Amniotic Fluid Of Women Research Articles

Evidence that intra-amniotic infections are often the result of an ascending invasion - a molecular microbiological study.

Background Microbial invasion of the amniotic cavity resulting in intra-amniotic infection is associated with obstetrical complications such as preterm labor with intact or ruptured membranes, cervical insufficiency, as well as clinical and histological chorioamnionitis. The most widely accepted pathway for intra-amniotic infection is the ascension of microorganisms from the lower genital tract. However, hematogenous dissemination of microorganisms from the oral cavity or intestine, retrograde seeding from the peritoneal cavity through the fallopian tubes, and introduction through invasive medical procedures have also been suggested as potential pathways for intra-amniotic infection. The primary reason that an ascending pathway is viewed as most common is that the microorganisms most often detected in the amniotic fluid are those that are typical inhabitants of the vagina. However, thus far, no studies have shown that microorganisms in the amniotic cavity are simultaneously present in the vagina of the woman from which they were isolated. The objective of the study was to determine the frequency with which microorganisms isolated from women with intra-amniotic infection are also present in the lower genital tract. Methods This was a cross-sectional study of women with intra-amniotic infection with intact membranes. Intra-amniotic infection was defined as a positive culture and elevated concentrations of interleukin-6 (IL-6) (>2.6 ng/mL) in amniotic fluid and/or acute histologic chorioamnionitis and funisitis. Microorganisms isolated from bacterial cultures of amniotic fluid were taxonomically identified through matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) and 16S ribosomal RNA (rRNA) gene sequencing. Vaginal swabs were obtained at the time of amniocentesis for the identification of microorganisms in the lower genital tract. The overall bacterial profiles of amniotic fluids and vaginal swabs were characterized through 16S rRNA gene sequencing. The bacterial profiles of vaginal swabs were interrogated for the presence of bacteria cultured from amniotic fluid and for the presence of prominent (>1% average relative abundance) operational taxonomic units (OTUs) within the overall 16S rRNA gene bacterial profiles of amniotic fluid. Results (1) A total of 75% (6/8) of women had bacteria cultured from their amniotic fluid that are typical residents of the vaginal ecosystem. (2) A total of 62.5% (5/8) of women with bacteria cultured from their amniotic fluid also had these bacteria present in their vagina. (3) The microorganisms cultured from amniotic fluid and also detected in the vagina were Ureaplasma urealyticum, Escherichia coli, and Streptococcus agalactiae. (4) 16S rRNA gene sequencing revealed that the amniotic fluid of women with intra-amniotic infection had bacterial profiles dominated by Sneathia, Ureaplasma, Prevotella, Lactobacillus, Escherichia, Gardnerella, Peptostreptococcus, Peptoniphilus, and Streptococcus, many of which had not been cultured from the amniotic fluid samples. (5) Seventy percent (7/10) of the prominent (>1% average relative abundance) OTUs found in amniotic fluid were also prominent in the vagina. Conclusion The majority of women with intra-amniotic infection had bacteria cultured from their amniotic fluid that were typical vaginal commensals, and these bacteria were detected within the vagina at the time of amniocentesis. Molecular microbiological interrogation of amniotic fluid from women with intra-amniotic infection revealed that the bacterial profiles of amniotic fluid were largely consistent with those of the vagina. These findings indicate that ascension from the lower genital tract is the primary pathway for intra-amniotic infection.

Pediatric Pulmonary Hypertension on the World Stage: Do We Need Separate Neonatal Guidelines?

Pediatric Pulmonary Hypertension on the World Stage: Do We Need Separate Neonatal Guidelines?

Effects of long working hours and shift work during pregnancy on obstetric and perinatal outcomes: A large prospective cohort study-Japan Environment and Children's Study.

BackgroundThe work patterns of pregnant women may be related to adverse obstetric and perinatal outcomes. This study aimed to clarify the effects of weekly working time according to frequencies of night shifts during pregnancy on adverse outcomes in Japan.MethodsThe Japan Environment and Children's Study, a prospective cohort study, was conducted in 15 regions nationwide in Japan. The study population included pregnant women with singleton pregnancies (n = 99 744). The mothers’ working hours and frequencies of night shifts during the first and the second/third trimesters were assessed using a self‐administered questionnaire. Outcome data were collected from medical transcripts.ResultsCompared with nonworking women, women who worked during pregnancy had significantly increased adjusted odds ratios (aORs) of threatened miscarriage (maximum aOR: 1.47, 95% confidence interval [95% CI]: 1.26‐1.73) and of threatened preterm labor (maximum aOR: 1.63, 95% CI: 1.41‐1.87). Increased aORs were observed for hypertensive disorders of pregnancy (maximum aOR: 2.02, 95% CI: 1.39‐2.93) in women working ≥36 hours per week with night shifts, for vacuum/forceps delivery (maximum aOR: 1.34, 95% CI: 1.22‐1.48) at ≥36 hours with or without night shifts, and for small‐for‐gestational‐age babies (aOR: 1.32, 95% CI: 1.10‐1.59) at ≥46 hours with night shifts. In contrast, lower aORs were observed for gestational diabetes and meconium‐stained amniotic fluid in women working without night shifts.ConclusionsWork during pregnancy slightly increased the risks of threatened miscarriage and threatened preterm labor. Long working hours increased the risks of hypertensive disorders of pregnancy, vacuum/forceps delivery, and small‐for‐gestational‐age babies.

Cellular immune responses in amniotic fluid of women with preterm labor and intra‐amniotic infection or intra‐amniotic inflammation

Preterm birth is commonly preceded by preterm labor, a syndrome that is causally linked to both intra-amniotic infection and intra-amniotic inflammation. However, the stereotypical cellular immune responses in these two clinical conditions are poorly understood. Amniotic fluid samples (n=26) were collected from women diagnosed with preterm labor and intra-amniotic infection (amniotic fluid IL-6 concentrations ≥2.6ng/mL and culturable microorganisms, n=10) or intra-amniotic inflammation (amniotic fluid IL-6 concentrations ≥2.6ng/mL without culturable microorganisms, n=16). Flow cytometry was performed to evaluate the phenotype and number of amniotic fluid leukocytes. Amniotic fluid concentrations of classical pro-inflammatory cytokines, type 1 and type 2 cytokines, and T-cell chemokines were determined using immunoassays. Women with spontaneous preterm labor and intra-amniotic infection had (a) a greater number of total leukocytes, including neutrophils and monocytes/macrophages, in amniotic fluid; (b) a higher number of total T cells and CD4+ T cells, but not CD8+ T cells or B cells, in amniotic fluid; and (c) increased amniotic fluid concentrations of IL-6, IL-1β, and IL-10, compared to those with intra-amniotic inflammation. However, no differences in amniotic fluid concentrations of T-cell cytokines and chemokines were observed between these two clinical conditions. The cellular immune responses observed in women with preterm labor and intra-amniotic infection are more severe than in those with intra-amniotic inflammation, and neutrophils, monocytes/macrophages, and CD4+ T cells are the main immune cells responding to microorganisms that invade the amniotic cavity. These findings provide insights into the intra-amniotic immune mechanisms underlying the human syndrome of preterm labor.

Presence of Chlamydia trachomatis DNA in the amniotic fluid in women with preterm prelabor rupture of membranes

Objective The primary aim of this study was to assess the rate and load of amniotic fluid Chlamydia trachomatis DNA and their associations with intra-amniotic infection and intra-uterine inflammatory complications in women with preterm prelabor rupture of membranes (PPROM). The secondary aim was to assess the short-term morbidity of newborns from PPROM pregnancies complicated by amniotic fluid C. trachomatis DNA. Methods A retrospective study of 788 women with singleton pregnancies complicated by PPROM between 24 + 0 and 36 + 6 weeks of gestation was performed. Transabdominal amniocenteses were performed at the time of admission. C. trachomatis DNA in the amniotic fluid was assessed by real-time polymerase chain reaction using a commercial AmpliSens® C. trachomatis/Ureaplasma/Mycoplasma hominis-FRT kit, and the level of Ct DNA was quantified. Results Amniotic fluid C. trachomatis DNA complicated 2% (16/788) of the PPROM pregnancies and was present in very low loads (median 57 copies DNA/mL). In addition to amniotic fluid C. trachomatis DNA, other bacteria were detected in 62% (10/16) of the C. trachomatis DNA-complicated PPROM pregnancies. Amniotic fluid C. trachomatis DNA was associated with intra-amniotic infection, histologic chorioamnionitis (HCA), and funisitis in 31%, 47%, and 33%, respectively. The presence of C. trachomatis DNA accompanied by Ureaplasma species in the amniotic fluid was associated with a higher rate of HCA than the presence of amniotic fluid C. trachomatis DNA alone. The composite neonatal morbidity in newborns from PPROM pregnancies with amniotic fluid C. trachomatis DNA was 31%. Conclusion The presence of C. trachomatis DNA in the amniotic fluid is a relatively rare condition in PPROM. Amniotic fluid C. trachomatis DNA in PPROM is not related to intensive intra-amniotic and intr-auterine inflammatory responses or adverse short-term neonatal outcomes.

Association of maternal age with fetal sex chromosome aneuploidies

To analyze the impact of maternal age on sex chromosome aneuploidies (SCA). Pregnant women who had karyotype analysis of amniotic fluid in Women's Hospital, Zhejiang University School of Medicine from January 2014 to July 2018 were recruited. The association of the maternal age with fetal SCAs was analyzed. The incidence of 45, X in age group >34-<38 was lower than that of ≤ 28 age group (P<0.05). For the incidences of total sex chromosome trisomy and 47, XXY in age groups 34-<38 and ≥38 were higher than age groups ≤28 and >28-34 (P<0.05 or P<0.01). The incidence of 47, XXX in age group ≥ 38 was higher than that in age group>28-34 (P<0.05). However, the incidence of 47, XYY had no differences among the four groups (P>0.05). After excluding the high risk of sex chromosome abnormalities by non-invasive prenatal testing (NIPT), we found that for 45, X, the incidences of two groups with advanced age were lower than that of ≤ 28 year-old group of age group (P<0.05 or P<0.01), and incidence in age group >34-<38 was also lower than that in age group >28-34 (P<0.05). The other results were consistent with those without excluding the high risk of sex chromosome abnormalities by NIPT. Advanced age decreases the incidence of 45, X, but increases the risk of sex chromosome trisomy, especially 47, XXX and 47, XXY.

Gasdermin D: in vivo evidence of pyroptosis in spontaneous labor at term

Objective: Pyroptosis is an inflammatory form of programmed cell death that is mediated by the activation of the inflammasome and depends on the pore-forming function of gasdermin D. Therefore, the detection of gasdermin D represents in vivo evidence of pyroptosis. We recently showed that there is intra-amniotic inflammasome activation in spontaneous labor at term; however, evidence of pyroptosis is lacking. The objectives of this study were to investigate (1) whether gasdermin D is detectable in the amniotic fluid of women who delivered at term; (2) whether amniotic fluid gasdermin D concentrations are associated with the process of spontaneous labor at term; and (3) whether gasdermin D is expressed in the chorioamniotic membranes from these patients.Methods: This retrospective cross-sectional study included amniotic fluid samples from 41 women who underwent spontaneous labor at term (n = 17) or delivered at term without labor (n = 24). As a readout of pyroptosis, gasdermin D was determined in amniotic fluid samples using a specific and sensitive ELISA kit. The 90th percentile of amniotic fluid gasdermin D concentrations was calculated among women without spontaneous labor at term (reference group). The association between high amniotic fluid gasdermin D concentrations (≥90th percentile in the reference group) and spontaneous labor at term was tested using the Fisher’s exact test. A p value <.05 was considered significant. Multiplex immunofluorescence staining and phenoptics (multispectral imaging) were performed to determine gasdermin D expression in the chorioamniotic membranes and to colocalize this protein with the inflammasome-related molecules caspase-1 and interleukin-1β.Results: (1) Gasdermin D is present in the amniotic fluid of women who delivered at term; (2) the 90th percentile of amniotic fluid gasdermin D concentrations in women who delivered at term without spontaneous labor was 3.4 ng/mL; (3) the proportion of women with amniotic fluid gasdermin D concentrations above the threshold was higher in those who underwent term labor than in those who delivered at term without labor; (4) amniotic fluid concentrations of gasdermin D > 3.4 ng/mL were significantly associated with the presence of spontaneous labor in women who delivered at term (odds ratio 6.0, p-value .048); and (5) the protein expression of gasdermin D is increased in the chorioamniotic membranes of women who underwent spontaneous labor at term and is colocalized with caspase-1 and IL-1β.Conclusions: Gasdermin D is increased in the amniotic fluid and chorioamniotic membranes of women who underwent spontaneous labor at term compared to those without labor. These data provide evidence implicating pyroptosis in the mechanisms that lead to the sterile inflammatory process of term parturition.

Associations between second-trimester amniotic fluid levels of ADAMTS4, ADAMTS5, IL-6, and TNF-α and spontaneous preterm delivery in singleton pregnancies.

Background We investigated the roles of inflammatory cytokines and the A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family in the etiopathogenesis of spontaneous preterm delivery by comparing the ADAMTS4, ADAMTS5, interleukin (IL)-6, and tumor necrosis factor-α (TNF-α) levels in second-trimester amniotic fluid between pregnant women with preterm birth and term controls. Methods All pregnant women who underwent second-trimester amniocentesis for genetic analysis between January 1, 2016, and January 1, 2018, were enrolled in this study. From this cohort, 22 patients who subsequently experienced spontaneous preterm delivery before 34 weeks of pregnancy formed the study group, and 22 age- and body mass index (BMI)-matched patients without preterm birth constituted the control group. Results No significant differences were observed between the preterm birth and control groups in terms of age, BMI, obstetric history of preterm delivery, gestational age at amniocentesis, or indication for amniocentesis. The mean amniotic fluid levels of ADAMTS4 and ADAMTS5 were significantly increased in the preterm birth group compared to the control group (248.3±22.6 and 182.4±19.8 pg/mL, P=0.012; and 198.6±21.6 and 159.1±21.7 pg/mL, P=0.035, respectively). Significantly increased IL-6 and TNF-α levels were also detected in the amniotic fluid of women who experienced spontaneous preterm delivery, relative to controls (142.1±16.2 and 95.8±16.4 pg/mL, P<0.001; and 139.4±12.5 and 89.6±11.2 pg/mL, P<0.001, respectively). Conclusion The results of this study imply that increased mid-trimester amniotic fluid levels of ADAMTS4, ADAMTS5, IL-6, and TNF-α play an important role in the pathophysiology of spontaneous preterm delivery.

Peculiarities of Changing Bioregulators and Organic Acids in the Serum of Blood and Amniothic Fluid of Women with Spontaneous Preterm Labor

Background: Premature birth is currently a serious problem for obstetrician-gynecologists. Most researcher consider this problem from the standpoint of metabolome disturbances.Aims: determination of the level of bioactive regulators and organic acids with physiological and complicated pregnancy, ending in timely and spontaneous preterm labor, in blood serum and amniotic fluid and revealing the presence of their relationship for the development of premature birth and the condition of newborns.Materials and methods: 72 women were examined, 40 of them with a physiological pregnancy (control group) and 32 with premature labor (the main group). All studies were carried out in the blood serum and amniotic fluid of women of these groups. The content of TGFβ was determined by the method of solid-phase enzyme immunoassay. Identification and quantitative determination of citric and succinic acids were carried out by the method of capillary electrophoresis.Results: TNFα production in the blood serum of women with preterm labor, decreased by 38%, and the level of citric and succinic acids by 33 and 30%, respectively, compared with the control group. In the amniotic fluid the level of TNFα was increased 2.5 times and TGFβ ― 2.2 times, citric acid ― 79%.Conclusions: The revealed imbalance in the level of cytokines and organic acids studied in that biological fluids can evidently affect the fetal condition, which can be considered as possible markers of perinatal injuries and prognostic indicators of the state of the newborn.

Is amniotic fluid of women with uncomplicated term pregnancies free of bacteria?

The "sterile womb" paradigm is debated. Recent evidence suggests that the offspring's first microbial encounter is before birth in term uncomplicated pregnancies. The establishment of a healthy microbiota early in life might be crucial for reducing the burden of diseases later in life. We aimed to investigate the presence of a microbiota in sterilely collected amniotic fluid in uncomplicated pregnancies at term in the Preventing Atopic Dermatitis and Allergies in children (PreventADALL) study cohort. Amniotic fluid was randomly sampled at cesarean deliveries in pregnant women in 1 out of 3 study sites included in the PreventADALL study. From 65 pregnancies at term, where amniotic fluid was successfully sampled, we selected 10 from elective (planned, without ongoing labor) cesarean deliveries with intact amniotic membranes and all 14 with prior rupture of membranes were included as positive controls. Amniotic fluid was analyzed by culture-independent and culture-dependent techniques. The median (min-max) concentration of prokaryotic DNA (16S rRNA gene copies/mL; digital droplet polymerase chain reaction) was low for the group with intact membranes [664 (544-748)]-corresponding to the negative controls [596 (461-679)], while the rupture of amniotic membranes group had >10-fold higher levels [7700 (1066-251,430)] (P= .0001, by Mann-Whitney U test). Furthermore, bacteria were detected in 50% of the rupture of amniotic membranes samples by anaerobic culturing, while none of the intact membranes samples showed bacterial growth. Sanger sequencing of the rupture of amniotic membrane samples identified bacterial strains that are commonly part of the vaginal flora and/or associated with intrauterine infections. We conclude that fetal development in uncomplicated pregnancies occurs in the absence of an amniotic fluid microbiota and that the offspring microbial colonization starts after uterine contractions and rupture of amniotic membrane.

Incidence of echogenic amniotic fluid at term pregnancy and its association with meconium

The presence of echogenic amniotic fluid at term gestation on sonography is uncommon. The aim of our study was to investigate the incidence of echogenic amniotic fluid at term pregnancy, and to determine how often echogenic amniotic fluid was associated with meconium. All singleton pregnant women at term who were admitted to our labor unit and who delivered within 24h of the ultrasound scan were included in the study. For each woman, gestational age, maternal age, gravidity, parity, the character of the amniotic fluid on ultrasound at admission (clear or echogenic), birth weight, and the character of the amniotic fluid on artificial or spontaneous rupture of membranes or on cesarean section (clear/with vernix/meconium-stained) were recorded. When amniotic fluid was assessed on ultrasound, among 278 patients, 9 (3.2%) patients' amniotic fluid was echogenic. When the amniotic fluid was assessed at delivery, the rates of meconium-stained amniotic fluid in women with and without echogenic amniotic fluid were 44.44% (4/9) and 9.3% (25/269), respectively; the difference was statistically significant (p=0.035). We found a sensitivity and specificity of 13.79 and 97.99%, and a positive and negative predictive value of 44.44 and 90.7%, respectively, for echogenic amniotic fluid seen on ultrasound in identifying meconium-stained amniotic fluid. The incidence of echogenic amniotic fluid at term gestation was found as 3.2 and 44.4% of cases of echogenic amniotic fluid was associated with meconium.

In vivo evidence of inflammasome activation during spontaneous labor at term

Objective: Upon inflammasome activation, the adaptor protein of the inflammasome ASC (apoptosis-associated speck-like protein containing a CARD) forms intracellular specks, which can be released into the extracellular space. The objectives of this study were to investigate whether (1) extracellular ASC is present in the amniotic fluid of women who delivered at term; (2) amniotic fluid ASC concentrations are greater in women who underwent spontaneous labor at term than in those who delivered at term in the absence of labor; and (3) amniotic epithelial and mesenchymal cells can form intracellular ASC specks in vitro.Methods: This retrospective cross-sectional study included amniotic fluid samples from 41 women who delivered at term in the absence of labor (n = 24) or underwent spontaneous labor at term (n = 17). Amniotic epithelial and mesenchymal cells were also isolated from the chorioamniotic membranes obtained from a separate group of women who delivered at term (n = 3), in which ASC speck formation was assessed by confocal microscopy. Monocytes from healthy individuals were used as positive controls for ASC speck formation (n = 3).Results: (1) The adaptor protein of the inflammasome ASC is detectable in the amniotic fluid of women who delivered at term; (2) amniotic fluid ASC concentration was higher in women who underwent spontaneous labor at term than in those who delivered at term without labor; and (3) amniotic epithelial and mesenchymal cells are capable of forming ASC specks and/or filaments in vitro.Conclusion: Amniotic fluid ASC concentrations are increased in women who undergo spontaneous labor at term. Amniotic epithelial and mesenchymal cells are capable of forming ASC specks, suggesting that these cells are a source of extracellular ASC in the amniotic fluid. These findings provide in vivo evidence that there is inflammasome activation in the amniotic cavity during the physiological process of labor at term.

The diagnostic performance of the beta-glucan assay in the detection of intra-amniotic infection with Candida species

Introduction: A bioassay based on the detection of beta-glucan, a constituent of the cell wall of fungi, has been successfully used to diagnose fungal infections in a variety of biological fluids but not yet in the amniotic fluid.Objective: To determine the diagnostic performance of a beta-glucan bioassay in the detection of Candida species in the amniotic fluid of women who either did or did not have an intrauterine contraceptive device (IUD) in place during an episode of spontaneous preterm parturition.Methods: The study population comprised women who had a singleton pregnancy without congenital or chromosomal abnormalities, who experienced preterm labor or preterm prelabor rupture of the fetal membranes, and who underwent a transabdominal amniocentesis for clinical indications. Samples of amniotic fluid were cultured for aerobic and anaerobic bacteria, genital mycoplasmas, and Candida species, and assayed for beta-glucan, using the (1→3)-beta-d-glucan-specific Limulus amebocyte lysate test (beta-glucan assay) in all cases. Amniotic fluid interleukin (IL)-6 assay results were also available for all cases. The beta-glucan assay takes about 1 hour to run: a concentration >80 pg/mL was considered positive for fungi. Sterile intra-amniotic inflammation of the amniotic cavity was defined by the presence of an amniotic fluid IL-6 concentration ≥2.6 ng/mL and a negative amniotic fluid culture.Results: (1) One hundred ninety-seven (197) women met the study criteria, of whom 58 (29.4%) had an IUD in place; (2) 20 (10.2%) women had a culture of proven intra-amniotic Candida species-related infection, 19 of whom had a positive beta-glucan assay [sensitivity, 95% (19/20; 95% confidence interval (CI): 75.1–99.9%)]; and (3) the specificity of the beta-glucan assay was 75.1% [133/177; 95% CI: 68.1–99.9%]. It was affected by the presence of nonfungal intra-amniotic infections and an IUD, but not by the presence of sterile intra-amniotic inflammation, and there was a significant interaction between the presence of an IUD and nonfungal intra-amniotic infections (estimated for the interaction effect = 2.1923, p value =.026). The assay’s specificity was reduced when nonfungal intra-amniotic infections were diagnosed but only in women who did not have an IUD. Among women without an IUD, the assay’s specificity was 91.4% (117/128); it was 93% (106/114) for those without intra-amniotic infection, and 78.6% (11/14) for those with a nonfungal intra-amniotic infection; the difference was not significant (p = .09). Among women with an IUD, the assay’s specificity was 32.7% (16/49); 42.9% (9/21) for those with a nonfungal intra-amniotic infection; and 25% (7/28) for those without intra-amniotic infection; and the difference was significant (p = .03).Conclusions: The beta-glucan assay is a sensitive, rapid, point-of-care test used to diagnose intra-amniotic Candida species-related infection, and it has a high specificity in pregnant women who did not have an IUD in place.

Amniotic fluid pentraxins: Potential early markers for identifying intra-amniotic inflammatory complications in preterm pre-labor rupture of membranes.

In this study, pentraxin 3 (PTX3), C-reactive protein (CRP), and serum amyloid P component (SAP) concentrations in the amniotic fluid of women with preterm pre-labor rupture of membranes (PPROM) were evaluated based on evidence of microbial invasion of the amniotic cavity (MIAC), intra-amniotic inflammation (IAI), and microbial-associated IAI. A total of 149 women with PPROM were included in this study. Amniotic fluid samples were obtained by transabdominal amniocentesis. Amniotic fluid PTX3, SAP, and CRP concentrations were assessed using enzyme-linked immunosorbent assay. PTX3 and CRP concentrations were higher in women with MIAC, IAI, and microbial-associated IAI than in women without these conditions. SAP concentrations were only higher in the presence of IAI and microbial-associated IAI. Amniotic fluid PTX3 concentrations of 11ng/mL were found to be the best value for identifying the presence of microbial-associated IAI and IAI in women with PPROM. To conclude, amniotic fluid pentraxins are involved in intra-amniotic inflammatory responses in pregnancies complicated by PPROM.

Intra-Amniotic Soluble Endoglin Impairs Lung Development in Neonatal Rats

Soluble endoglin (sENG) is increased in the amniotic fluid of women with preeclampsia and chorioamnionitis. Preterm infants born to women with these disorders have an increased risk of aberrant lung development. Whether this increased risk is secondary to elevated sENG levels is unclear. The objective of this study was to determine whether intrauterine exposure to an adenovirus overexpressing sENG impairs neonatal lung angiogenesis by modulating lung endothelial nitric oxide synthase (eNOS) signaling. Pregnant Sprague-Dawley rats were randomly assigned to receive ultrasound-guided intra-amniotic injections of adenovirus overexpressing sENG (Ad-sENG) or control adenovirus (Ad-control) on embryonic day 17. After this exposure, rat pups were maintained in normoxia and evaluated on postnatal day 14. Intra-amniotic Ad-sENG decreased lung vascular endothelial growth factor receptor 2 and eNOS expression in rat pups. This was accompanied by a marked decrease in lung angiogenesis and alveolarization. Ad-sENG-exposed pups also had an increase in right ventricular systolic pressure, weight ratio of right ventricle to left ventricle plus septum, and pulmonary vascular remodeling. In addition, exposure of human pulmonary artery endothelial cells to recombinant sENG reduced endothelial tube formation and protein levels of eNOS, phosphorylated eNOS, and phosphorylated Smad1/5. Together, our findings demonstrate that intrauterine exposure to an adenovirus overexpressing sENG disrupts lung development by impairing Smad1/5-eNOS signaling. We speculate that sENG-mediated dysregulation of Smad1/5-eNOS signaling contributes to impaired lung development and potentially primes the developing lung for further postnatal insults. Further studies exploring the relationship between amniotic fluid sENG levels and preterm respiratory outcomes will be necessary.

Are amniotic fluid neutrophils in women with intraamniotic infection and/or inflammation of fetal or maternal origin?

Neutrophils are the most abundant white blood cells found in the amniotic cavity of women with intraamniotic infection and/or inflammation. The current belief is that these neutrophils are of fetal origin. However, abundant neutrophils have been found in the amniotic fluid of women with a severe acute maternal inflammatory response but without a severe fetal inflammatory response in the placenta, suggesting that these innate immune cells can also be of maternal origin or a mixture of both fetal and maternal neutrophils. We sought to investigate the origin of amniotic fluid neutrophils from women with intraamniotic infection and/or inflammation and to correlate these findings with acute histologic maternal and fetal inflammatory responses in the placenta. Amniotic fluid was collected from 15 women with suspected intraamniotic infection and/or inflammation (positive microbiological cultures and/or interleukin-6 concentrations ≥2.6 ng/mL). Amniotic fluid neutrophils were purified by fluorescence-activated cell sorting, DNA was extracted, and DNA fingerprinting was performed. DNA fingerprinting was also performed in the umbilical cord and maternal blood DNA. Fluorescence in situ hybridization was assayed in women with male neonates. Blinded placental histopathological evaluations were conducted. First, DNA fingerprinting revealed that 43% (6/14) of women who underwent a single amniocentesis had mostly fetal neutrophils in the amniotic fluid. Second, DNA fingerprinting showed that 36% (5/14) of the women who underwent a single amniocentesis had predominantly maternal neutrophils in the amniotic fluid. Third, DNA fingerprinting indicated that 21% (3/14) of the women who underwent a single amniocentesis had an evident mixture of fetal and maternal neutrophils in the amniotic fluid. Fourth, DNA fingerprinting revealed that a woman who underwent 2 amniocenteses (patient 15) had fetal neutrophils first, and as infection progressed, abundant maternal neutrophils invaded the amniotic cavity. Fifth, fluorescence in situ hybridization confirmed DNA fingerprinting results by showing that both fetal and maternal neutrophils were present in the amniotic fluid. Sixth, most of the women who had predominantly amniotic fluid neutrophils of fetal origin at the time of collection delivered extremely preterm neonates (71% [5/7]). Seventh, all of the women who had predominantly amniotic fluid neutrophils of maternal origin at the time of collection delivered term or late preterm neonates (100% [6/6]). Eighth, 2 of the women who had an evident mixture of fetal and maternal neutrophils in the amniotic fluid at the time of collection delivered extremely preterm neonates (67% [2/3]), and the third woman delivered a term neonate (33% [1/3]). Finally, most of the women included in this study presented acute maternal and fetal inflammatory responses in the placenta (87% [13/15]). Amniotic fluid neutrophils can be either predominantly of fetal or maternal origin, or a mixture of both fetal and maternal origin, in women with intraamniotic infection and/or inflammation. The findings herein provide evidence that both fetal and maternal neutrophils can invade the amniotic cavity, suggesting that both the fetus and the mother participate in the host defense mechanisms against intraamniotic infection.

Neutrophil Extracellular Traps in the Amniotic Cavity of Women with Intra-Amniotic Infection: A New Mechanism of Host Defense.

Neutrophil extracellular traps (NETs) control microbial infections through their antimicrobial activities attributed to DNA, histones, granules, and cytoplasmic proteins (eg, elastase). Intra-amniotic infection is characterized by the influx of neutrophils into the amniotic cavity; therefore, the aim of this study was to determine whether amniotic fluid neutrophils form NETs in this inflammatory process. Amniotic fluid samples from women with intra-amniotic infection (n = 15) were stained for bacteria detection using fluorescent dyes. Amniotic fluid neutrophils were purified by filtration. As controls, neutrophils from maternal blood samples (n = 3) were isolated by density gradients. Isolated neutrophils were plated onto glass cover slips for culture with and without 100 nM of phorbol-12-myristate-13-acetate (PMA). NET formation was assessed by 4',6-diamidino-2-phenylindole (DAPI) staining and scanning electron microscopy. Different stages of NET formation were visualized using antibodies against elastase and histone H3, in combination with DAPI staining, by confocal microscopy. Finally, maternal or neonatal neutrophils were added to amniotic fluid samples from women without intra-amniotic infection (n = 4), and NET formation was evaluated by DAPI staining. (1) NETs were present in the amniotic fluid of women with intra-amniotic infection; (2) all of the amniotic fluid samples had detectable live and dead bacteria associated with the presence of NETs; (3) in contrast to neutrophils from the maternal circulation, amniotic fluid neutrophils did not require PMA stimulation to form NETs; (4) different stages of NET formation were observed by co-localizing elastase, histone H3, and DNA in amniotic fluid neutrophils; and (5) neither maternal nor neonatal neutrophils form NETs in the amniotic fluid of women without intra-amniotic infection. NETs are detectable in the amniotic fluid of women with intra-amniotic infection.

Measurement of Interleukin 8 in Cervicovaginal Fluid in Women With Preterm Premature Rupture of Membranes: A Comparison of Amniotic Fluid Samples.

Cervicovaginal fluid (CVF) samples may be a feasible alternative to amniotic fluid (AF) sampling in women with preterm premature rupture of the membranes (PPROMs), because PPROM causes AF to spill into the CVF. We aimed to assess the correlation and limits of agreement of interleukin 8 (IL-8) levels between CVF and AF in women with PPROM and to compare the clinical value of CVF IL-8 to AF IL-8 for the prediction of microbial invasion of amniotic cavity (MIAC). A retrospective cohort observational study was conducted on 85 women with singleton pregnancies (24-34 weeks) presenting with PPROM. The CVF samples were obtained simultaneously with AF samples retrieved by transabdominal amniocentesis. The levels of IL-8 in paired CVF and AF samples were measured with enzyme-linked immunosorbent assay in the same plate in duplicate using the same dilutions. The prevalence of a positive AF culture was 40% (34 of 85). The CVF IL-8 levels were significantly and positively correlated with AF IL-8 levels ( r = 0.778). However, the level of agreement between CVF and AF IL-8 levels yielded a Cohen κ statistic of 0.276. Paired Student t test revealed that the difference between CVF and AF IL-8 levels was statistically significant. The area under the curve for AF IL-8 was significantly higher than that for CVF IL-8 ( P = .013). In women with PPROM, IL-8 levels in CVF were significantly correlated with, but were significantly different from, those in AF samples. The CVF IL-8 has moderate predictive capability for the risk of MIAC, but this is inferior to AF IL-8.

Development of a new panel of 9 steroids in amniotic fluid in LC–MSMS

Background Since 1980 (Forest et al. [1] ) steroid analysis in amniotic fluid (AF) by radioimmunoassay were used in our laboratory as a tool for the prenatal diagnosis of 21 hydroxylase deficiency or other disorder of sex development (DSD). LC–MSMS became reference method for several reasons: analytical performance, possibility of studying a panel of several steroids. Objective Establishment of reference values for 21-deoxycortisol (21DF), deoxycorticosterone (DOC), corticosterone (Cortico), 17-hydroxyprogesterone (17OHP), 11-deoxycortisol (CS), dehydroepiandrosterone (DHEA), delta4-androstenedione (D4), testosterone (T) and dihydrotestosterone (DHT) in AF by LC–MS/MS method according to sex and term of pregnancy. Method Liquid chromatography was performed on an Agilent Technologies UPLC 1290 Infinity and selected steroids were analyzed with an Agilent Technologies 6460 triple quadrupole. Internal standard were added in 1 mL of the sample before supported liquid extraction. Materials After informed consent, we included 193 pregnant women AF performed from 14 to 36 weeks of amenorrhea, for increased maternal serum markers. All the samples associated with fetus ultrasound morphological, karyotypic or neonatal abnormalities were excluded. Results We validate the method allowing quantification of the 9 steroids in amniotic fluid with variability coefficient less than 10% intra- and inter-assay. The average concentration of these steroids was similar in both sex for 21DF( Conclusion LC–MS/MS is a rapid, sensitive method for steroid analysis in AF. References values will be very useful for the management of a mismatch between external genitalia observed by ultrasound and karyotype.

High Mobility Group-Box 1 (HMGB1) levels are increased in amniotic fluid of women with intra-amniotic inflammation-determined preterm birth, and the source may be the damaged fetal membranes

BackgroundHigh Mobility Group Box-1 (HMGB1) is considered a prototype alarmin molecule. Upon its extracellular release, HMGB1 engages pattern recognition receptors and the Receptor for Advanced Glycation End-products (RAGE) followed by an outpouring of inflammatory cytokines, including interleukin (IL)-6. MethodsWe assayed the amniotic fluid (AF) levels of HMGB1 and IL-6 in 255 women that either had a normal pregnancy outcome or delivered preterm. Immunohistochemistry on fetal membranes was used for cellular localization and validation of immunoassay findings. HMGB1 also was analyzed in amniochorion tissue explants subjected to endotoxin. ResultsAF HMGB1 levels are not gestational age regulated but are increased in women with intra-amniotic inflammation and preterm birth. The likely source is the damaged amniochorion, as demonstrated by immunohistochemistry and explant experiments. ConclusionsOur research supports a role for HMGB1 in the inflammatory response leading to preterm birth. As a delayed phase cytokine, in utero exposure to elevated AF HMGB1 levels may have an impact on the newborn beyond the time of birth.