further research is needed, strategies that improve the nutritional status of African-American women may be a means of addressing a portion of the racial disparity in PTB.

To investigate differences in pre-pregnancy BMI status in patients with spontaneous preterm birth (PTB) compared with term birth and assess the role of ethnicity as a risk modifier in BMI-associated PTB.A case-control study involving self-reported African American and Caucasian women delivering singletons in Nashville, TN, USA, 2003-2009. Maternal pre-pregnancy BMI was recorded in 447 PTB-cases (African American = 145, Caucasian = 302) and 1315 term-birth controls (African American = 522; Caucasian = 793). Crude and adjusted odds ratio (OR and AOR) for PTB were calculated using normal BMI (18.5-24.9 kg/m(2)) as reference. Age, education, marital status, income, smoking, parity, previous PTB and pregnancy weight gain were included as covariates in logistic regression.No significant differences were noted in the OR for PTB among different BMI categories when women of different ethnicity were combined. Odds of PTB were greater in obese than in normal weight Caucasian women, even after adjusting for confounders (AOR = 1.84, 95%CI [1.15, 2.95]). Obese African American women had a decreased crude OR for PTB, although this was not significant after adjusting for confounders (AOR = 0.72, 95%CI [0.38, 1.40]). The odds for early PTB (32 weeks) were decreased in obese compared with normal weight African American women (OR = 0.23, 95%CI [0.08, 0.70]), whereas they were increased in obese compared with normal weight Caucasian women (OR = 2.30, 95%CI [1.32, 4.00]).The risk for PTB in women with different pre-pregnancy BMI categories differs according to ethnicity.

Fetal membranes overlying the cervix (i.e. supracervical site, SCS) are characterised by increased extracellular matrix (ECM) degradation. In non-gestational tissues, the mitogen activated protein kinase (MAPK) and activator protein (AP)-1 family are involved in the regulation of the ECM degrading enzyme metalloproteinase (MMP)-9. The aims of this study were (i) to compare the expression of AP-1 proteins in fetal membranes from the SCS and a distal site (DS), and (ii) determine if the MAPK/AP-1 pathway is involved in the regulation of MMP-9. Fetal membranes overlying the cervix were identified in situ in women undergoing term elective Caesarean section. Immunohistochemistry (n = 6) was used to localise the expression of the MAPK proteins ERK (total and phosphorylated), JNK (total and phosphorylated) and p38 MAPK (total and phosphorylated), and the AP-1 proteins JunB, cJun (total and phosphorylated), JunD, cFos and FosD. There was no difference in JNK, p38 MAPK, FosB, cJun and JunD protein expression between SC and distal fetal membranes. However, when compared to DS, the intensity and/or extent of staining of ERK, p-ERK, p-JNK, p-p38 MAPK, cFos, JunB and p-cJun were greater in amnion and chorion obtained from the SCS. In order to elucidate a role for these proteins in ECM degradation, pharmacological inhibitors of MAPK protein activation were utilised in primary amnion cells. The ERK inhibitor U0126, JNK inhibitor SP600125 and p38 MAPK inhibitor SB202190 all significantly decreased IL-β-induced MMP-9 gene expression and pro MMP-9 in human primary amnion cells. In summary, at term, non laboured SC fetal membranes are characterised by increased expression of MAPK and AP-1 proteins. MMP-9 expression and production was significantly suppressed by inhibitors of three key enzymes in the signalling cascades leading to AP-1 formation, ERK 1/2, JNK and p38 MAPK. Thus, the MAPK/AP-1 pathway may play a role in the degradation of the ECM at the SCS making it more susceptible to membrane rupture.

Background Although adolescent idiopathic scoliosis affects approximately 3% of adolescents, the genetic contributions have proven difficult to identify. Work in model organisms, including zebrafish, chickens, and mice, has implicated the lysyl oxidase family of enzymes in the development of scoliosis. We hypothesized that common polymorphisms in the five human lysyl oxidase genes (LOX, LOXL1, LOXL2, LOXL3, and LOXL4) may be associated with the phenotype of adolescent idiopathic scoliosis. Methods This was a case-control genetic association study. A total of 112 coding and tag SNPs in LOX, LOXL1, LOXL2, LOXL3, and LOXL4 were genotyped in a discovery cohort of 138 cases and 411 controls. Genotypes were tested for association with adolescent idiopathic scoliosis by logistic regression with a two degree of freedom genotypic model and gender as a covariate. Fourteen SNPs with p < 0.1 in the discovery phase were genotyped in an independent replication cohort of 400 cases and 506 controls. Results No evidence for significant association was found between coding or tag SNPs in LOX, LOXL1, LOXL2, LOXL3, and LOXL4 and the phenotype of adolescent idiopathic scoliosis. Conclusions Despite suggestive evidence in model organisms, common variants and known coding SNPs in the five human lysyl oxidase genes do not confer increased genotypic risk for adolescent idiopathic scoliosis. The above methodology does not address rare variants or individually private mutations in these genes, and future research may focus on this area.

Infection has been hypothesized to be one of the factors associated with spontaneous preterm birth (PTB) and with the racial disparity in rates of PTB between African American and Caucasian women. However, recent findings refute the generalizability of the role of infection and inflammation. African Americans have an increased incidence of PTB in the setting of intraamniotic infection, periodontal disease, and bacterial vaginosis compared to Caucasians. Herein we report variability in infection- and inflammation-related factors based on race/ethnicity. For African American women, an imbalance in the host proinflammatory response seems to contribute to infection-associated PTB, as evidenced by a greater presence of inflammatory mediators with limited or reduced presence of immune balancing factors. This may be attributed to differences in the genetic variants associated with PTB between African Americans and Caucasians. We argue that infection may not be a cause of racial disparity but in association with other risk factors such as stress, nutritional deficiency, and differences in genetic variations in PTB, pathways and their complex interactions may produce differential inflammatory responses that may contribute to racial disparity.