Questioning the fetal microbiome illustrates pitfalls of low-biomass microbial studies.

Whether the human fetus and the prenatal intrauterine environment (amniotic fluid and placenta) are stably colonized by microbial communities in a healthy pregnancy remains a subject of debate. Here we evaluate recent studies that characterized microbial populations in human fetuses from the perspectives of reproductive biology, microbial ecology, bioinformatics, immunology, clinical microbiology and gnotobiology, and assess possible mechanisms by which the fetus might interact with microorganisms. Our analysis indicates that the detected microbial signals are likely the result of contamination during the clinical procedures to obtain fetal samples or during DNA extraction and DNA sequencing. Furthermore, the existence of live and replicating microbial populations in healthy fetal tissues is not compatible with fundamental concepts of immunology, clinical microbiology and the derivation of germ-free mammals. These conclusions are important to our understanding of human immune development and illustrate common pitfalls in the microbial analyses of many other low-biomass environments. The pursuit of a fetal microbiome serves as a cautionary example of the challenges of sequence-based microbiome studies when biomass is low or absent, and emphasizes the need for a trans-disciplinary approach that goes beyond contamination controls by also incorporating biological, ecological and mechanistic concepts.

Antibody microarray analysis of amniotic fluid proteomes in women with cervical insufficiency and short cervix, and their association with pregnancy latency length.

INTRODUCTION: This study aimed to investigate amniotic fluid (AF) proteins that were differentially expressed between patients with cervical insufficiency (CI) and asymptomatic short cervix (SCX, ≤ 25 mm), and whether these proteins could be predictive of spontaneous preterm birth (SPTB) in these patients. METHOD: This was a retrospective cohort study of 129 singleton pregnant women with CI (n = 80) or SCX (n = 49) at 17 to 26 weeks who underwent amniocentesis. An antibody microarray was used to perform comparative proteomic profiling of AF from matched CI (n = 20) and SCX (n = 20) pregnancies. In the total cohort, an ELISA validation study was performed for 15 candidate proteins of interest. Subgroup analyses of patients with CI and SCX were conducted to evaluate the association between the 15 proteins and SPTB at < 32 weeks of gestation. RESULTS: Eighty-six proteins showed intergroup differences. ELISA validation confirmed significantly higher levels of AF EN-RAGE, IL-8, lipocalin-2, MMP-9, S100A8/A9, thrombospondin-2, and TNFR2 in patients with CI than in those with SCX. Multivariable analysis showed that increased AF levels of EN-RAGE, S100A8/A9, and uPA were independently associated with SPTB at < 32 weeks in patients with CI; whereas in patients with SCX, high AF levels of APRIL, EN-RAGE, LBP, and TNFR2 were independently associated with SPTB at < 32 weeks. CONCLUSIONS: Multiple AF proteins show altered expression in patients with CI compared with SCX controls. Moreover, several novel mediators involved in inflammation were identified as potential biomarkers for predicting SPTB after the diagnosis of CI and SCX. These results provide new insights into target-specific molecules for targeted therapies to prevent SPTB in patients with CI/SCX.

The Role of Innate Immune System in the Human Amniotic Membrane and Human Amniotic Fluid in Protection Against Intra-Amniotic Infections and Inflammation.

Intra-amniotic infection and inflammation (IAI) affect fetal development and are highly associated with preterm labor and premature rupture of membranes, which often lead to adverse neonatal outcomes. Human amniotic membrane (hAM), the inner part of the amnio-chorionic membrane, protects the embryo/fetus from environmental dangers, including microbial infection. However, weakened amnio-chorionic membrane may be breached or pathogens may enter through a different route, leading to IAI. The hAM and human amniotic fluid (hAF) respond by activation of all components of the innate immune system. This includes changes in 1) hAM structure, 2) presence of immune cells, 3) pattern recognition receptors, 4) cytokines, 5) antimicrobial peptides, 6) lipid derivatives, and 7) complement system. Herein we provide a comprehensive and integrative review of the current understanding of the innate immune response in the hAM and hAF, which will aid in design of novel studies that may lead to breakthroughs in how we perceive the IAI.

High Mobility Group Box 1 in Pig Amniotic Membrane Experimentally Infected with E. coli O55.

Intra-amniotic infections (IAI) are one of the reasons for preterm birth. High mobility group box 1 (HMGB1) is a nuclear protein with various physiological functions, including tissue healing. Its excessive extracellular release potentiates inflammatory reaction and can revert its action from beneficial to detrimental. We infected the amniotic fluid of a pig on the 80th day of gestation with 1 × 104 colony forming units (CFUs) of E. coli O55 for 10 h, and evaluated the appearance of HMGB1, receptor for glycation endproducts (RAGE), and Toll-like receptor (TLR) 4 in the amniotic membrane and fluid. Sham-infected amniotic fluid served as a control. The expression and release of HMGB1 were evaluated by Real-Time PCR, immunofluorescence, immunohistochemistry, and ELISA. The infection downregulated HMGB1 mRNA expression in the amniotic membrane, changed the distribution of HMGB1 protein in the amniotic membrane, and increased its level in amniotic fluid. All RAGE mRNA, protein expression in the amniotic membrane, and soluble RAGE level in the amniotic fluid were downregulated. TLR4 mRNA and protein expression and soluble TLR4 were all upregulated. HMGB1 is a potential target for therapy to suppress the exaggerated inflammatory response. This controlled expression and release can, in some cases, prevent the preterm birth of vulnerable infants. Studies on suitable animal models can contribute to the development of appropriate therapy.

Bacteria in the amniotic fluid without inflammation: early colonization vs. contamination.

OBJECTIVES: Intra-amniotic infection, defined by the presence of microorganisms in the amniotic cavity, is often accompanied by intra-amniotic inflammation. Occasionally, laboratories report the growth of bacteria or the presence of microbial nucleic acids in amniotic fluid in the absence of intra-amniotic inflammation. This study was conducted to determine the clinical significance of the presence of bacteria in amniotic fluid samples in the absence of intra-amniotic inflammation. METHODS: A retrospective cross-sectional study included 360 patients with preterm labor and intact membranes who underwent transabdominal amniocentesis for evaluation of the microbial state of the amniotic cavity as well as intra-amniotic inflammation. Cultivation techniques were used to isolate microorganisms, and broad-range polymerase chain reaction coupled with electrospray ionization mass spectrometry (PCR/ESI-MS) was utilized to detect the nucleic acids of bacteria, viruses, and fungi. RESULTS: Patients whose amniotic fluid samples evinced microorganisms but did not indicate inflammation had a similar perinatal outcome to those without microorganisms or inflammation [amniocentesis-to-delivery interval (p=0.31), spontaneous preterm birth before 34 weeks (p=0.83), acute placental inflammatory lesions (p=1), and composite neonatal morbidity (p=0.8)]. CONCLUSIONS: The isolation of microorganisms from a sample of amniotic fluid in the absence of intra-amniotic inflammation is indicative of a benign condition, which most likely represents contamination of the specimen during the collection procedure or laboratory processing rather than early colonization or infection.

Imbalance between inflammatory and regulatory cord blood B cells following pre-term birth.

Preterm birth (PTB) is one of the most frequent pregnancy complications. It affects millions of babies each year worldwide and is associated with increased morbidity and mortality. PTB-associated alterations in the maternal immune response may have a direct effect on the developing fetal immune system. Having recently shown that B regulatory (Breg) cells are decreased in number and functionally impaired in maternal blood from women delivering preterm, we now addressed the question whether the adaptive immune system is also altered in cord blood (CB) after the onset of PTB. PTB was associated with increased concentrations of IL-6, TNF-α and IL-21 in CB and enhanced IL-6, but decreased IFN-γ and IL-4 in amniotic fluid (AF) samples compared to term delivery (TD). We found no differences in the frequency of CD19 + B cells, CD4 + T cells or CD4+Foxp3+CD25+ T regulatory (Treg) cells in CB cells in PTB vs TD. The frequency of CD86 + B cells was increased, while the percentage of CD24hiCD38hiCD19 + Breg and CD1dhiCD5+ Breg cells and the ability of B cells to convert into Breg cells was diminished in PTB compared to TD. CB B cells from PTB secreted more IL-6, TNF-α, IL-9 and IL-2 compared to B cells obtained from term samples. We conclude that, after PTB onset, a shift from immunoregulation towards inflammation takes place in CB cells that are reportedly representative of the fetal compartment. B cells have a substantial contribution herein. This phenomenon might account for the observed enhanced mortality and morbidity in prematurely born infants. Further studies will clarify how to employ this easy-to-obtain information for closely monitoring newborns at risk.

Amniotic Fluid Proteasome and Immunoproteasome in the Setting of Intra-Amniotic Infection, Inflammation, and Preterm Birth.

Preterm birth is an important determinant of neonatal morbidity and mortality and intra-amniotic infection (IAI) and inflammation play a causative role. The constitutive proteasome and immunoproteasome are key players in maintenance of proteostasis and their alteration outside pregnancy has been linked to pathogenesis of numerous inflammatory diseases. Our goal was to evaluate the levels, activities, and potential origin of amniotic fluid (AF) proteasome in women with preterm birth induced by infection and/or inflammation. Total proteasome and immunoproteasome concentrations were measured in AF retrieved by trans-abdominal amniocentesis from 155 pregnant women. Proteasome activities were measured with fluorogenic substrates targeting caspase-like (CAS-L), trypsin-like (TRY-L), or chymotrypsin-like (CHE-L) lytic activities. We found that IAI significantly upregulated AF concentrations of total proteasome and of the immunoproteasome (P<0.001 for both) with no differences based on gestational age. Based on substrate preference and profile of pharmacologic inhibition, we identified the CHE-L activity of the immunoproteasome as the primary lytic activity upregulated in AF of pregnancies complicated by IAI. When compared with matched maternal blood and cord blood, proteasome activity was by far the highest in AF and this was further elevated in IAI. Western blot confirmed ß5 (PSMB5) and ß5i (PSMB8) subunits of the constitutive proteasome and immunoproteasome are present in AF and IHC staining of fetal membranes pointed to chorio-decidua as a potential source. In conclusion, IAI is associated with increased AF immunoproteasome activity that by analogy with other inflammatory diseases may generate antigenic oligopeptides and may play a role in triggering preterm birth.

Daily monitoring of vaginal interleukin 6 as a predictor of intraamniotic inflammation after preterm premature rupture of membranes - a new method of sampling studied in a prospective multicenter trial.

OBJECTIVES: (A) To introduce a new technique for vaginal fluid sampling (biocompatible synthetic fiber sponge) and (B) evaluate the collected vaginal fluid interleukine-6 (IL-6vag)-concentration as a new diagnostic tool for daily monitoring of intrauterine inflammation after preterm premature rupture of membranes (PPROM). Secondary objectives were to compare the potential to predict an intrauterine inflammation with established inflammation parameters (e.g., maternal white blood cell count). METHODS: This prospective clinical case-control diagnostic accuracy multicenter study was performed with women after PPROM (gestational age 24.0/7 - 34.0/7 weeks). Sampling of vaginal fluid was performed once daily. IL-6vag was determined by electrochemiluminescence-immunoassay-kit. Neonatal outcome and placental histology results were used to retrospectively allocate the cohort into two subgroups: 1) inflammation and 2) no inflammation (controls). RESULTS: A total of 37 cases were included in the final analysis. (A): Measurement of IL-6 was successful in 86% of 172 vaginal fluid samples. (B): Median concentration of IL-6vag in the last vaginal fluid sample before delivery was significantly higher within the inflammation group (17,085 pg/mL) compared to the controls (1,888 pg/mL; p=0.01). By Youden's index an optimal cut-off for prediction an intrauterine inflammation was: 6,417 pg/mL. Two days before delivery, in contrast to all other parameters IL-6vag remained the only parameter with a sufficient AUC of 0.877, p<0.001, 95%CI [0.670-1.000]. CONCLUSIONS: This study established a new technique for vaginal fluid sampling, which permits assessment of IL-6vag concentration noninvasively in clinical daily routine monitoring.

Clinical chorioamnionitis at term X: microbiology, clinical signs, placental pathology, and neonatal bacteremia - implications for clinical care.

OBJECTIVES: Clinical chorioamnionitis at term is considered the most common infection-related diagnosis in labor and delivery units worldwide. The syndrome affects 5-12% of all term pregnancies and is a leading cause of maternal morbidity and mortality as well as neonatal death and sepsis. The objectives of this study were to determine the (1) amniotic fluid microbiology using cultivation and molecular microbiologic techniques; (2) diagnostic accuracy of the clinical criteria used to identify patients with intra-amniotic infection; (3) relationship between acute inflammatory lesions of the placenta (maternal and fetal inflammatory responses) and amniotic fluid microbiology and inflammatory markers; and (4) frequency of neonatal bacteremia. METHODS: This retrospective cross-sectional study included 43 women with the diagnosis of clinical chorioamnionitis at term. The presence of microorganisms in the amniotic cavity was determined through the analysis of amniotic fluid samples by cultivation for aerobes, anaerobes, and genital mycoplasmas. A broad-range polymerase chain reaction coupled with electrospray ionization mass spectrometry was also used to detect bacteria, select viruses, and fungi. Intra-amniotic inflammation was defined as an elevated amniotic fluid interleukin-6 (IL-6) concentration ≥2.6 ng/mL. RESULTS: (1) Intra-amniotic infection (defined as the combination of microorganisms detected in amniotic fluid and an elevated IL-6 concentration) was present in 63% (27/43) of cases; (2) the most common microorganisms found in the amniotic fluid samples were Ureaplasma species, followed by Gardnerella vaginalis; (3) sterile intra-amniotic inflammation (elevated IL-6 in amniotic fluid but without detectable microorganisms) was present in 5% (2/43) of cases; (4) 26% of patients with the diagnosis of clinical chorioamnionitis had no evidence of intra-amniotic infection or intra-amniotic inflammation; (5) intra-amniotic infection was more common when the membranes were ruptured than when they were intact (78% [21/27] vs. 38% [6/16]; p=0.01); (6) the traditional criteria for the diagnosis of clinical chorioamnionitis had poor diagnostic performance in identifying proven intra-amniotic infection (overall accuracy, 40-58%); (7) neonatal bacteremia was diagnosed in 4.9% (2/41) of cases; and (8) a fetal inflammatory response defined as the presence of severe acute funisitis was observed in 33% (9/27) of cases. CONCLUSIONS: Clinical chorioamnionitis at term, a syndrome that can result from intra-amniotic infection, was diagnosed in approximately 63% of cases and sterile intra-amniotic inflammation in 5% of cases. However, a substantial number of patients had no evidence of intra-amniotic infection or intra-amniotic inflammation. Evidence of the fetal inflammatory response syndrome was frequently present, but microorganisms were detected in only 4.9% of cases based on cultures of aerobic and anaerobic bacteria in neonatal blood.

Maternal environmental enrichment modulates the immune response against an inflammatory challenge during gestation and protects the offspring.

The production of pro-inflammatory cytokines during inflammatory processes has been associated with preterm birth (PTB) and fetal injury in humans and mice. We previously demonstrated that exposition to an enriched environment (EE), defined as a noninvasive and biological significant stimulus of the sensory pathway combined with voluntary physical activity, prevented PTB and perinatal death induced by the systemic administration of bacterial lipopolysaccharide (LPS) in mice. This work aimed to analyze whether EE modulates the immune response to the inflammatory process induced by LPS in peripheral blood and the amniotic fluid (AF). We observed that EE modulated maternal white blood cell count and its response to LPS. Furthermore, we found higher levels of IL-10 and a higher percentage of B cells in AF from EE exposed mothers compared to controls. Albeit LPS significantly increased IL-6 levels in AF from both groups, it was 3.6 times higher in control environment (CE) exposed group when compared to EE. Similarly, levels of IL-22 were significantly increased by LPS in both groups, but it was 6.7 times higher in EE group. Interestingly, levels of PGE2 in AF were only increased in the EE-LPS treated group, and a positive correlation between IL-22 and PGE2 levels was observed. During lactation, EE prevented LPS-induced delay in physical landmarks analyzed to assess offspring development. Our results suggest that EE modulates the immune response to systemic LPS-administration protecting the offspring. We propose that an EE-like protocol could be designed for pregnant women aiming at preventing the sequelae present in premature children.

Interleukin-6 pathobiology in equine placental infection.

PROBLEM: Ascending placentitis is the leading cause of abortion in the horse. Interleukin (IL)-6 is considered predictive of placental infection in other species, but little is understood regarding its role in the pathophysiology of ascending placentitis. METHOD OF STUDY: Sub-acute ascending placentitis was induced via trans-cervical inoculation of S zooepidemicus, and various fluids/serum/tissues collected 8 days later. Concentrations of IL-6 were detected within fetal fluids and serum in inoculated (n = 6) and control (n = 6) mares. RNASeq was performed on the placenta (endometrium and chorioallantois) to assess transcripts relating to IL-6 pathways. IHC was performed for immunolocalization of IL-6 receptor (IL-6R) in the placenta. RESULTS: IL-6 concentrations increased in allantoic fluid following inoculation, with a trend toward an increase in amniotic fluid. Maternal serum IL-6 was increased in inoculated animals, while no changes were noted in fetal serum. mRNA expression of IL-6-related transcripts within the chorioallantois indicates that IL-6 is activating the classical JAK/STAT pathway, thereby acting as anti-inflammatory, anti-apoptotic, and pro-survival. The IL-6R was expressed within the chorioallantois, indicating a paracrine signaling pathway of maternal IL-6 to fetal IL-6R. CONCLUSION: IL-6 plays a crucial role in the placental response to induction of sub-acute equine ascending placentitis, and this could be noted in amniotic fluid, allantoic fluid, and maternal serum. Additionally, IL-6 is acting as anti-inflammatory in this disease, potentially altering disease progression, impeding abortion signals, and assisting with the production of a viable neonate.

Extracellular vesicles in spontaneous preterm birth.

Feto-maternal communication helps to maintain pregnancy and contributes to parturition at term and preterm. Endocrine and immune factor are well-reported communication mediators. Recent advances in extracellular vesicle (EV) biology have introduced them as major communication channels between the mother and fetus. EVs are round structures with a lipid bilayer membrane. EVs are generally categorized based on their size and mode of biogenesis. The most commonly reported EVs are exosomes with a size range of 30-160 nm that are formed inside the intraluminal vesicles of multivesicular body. Microvesicles (MVs) are larger than > 200 nm and formed by outward budding of plasma membrane. Vesicles are released from all cells and carry various factors that reflect the physiologic state of cell at the time of their release. Analysis of vesicle provides a snapshot of origin cell. Recent studies in perinatal medicine have shown that exosomes are key communicators between feto-maternal units, and they can cross placenta. Fetal-derived exosomes released under term labor-associated conditions can cause parturition-associated changes in maternal uterine tissues. Exosomes carrying inflammatory cargo can cause preterm birth in animal models suggesting their functional role in parturition. A few reports have profiled differences between exosome cargos from term and preterm pregnancies and indicated their biomarker potential to predict high-risk pregnancy status. There are hardly any reports on MVs and their functional roles in reproduction. Herein, we review of EVs and MVs, their characteristics, function, and usefulness predicting adverse pregnancy complications such as preterm birth.

RNA Sequencing Reveals Diverse Functions of Amniotic Fluid Neutrophils and Monocytes/Macrophages in Intra-Amniotic Infection.

Intra-amniotic infection, the invasion of microbes into the amniotic cavity resulting in inflammation, is a clinical condition that can lead to adverse pregnancy outcomes for the mother and fetus as well as severe long-term neonatal morbidities. Despite much research focused on the consequences of intra-amniotic infection, there remains little knowledge about the innate immune cells that respond to invading microbes. We performed RNA-seq of sorted amniotic fluid neutrophils and monocytes/macrophages from women with intra-amniotic infection to determine the transcriptomic differences between these innate immune cells. Further, we sought to identify specific transcriptomic pathways that were significantly altered by the maternal or fetal origin of amniotic fluid neutrophils and monocytes/macrophages, the presence of a severe fetal inflammatory response, and pregnancy outcome (i.e., preterm or term delivery). We show that significant transcriptomic differences exist between amniotic fluid neutrophils and monocytes/macrophages from women with intra-amniotic infection, indicating the distinct roles these cells play. The transcriptome of amniotic fluid immune cells varies based on their maternal or fetal origin, and the significant transcriptomic differences between fetal and maternal monocytes/macrophages imply that those of fetal origin exhibit impaired functions. Notably, transcriptomic changes in amniotic fluid monocytes/macrophages suggest that these immune cells collaborate with neutrophils in the trafficking of fetal leukocytes throughout the umbilical cord (i.e., funisitis). Finally, amniotic fluid neutrophils and monocytes/macrophages from preterm deliveries display enhanced transcriptional activity compared to those from term deliveries, highlighting the protective role of these cells during this vulnerable period. Collectively, these findings demonstrate the underlying complexity of local innate immune responses in women with intra-amniotic infection and provide new insights into the functions of neutrophils and monocytes/macrophages in the amniotic cavity.

Cellular immune responses in amniotic fluid of women with a sonographic short cervix.

Objectives A sonographic short cervix is one of the strongest predictors of preterm delivery. However, the cellular immune composition of amniotic fluid in women with a short cervix has not yet been described. Herein, we determined cellular and soluble immune responses in amniotic fluid from pregnant women with a mid-trimester asymptomatic short cervix. Methods Amniotic fluid samples (n=77) were collected from asymptomatic women with a cervical length between 15 and 25 mm (n=36, short cervix) or ≤15 mm (n=41, severely short cervix) diagnosed by ultrasound. Flow cytometry and multiplex measurement of cytokines/chemokines were performed. Results (1) The cellular immune composition of amniotic fluid did not differ between women with a severely short cervix (≤15 mm) and those with a short cervix 15-25 mm; (2) amniotic fluid concentrations of multiple cytokines/chemokines were higher in women with a severely short cervix (≤15 mm) than in those with a short cervix 15-25 mm; (3) the cellular immune composition of amniotic fluid did not differ between women with a severely short cervix (≤15 mm) who ultimately underwent preterm delivery and those who delivered at term; and (4) amniotic fluid concentrations of IL-2, but not other immune mediators, were increased in women with a severely short cervix (≤15 mm) who ultimately delivered preterm compared to those who delivered at term. Conclusions Women with a severely short cervix (≤15 mm) have increased concentrations of pro-inflammatory mediators in the amniotic cavity; yet, these do not translate to changes in the cellular immune response.

Exposure to Gestational Diabetes Enriches Immune-Related Pathways in the Transcriptome and Methylome of Human Amniocytes.

CONTEXT: Gestational diabetes (GDM) has profound effects on the intrauterine metabolic milieu and is linked to obesity and diabetes in offspring, but the mechanisms driving these effects remain largely unknown. Alterations in DNA methylation and gene expression in amniocytes exposed to GDM in utero represent a potential mechanism leading to metabolic dysfunction later in life. OBJECTIVE: To profile changes in genome-wide DNA methylation and expression in human amniocytes exposed to GDM. DESIGN: A nested case-control study (n = 14 pairs) was performed in amniocytes matched for offspring sex, maternal race/ethnicity, maternal age, gestational age at amniocentesis, and gestational age at birth. Sex-specific genome-wide DNA methylation analysis and RNA-sequencing were completed and differentially methylated regions (DMRs) and gene expression changes were identified. Ingenuity pathway analysis identified biologically relevant pathways enriched after GDM exposure. In silico high-throughput chromosome conformation capture (Hi-C) analysis identified potential chromatin interactions with DMRs. RESULTS: Expression of interferon-stimulated genes was increased in GDM amniocytes, accounting for 6 of the top 10 altered genes (q < 0.05). Enriched biological pathways in GDM amniocytes included pathways involving inflammation, the interferon response, fatty liver disease, monogenic diabetes, and atherosclerosis. Forty-two DMRs were identified in male GDM-exposed amniocytes and 20 in female amniocyte analysis (q < 0.05). Hi-C analysis identified interactions between DMRs and 11 genes with significant expression changes in male amniocytes and 9 in female amniocytes (P < .05). CONCLUSION: In a unique repository of human amniocytes exposed to GDM in utero, transcriptome analysis identified enrichment of inflammation and interferon-related pathways and novel DMRs with potential distal regulatory functions.

Inflammation with the participation of arachidonic (AA) and linoleic acid (LA) derivatives (HETEs and HODEs) is necessary in the course of a normal reproductive cycle and pregnancy.

Data on arachidonic (AA) and linoleic (LA) acid derivatives and their role in the reproductive cycle are limited. In order to systematize these reports, 54 scientific investigations were analyzed, which revealed the important role of AA and LA in the planning and course of pregnancy. Ovulation, menstruation, pregnancy, and childbirth are strongly related to the occurrence of physiological inflammatory reactions. Ovulation and menstruation are cyclic tissue remodeling processes that cause changes in the synthesis of inflammation mediators, such as prostaglandins and leukotrienes. Thus, the cyclooxygenase (COX) and lipoxygenase-5 (5-LOX) pathway for AA transformation is activated. Only the absence of neutrophils during this process differentiates an embryo implantation from a standard inflammatory response. It has been found that in COX-2 deficiency conditions, incorrect embryo implantation and decidual reaction occur; therefore, the mechanism associated with the activation of the nuclear factor (NF)-κB pathway seems to play an important role in the course of embryo implantation. In addition, 12/15-LOX may be key modulators of uterine activity during the implantation process. According to the current state of knowledge, AA derivatives synthesized throughout the cytochrome P450 (CYP) and LOX pathways play a special role in the late pregnancy period. Decreased 5-HETE levels have been related to slowing down the progression of labor, while 11-HETE and 15-HETrE to its acceleration. It has been also proven that renal 20-HETE contents undergo significant changes in the late pregnancy period, which are caused by an increase in their adrenal medulla and vascular synthesis, leading to decrease of blood pressure and an increase of sodium excretion, finally conditioning a normal course of labor.

Erythropoietin prevents LPS-induced preterm birth and increases offspring survival.

PROBLEM: Preterm delivery is the leading cause of neonatal mortality and contributes to delayed physical and cognitive development in children. At present, there is no efficient therapy to prevent preterm labor. A large body of evidence suggests that infections might play a significant and potentially preventable cause of premature birth. This work assessed the effects of erythropoietin (EPO) in a murine model of inflammation-associated preterm delivery, which mimics central features of preterm infections in humans. METHOD OF STUDY: BALB/c mice were injected i.p. with 20 000 IU/kg EPO or normal saline twice on gestational day (GD) 15, with a 3 hours time interval between injections. An hour after the first EPO or normal saline injection, all mice received two injections of 50 µg/kg LPS, also given 3 hours apart. RESULTS: EPO significantly prevented preterm labor and increased offspring survival in an LPS induced preterm delivery model. EPO prevented LPS-induced leukocyte infiltration into the placenta. Moreover, EPO inhibited the expression of pro-inflammatory cytokines, interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and tumour necrosis factor-α (TNF-α) in maternal serum and amniotic fluid. EPO also prevented LPS-induced increase in placental prostaglandin (PG)E2 and uterine inducible nitric oxide synthase (iNOS) production, while decreasing nuclear factor kappa-B (NF-κß) activity in the myometrium. EPO also increased the gene expression of placental programmed cell death ligand 1 (PD-L1) in LPS-treated mice. CONCLUSIONS: Our results suggest that EPO could be a potential novel therapeutic strategy to tackle infection-related preterm labor.

Complement and other immune-related factors in cervicovaginal fluid associated with intra-amniotic infection/inflammation and spontaneous preterm delivery in women with preterm labor.

PURPOSE: To investigate whether complement and other immune-related proteins in cervicovaginal fluid (CVF) can predict intra-amniotic infection and/or inflammation (IAI) and spontaneous preterm delivery (SPTD, < 34.0 weeks) in women with preterm labor (PTL) and to compare the predictive abilities of these biomarkers with that of amniotic fluid (AF) white blood cells (WBCs). METHODS: We designed a retrospective cohort study of 145 women with PTL at 23.0-33.6 weeks who underwent amniocentesis. AF was cultured and assayed for WBC count and interleukin-6 (IL-6). CVF samples were obtained at the time of amniocentesis. CVF was assayed for complement C3a and C5a, IGFBP-1, and MMP-9 by ELISA. RESULTS: In the multivariate analysis, elevated CVF levels of C5a and IGFBP-1 were significantly associated with IAI and SPTD at < 34 weeks, while those of C3a were associated with IAI, but not SPTD, even after adjusting for other baseline confounders. For C3a, C5a, and IGFBP-1 in the CVF, area under the curve (AUC) values were statistically similar to that of AF WBCs for detecting IAI, whereas these CVF biomarkers had similar or higher AUC values than AF WBCs for predicting SPTD at < 34 weeks. However, univariate analysis showed no significant correlation between high CVF MMP-9 and IAI or SPTD at < 34 weeks. CONCLUSIONS: In women with PTL, the CVF levels of C3a, C5a, and IGFBP-1 may be useful as novel non-invasive predictors of IAI and SPTD at < 34 weeks. These biomarkers (especially IGFBP-1) have similar or better diagnostic performance compared to AF WBCs.

Successful use of an artificial placenta-based life support system to treat extremely preterm ovine fetuses compromised by intrauterine inflammation.

BACKGROUND: Ex vivo uterine environment therapy is an experimental intensive care strategy for extremely preterm infants born between 21 and 24 weeks of gestation. Gas exchange is performed by membranous oxygenators connected by catheters to the umbilical vessels. The fetus is submerged in a bath of synthetic amniotic fluid. The lungs remain fluid filled, and pulmonary respiration does not occur. Intrauterine inflammation is strongly associated with extremely preterm birth and fetal injury. At present, there are no data that we are aware of to show that artificial placenta-based systems can be used to support extremely preterm fetuses compromised by exposure to intrauterine inflammation. OBJECTIVE: To evaluate the ability of our ex vivo uterine environment therapy platform to support extremely preterm ovine fetuses (95-day gestational age; approximately equivalent to 24 weeks of human gestation) exposed to intrauterine inflammation for a period of 120 hours, the following primary endpoints were chosen: (1) maintenance of key physiological variables within normal ranges, (2) absence of infection and inflammation, (3) absence of brain injury, and (4) gross fetal growth and cardiovascular function matching that of age-matched in utero controls. STUDY DESIGN: Ten ewes with singleton pregnancies were each given a single intraamniotic injection of 10-mg Escherichia coli lipopolysaccharides under ultrasound guidance 48 hours before undergoing surgical delivery for adaptation to ex vivo uterine environment therapy at 95-day gestation (term=150 days). Fetuses were adapted to ex vivo uterine environment therapy and maintained for 120 hours with constant monitoring of key vital parameters (ex vivo uterine environment group) before being killed at 100-day equivalent gestational age. Umbilical artery blood samples were regularly collected to assess blood gas data, differential counts, biochemical parameters, inflammatory markers, and microbial load to exclude infection. Ultrasound was conducted at 48 hours after intraamniotic lipopolysaccharides (before surgery) to confirm fetal viability and at the conclusion of the experiments (before euthanasia) to evaluate cardiac function. Brain injury was evaluated by gross anatomic and histopathologic investigations. Eight singleton pregnant control animals were similarly exposed to intraamniotic lipopolysaccharides at 93-day gestation and were killed at 100-day gestation to allow comparative postmortem analyses (control group). Biobanked samples from age-matched saline-treated animals served as an additional comparison group. Successful instillation of lipopolysaccharides into the amniotic fluid exposure was confirmed by amniotic fluid analysis at the time of administration and by analyzing cytokine levels in fetal plasma and amniotic fluid. Data were tested for mean differences using analysis of variance. RESULTS: Six of 8 lipopolysaccharide control group (75%) and 8 of 10 ex vivo uterine environment group fetuses (80%) successfully completed their protocols. Six of 8 ex vivo uterine environment group fetuses required dexamethasone phosphate treatment to manage profound refractory hypotension. Weight and crown-rump length were reduced in ex vivo uterine environment group fetuses at euthanasia than those in lipopolysaccharide control group fetuses (P<.05). There were no biologically significant differences in cardiac ultrasound measurement, differential leukocyte counts (P>.05), plasma tumor necrosis factor α, monocyte chemoattractant protein-1 concentrations (P>.05), or liver function tests between groups. Daily blood cultures were negative for aerobic and anaerobic growth in all ex vivo uterine environment group animals. No cases of intraventricular hemorrhage were observed. White matter injury was identified in 3 of 6 lipopolysaccharide control group fetuses and 3 of 8 vivo uterine environment group fetuses. CONCLUSION: We report the use of an artificial placenta-based system to support extremely preterm lambs compromised by exposure to intrauterine inflammation. Our data highlight key challenges (refractory hypotension, growth restriction, and white matter injury) to be overcome in the development and use of artificial placenta technology for extremely preterm infants. As such challenges seem largely absent from studies based on healthy pregnancies, additional experiments of this nature using clinically relevant model systems are essential for further development of this technology and its eventual clinical application.

Maternal azithromycin therapy for Ureaplasma parvum intraamniotic infection improves fetal hemodynamics in a nonhuman primate model.

BACKGROUND: Ureaplasma parvum infection is a prevalent cause of intrauterine infection associated with preterm birth, preterm premature rupture of membranes, fetal inflammatory response syndrome, and adverse postnatal sequelae. Elucidation of diagnostic and treatment strategies for infection-associated preterm labor may improve perinatal and long-term outcomes for these cases. OBJECTIVE: This study assessed the effect of intraamniotic Ureaplasma infection on fetal hemodynamic and cardiac function and the effect of maternal antibiotic treatment on these outcomes. STUDY DESIGN: Chronically catheterized pregnant rhesus monkeys were assigned to control (n=6), intraamniotic inoculation with Ureaplasma parvum (107 colony-forming units/mL, n=15), and intraamniotic infection plus azithromycin treatment (12.5 mg/kg twice a day intravenously, n=8) groups. At approximately 135 days' gestation (term=165 days), pulsed and color Doppler ultrasonography was used to obtain measurements of fetal hemodynamics (pulsatility index of umbilical artery, ductus venosus, descending aorta, ductus arteriosus, aortic isthmus, right pulmonary artery, middle cerebral artery and cerebroplacental ratio, and left and right ventricular cardiac outputs) and cardiac function (ratio of peak early vs late transmitral flow velocity [marker of ventricular function], Tei index [myocardial performance index]). These indices were stratified by amniotic fluid proinflammatory mediator levels and cardiac histology. RESULTS: Umbilical and fetal pulmonary artery vascular impedances were significantly increased in animals from the intraamniotic inoculation with Ureaplasma parvum group (P<.05). Azithromycin treatment restored values to control levels. Amniotic fluid prostaglandin F2 alpha levels were significantly higher in animals with abnormal umbilical artery pulsatility index (>1.1) than in those with normal blood flow (P<.05; Spearman ρ=0.6, P<.05). In the intraamniotic inoculation with Ureaplasma parvum group, left ventricular cardiac output was significantly decreased (P<.001), and more animals had abnormal right-to-left ventricular cardiac output ratios (defined as >1.6, P<.05). Amniotic fluid interleukin-6 concentrations were elevated in cases of abnormal right-to-left ventricular cardiac output ratios compared with those in normal cases (P<.05). CONCLUSION: Fetal hemodynamic alterations were associated with intraamniotic Ureaplasma infection and ameliorated after maternal antibiotic treatment. Doppler ultrasonographic measurements merit continuing investigation as a diagnostic method to identify fetal cardiovascular and hemodynamic compromise associated with intrauterine infection or inflammation and in the evaluation of therapeutic interventions or clinical management of preterm labor.