Amniotic Fluid Of Patients Research Articles

Anatomical and physiological aspects of the mutual influence of circulatory hypoxia of the myometrium and non-progressive labour

Non-progressive labour occurs in almost 10% of deliveries and is a main reason for unplanned cesarean sections and operative vaginal births using obstetric forceps or vacuum extraction. This article describes the morphological transformation of the myometrium and uterine blood vessels that occurs during pregnancy. It is shown that the myometrium inevitably experiences hypoxia at the microstructural level during each normal or pathological labour. We analyzed recent studies of the mutual influence of myometrial hypoxia and labour dystocia. Experimental and clinical studies showed relationship between the pH values and lactate levels determined in the myometrium on its contractility. Further research is justified, including the study of the pH and lactate values in amniotic fluid in patients with non-progressive labor. Finally, that will allow for clarifying the conditions and timing of labour stimulation with oxytocin and identifying a group of patients for whom oxytocin administration is contraindicated or hopeless.

Are bacteria, fungi, and archaea present in the midtrimester amniotic fluid?

This study was conducted to determine whether bacteria, fungi, or archaea are detected in the amniotic fluid of patients who underwent midtrimester amniocentesis for clinical indications. Amniotic fluid samples from 692 pregnancies were tested by using a combination of culture and end-point polymerase chain reaction (PCR) techniques. Intra-amniotic inflammation was defined as an interleukin-6 concentration >2,935 pg/mL. Microorganisms were detected in 0.3% (2/692) of cases based on cultivation, 1.73% (12/692) based on broad-range end-point PCR, and 2% (14/692) based on the combination of both methods. However, most (13/14) of these cases did not have evidence of intra-amniotic inflammation and delivered at term. Therefore, a positive culture or end-point PCR in most patients appears to have no apparent clinical significance. Amniotic fluid in the midtrimester of pregnancy generally does not contain bacteria, fungi, or archaea. Interpretation of amniotic fluid culture and molecular microbiologic results is aided by the assessment of the inflammatory state of the amniotic cavity. The presence of microorganisms, as determined by culture or a microbial signal in the absence of intra-amniotic inflammation, appears to be a benign condition.

Изменение аминокислотного профиля плазмы пуповинной крови и амниотической жидкости от матерей с COVID-19

Neonates born to mothers with COVID-19 are at risk for infection, they may have high risk of complications during the neonatal period, and long-term health consequences. The study was aimed to define the amino acid profile of blood plasma and amniotic fluid in patients with COVID-19 in order to assess the relationship between the COVID-19 infection during the antenatal period, and metabolomic alterations in the “intrauterine” patient. The levels of 31 amino acids in the samples of amniotic fluid and cord blood plasma of pregnant women with COVID-19, obtained during delivery, were assessed by high-performance liquid chromatography-mass spectrometry. The index group included 29 patients with confirmed diagnosis of COVID-19, and the control group included 17 healthy women with uncomplicated pregnancies. There were significant (p < 0.05) differences in the concentrations of eight amino acids between the studied groups. Logistic regression models were developed (sensitivity 0.84; specificity 1) making it possible to define, whether the assessed amniotic fluid was obtained from COVID-19 patients. Significant differences in the concentrations of four amino acids were observed in the umbilical cord blood. The models developed made it possible to define whether the studied cord blood plasma belonged to controls or to COVID-19 patients (sensitivity and specificity 1). Three amino acids were detected, and their levels were significantly different in COVID-19 patients simultaneously in two points (amniotic fluid and cord blood plasma), depicting the fetal metabolome in a holistic manner. The impact of the virus on those infected results in pronounced metabolomic alterations in the amniotic fluid and the fetal cord blood plasma, which may lead to impaired programming of protein production, but never show up at birth.

Pharmacological treatment in pregnant women with moderate symptoms of coronavirus disease 2019 (COVID-19) pneumonia

Objective Pregnant women with moderate symptoms of COVID-19 are at risk for progressing to severe or critical illness. While there are limited data on the management of severe COVID-19 during pregnancy, information on pharmacological treatments of moderate COVID-19 is lacking. We report clinical outcomes of pregnant women hospitalized due to moderate COVID-19 illness treated with a 5-day course of remdesivir, antibiotics, and/or glucocorticoids. Materials and methods Case series of pregnant women hospitalized with moderate symptoms of COVID −19 pneumonia at two INOVA Health System hospitals from April 1 to December 31, 2020. Primary outcome was clinical recovery (breathing on ambient air and/or hospital discharge) on hospital day 7 (HD7). Cox regression analysis was performed to evaluate which variables were associated with the primary outcome. Results Out of 748 pregnant women with confirmed infection by reverse transcriptase polymerase chain reaction, 35 were hospitalized due to moderate symptoms of COVID-19 pneumonia (median gestational age 29 weeks). There was no maternal death. Seventeen patients received remdesivir within 48 hours of hospitalization: 15 remained with moderate symptoms and 2 (who also received glucocorticoids) had progressed to critical COVID-19 at remdesivir initiation; all 17 women in this group achieved clinical recovery on HD7. Seven women received remdesivir >48 hours following admission after they began treatment with glucocorticoids ± antibiotics and worsened to severe or critical disease; they all required supplemental oxygen on HD7. Eleven women were treated with antibiotics ± glucocorticoids but no remdesivir; on HD7, 3/11 achieved clinical recovery. Clinical recovery was significantly different among treatment groups; p < 0.001. When analyzing only women who remained with moderate symptoms at pharmacological treatments initiation, all 15 on remdesivir and only 3 of 11 on antibiotics achieved clinical recovery on HD7; p < 0.001. Delaying remdesivir for >48 hours after admission (HR 2.32, 95% CI 1.45–4.16) and >4-day duration of symptoms prior to hospitalization (HR 1.65, 95% CI 1.27–3.50) had an inverse association with clinical recovery. Incidental oligohydramnios was seen in 3/24 (12.5%) of women within 5 days of completing remdesivir treatment. Elevated transaminases was prevalent in women treated with remdesivir (8/24, 33.3%). Conclusion In our cohort, prompt initiation of remdesivir in pregnant women hospitalized with moderate symptoms of COVID-19 pneumonia within 48 hours of admission prevented worsening and allowed a fast clinical recovery by HD7. Deferring remdesivir for >48 hours after hospitalization and duration of symptoms >4 days before admission were independently associated with delayed clinical recovery and longer hospital admission. Ultrasound evaluation of the amniotic fluid in patients recovering from COVID-19 hospitalization should be considered.

Antibiotic administration reduces the rate of intraamniotic inflammation in preterm prelabor rupture of the membranes

Preterm prelabor rupture of the membranes (PPROM) is frequently complicated by intraamniotic inflammatory processes such as intraamniotic infection and sterile intraamniotic inflammation. Antibiotic therapy is recommended to patients with PPROM to prolong the interval between this complication and delivery (latency period), reduce the risk of clinical chorioamnionitis, and improve neonatal outcome. However, there is a lack of information regarding whether the administration of antibiotics can reduce the intensity of the intraamniotic inflammatory response or eradicate microorganisms in patients with PPROM. The first aim of the study was to determine whether antimicrobial agents can reduce the magnitude of the intraamniotic inflammatory response in patients with PPROM by assessing the concentrations of interleukin-6 in amniotic fluid before and after antibiotic treatment. The second aim was to determine whether treatment with intravenous clarithromycin changes the microbial load of Ureaplasma spp DNA in amniotic fluid. A retrospective cohort study included patients who had (1) a singleton gestation, (2) PPROM between 24+0 and 33+6 weeks, (3) a transabdominal amniocentesis at the time of admission, and (4) intravenous antibiotic treatment (clarithromycin for patients with intraamniotic inflammation and benzylpenicillin/clindamycin in the cases of allergy in patients without intraamniotic inflammation) for 7 days. Follow-up amniocenteses (7th day after admission) were performed in the subset of patients with a latency period lasting longer than 7 days. Concentrations of interleukin-6 were measured in the samples of amniotic fluid with a bedside test, and the presence of microbial invasion of the amniotic cavity was assessed with culture and molecular microbiological methods. Intraamniotic inflammation was defined as a bedside interleukin-6 concentration ≥745 pg/mL in the samples of amniotic fluid. Intraamniotic infection was defined as the presence of both microbial invasion of the amniotic cavity and intraamniotic inflammation; sterile intraamniotic inflammation was defined as the presenceof intraamniotic inflammation without microbial invasion of the amniotic cavity. A total of 270 patients with PPROM were included in this study: 207 patients delivered within 7 days and 63 patients delivered after 7 days of admission. Of the 63 patients who delivered after 7 days following the initial amniocentesis, 40 underwent a follow-up amniocentesis. Patients with intraamniotic infection (n= 7) and sterile intraamniotic inflammation (n= 7) were treated with intravenous clarithromycin. Patients without either microbial invasion of the amniotic cavity or intraamniotic inflammation (n= 26) were treated with benzylpenicillin or clindamycin. Treatment with clarithromycin decreased the interleukin-6 concentration in amniotic fluid at the follow-up amniocentesis compared to the initial amniocentesis in patients with intraamniotic infection (follow-up: median, 295 pg/mL, interquartile range [IQR], 72-673 vs initial: median, 2973 pg/mL, IQR, 1750-6296; P= .02) and in those with sterile intraamniotic inflammation (follow-up: median, 221 pg/mL, IQR 118-366 pg/mL vs initial: median, 1446 pg/mL, IQR, 1300-2941; P= .02). Samples of amniotic fluid with Ureaplasma spp DNA had a lower microbial load at the time offollow-up amniocentesis compared to the initial amniocentesis (follow-up: median, 1.8× 104 copies DNA/mL, 2.9× 104 to 6.7× 108 vs initial: median, 4.7× 107 copies DNA/mL, interquartile range, 2.9× 103 to 3.6× 107; P= .03). Intravenous therapy with clarithromycin was associated with a reduction in the intensity of the intraamniotic inflammatory response in patients with PPROM with either intraamniotic infection or sterile intraamniotic inflammation. Moreover, treatment with clarithromycin was related to a reduction in the load of Ureaplasma spp DNA in the amniotic fluid of patients with PPROM <34 weeks of gestation.

ITRAQ-based proteomics and in vitro experiments reveals essential roles of ACE and AP-N in the renin–angiotensin system-mediated congenital ureteropelvic junction obstruction

ObjectiveUreteropelvic junction obstruction (UPJO) is a common renal obstructive disorder, but its pathogenic mechanisms remain largely unclear. We aimed to investigate the potential involvement of the renin–angiotensin system in congenital UPJO pathogenesis. MethodsDifferentially expressed proteins in exosomes isolated from amniotic fluid of patients with congenital UPJO were characterized using iTRAQ (isobaric tags for relative and absolute quantification)-based proteomics. The expressions of angiotensin-converting enzyme (ACE) and aminopeptidase N (AP-N) in HK2 cells were inhibited by quinapril and siRNA, respectively. Cell proliferation and reactive oxygen species were measured by EdU staining and flow cytometry, respectively. Gene expression was detected by Western blot or qRT-PCR. The inflammatory factors were measured through ELISA. Mice that underwent unilateral ureteral obstruction were used as the animal model. ResultsThe identity of exosomes from amniotic fluids was confirmed by the expression of CD9 and CD26. In total, 633 differentially expressed proteins were identified in the amniotic fluid-derived exosomes from patients with UPJO, including 376 up- and 257 down-regulated proteins associated with multiple biological processes. Of them, ACE and AP-N were significantly decreased in the amniotic fluid exosomes. Inhibition of ACE and AP-N resulted in suppressed cell proliferation; repressed IARP, AT1R, and MAS1 expression; elevated ROS production; and increased IL-1β, TNF-α, and IL-6 levels in HK2 cells. Decreased ACE expression and elevated IL-1β levels were also observed in the mouse model. ConclusionSuppression of ACE and AP-N expression mediates congenital UPJO pathogenesis by repressing renal tubular epithelial proliferation, promoting ROS production, and enhancing inflammatory factor expression.

Microbial burden and inflammasome activation in amniotic fluid of patients with preterm prelabor rupture of membranes.

Background Intra-amniotic inflammation, which is associated with adverse pregnancy outcomes, can occur in the presence or absence of detectable microorganisms, and involves activation of the inflammasome. Intra-amniotic inflammasome activation has been reported in clinical chorioamnionitis at term and preterm labor with intact membranes, but it has not yet been investigated in women with preterm prelabor rupture of membranes (preterm PROM) in the presence/absence of detectable microorganisms. The aim of this study was to determine whether, among women with preterm PROM, there is an association between detectable microorganisms in amniotic fluid and intra-amniotic inflammation, and whether intra-amniotic inflammasome activation correlates with microbial burden. Methods Amniotic fluids from 59 cases of preterm PROM were examined for the presence/absence of microorganisms through culture and 16S ribosomal RNA (rRNA) gene quantitative real-time polymerase chain reaction (qPCR), and concentrations of interleukin-6 (IL-6) and ASC [apoptosis-associated spec-like protein containing a caspase recruitment domain (CARD)], an indicator of inflammasome activation, were determined. Results qPCR identified more microbe-positive amniotic fluids than culture. Greater than 50% of patients with a negative culture and high IL-6 concentration in amniotic fluid yielded a positive qPCR signal. ASC concentrations were greatest in patients with high qPCR signals and elevated IL-6 concentrations in amniotic fluid (i.e. intra-amniotic infection). ASC concentrations tended to increase in patients without detectable microorganisms but yet with elevated IL-6 concentrations (i.e. sterile intra-amniotic inflammation) compared to those without intra-amniotic inflammation. Conclusion qPCR is a valuable complement to microbiological culture for the detection of microorganisms in the amniotic cavity in women with preterm PROM, and microbial burden is associated with the severity of intra-amniotic inflammatory response, including inflammasome activation.

Contemporary placentography: harm or benefit?

Currently, pediatricians, neonatologists of maternity hospitals often take part in perinatal consultations. One of the indications for early delivery may be chronic placental insufficiency diagnosed during an ultrasound examination.&#x0D; Objective: to evaluate the effectiveness of the main method of diagnosis of placental insufficiency at the present time – ultrasound evaluation of the placenta.&#x0D; Materials and methods. An analysis was made of 357 birth histories, exchange cards, protocols for screening ultrasound in the third trimester of pregnancy and conclusions of pathoanatomical investigation.&#x0D; Results. To substantiate the need for prenatal diagnosis of chronic kidney disease, a comparison was made of the evaluation of newborn babies on the Apgar scale and the state of the placenta according to histological data. With compensated chronic placental insufficiency, there was no difference in Apgar scores at the first and fifth minutes, whereas in the subcompensated stage, Apgar score on the Apgar scale was significantly lower in the first and fifth minutes than in the control group. To identify possible in practical use of the objective parameters of diagnosis of chronic kidney disease, the thickness of the placenta was measured during the screening ultrasound in the third trimester of pregnancy, as well as the thickness of the placenta after its separation. Significant differences in the thickness of the placenta or in the screening ultrasound, or when measuring the placenta after separation in patients with and without chronic placental insufficiency was not detected. When assessing a violation of the rate of maturation of the placenta in the third trimester of pregnancy, no significant differences were found in the presence and absence of placental insufficiency. An analysis of the amniotic index was carried out to determine the amount of amniotic fluid in patients of the main and control groups, which revealed no significant differences between the groups.&#x0D; Conclusions. The authors believe that the main indicators currently used for diagnosis of chronic placental insufficiency (thickness of the placenta, degree of maturity of the placenta, appearance of structural changes in the placenta, change in the amount of amniotic fluid) are uninformative and modern placenography does more harm than benefit. The only parameter determined during the screening study in the third trimester of pregnancy and having significant differences in the main and control groups is fetal hypotrophy.

Comparison of two identification and susceptibility test kits for Ureaplasma spp and Mycoplasma hominis in amniotic fluid of patients at high risk for intra-amniotic infection

Objective: Ureaplasma urealyticum and Mycoplasma hominis are the most common microorganisms found in the amniotic fluid of patients at risk for preterm delivery. However, culture techniques for genital mycoplasms require special conditions, are barely considered as part of the evaluation of suspected intra-amniotic infection (IAI) and the results are available within 2 and 7 days. The objectives of this study are to validate the use of two commercially available kits (Mycoplasma IES y MYCOFAST® RevolutioN) for the identification of Ureaplasma spp. and Mycoplasma hominis in amniotic fluid, to compare the results of these kits with those obtained by culture and real-time polymerase chain reaction (qPCR) and to report the antibiotic sensitivity profile of the genital mycoplasms identified.Methods: This is a prospective cohort study including women with singleton and twin gestations between 16 and 36 weeks. Patients were admitted to perform an amniocentesis due to pregnancy complications considered at high risk for IAI (e.g. preterm labor with intact membranes, preterm prelabour rupture of membranes, short cervix, etc.), treatment of polyhydramnios, and for the assessment of fetal death and fever without a focus.Results: Overall, 93 patients underwent amniocentesis and 63 had results available for all tests. The prevalence of a positive culture was 6% (4/63). There were four cases of Ureaplasma spp. and none of Mycoplasma hominis. The qPCR identified one case as Ureaplasma spp., one case as Ureaplasma parvum and two cases as Ureaplasma urealyticum. For all tests, the diagnostic performance was as follows: sensitivity 100% [95% CI (39.8–100%)], specificity 100% [95% CI (93.9–100%)], positive predictive value 100% [95% CI (39.8–100%)] and negative predictive value 100% [95% CI (93.9–100%)]. In this cohort, Ureaplasma spp. showed low resistance to erythromycin, but a high resistance to clindamycin and clarithromycin that may change according to the antibiotic concentration.Conclusions: To our knowledge, this is the first study that validates the use of the Mycoplasma IES and MYCOFAST® RevolutioN kits for the identification of genital mycoplasmas in amniotic fluid. The results of these kits are mostly available within 24 hours, have an excellent correlation with those from broth cultures and qPCR and characterize the antibiotic sensitivity profile of the genital mycoplasms identified, providing an opportunity for specific treatment in cases of IAI. Further validation studies in other populations are needed.

Clinical chorioamnionitis at term IX: in vivo evidence of intra-amniotic inflammasome activation.

Background The inflammasome has been implicated in the mechanisms that lead to spontaneous labor at term. However, whether the inflammasome is activated in the amniotic cavity of women with clinical chorioamnionitis at term is unknown. Herein, by measuring extracellular ASC [apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (CARD)], we investigated whether there is in vivo inflammasome activation in amniotic fluid of patients with clinical chorioamnionitis at term with sterile intra-amniotic inflammation and in those with intra-amniotic infection. Methods This was a retrospective cross-sectional study that included amniotic fluid samples collected from 76 women who delivered after spontaneous term labor with diagnosed clinical chorioamnionitis. Intra-amniotic inflammation was defined as an elevated amniotic fluid interleukin (IL)-6 concentration ≥2.6 ng/mL, and intra-amniotic infection was diagnosed by the presence of microbial invasion of the amniotic cavity (MIAC) accompanied by intra-amniotic inflammation. Patients were classified into the following groups: (1) women without intra-amniotic inflammation or infection (n=16); (2) women with MIAC but without intra-amniotic inflammation (n=5); (3) women with sterile intra-amniotic inflammation (n=15); and (4) women with intra-amniotic infection (n=40). As a readout of in vivo inflammasome activation, extracellular ASC was measured in amniotic fluid by enzyme-linked immunosorbent assay. Acute inflammatory responses in the amniotic fluid and placenta were also evaluated. Results In clinical chorioamnionitis at term: (1) amniotic fluid concentrations of ASC (extracellular ASC is indicative of in vivo inflammasome activation) and IL-6 were greater in women with intra-amniotic infection than in those without intra-amniotic inflammation, regardless of the presence of MIAC; (2) amniotic fluid concentrations of ASC and IL-6 were also higher in women with sterile intra-amniotic inflammation than in those without intra-amniotic inflammation, regardless of the presence of MIAC; (3) amniotic fluid concentrations of IL-6, but not ASC, were more elevated in women with intra-amniotic infection than in those with sterile intra-amniotic inflammation; (4) a positive and significant correlation was observed between amniotic fluid concentrations of ASC and IL-6; (5) no differences were observed in amniotic fluid ASC and IL-6 concentrations between women with and without MIAC in the absence of intra-amniotic inflammation; (6) women with intra-amniotic infection had elevated white blood cell counts and reduced glucose levels in amniotic fluid compared to the other three study groups; and (7) women with intra-amniotic infection presented higher frequencies of acute maternal and fetal inflammatory responses in the placenta than those with sterile intra-amniotic inflammation. Conclusion The intra-amniotic inflammatory response, either induced by alarmins or microbes, is characterized by the activation of the inflammasome - as evidenced by elevated amniotic fluid concentrations of extracellular ASC - in women with clinical chorioamnionitis at term. These findings provide insight into the intra-amniotic inflammatory response in women with clinical chorioamnionitis at term.

A clinical retrospective study on 160 cases of multiple umbilical cord around the neck

Objective: To compare the neonatal and maternal outcomes between the patients with umbilical cord around the neck (≥3 loops) and with (1 or 2 loops). Methods: A retrospective analysis was conducted on the clinical data of 160 cases with multiple umbilical cord around the neck (≥3 loops) in the Department of Obstetrics, Women's Hospital School of Medicine Zhejiang University between January 2014 and April 2017.For each case, two control women who gave birth at the same day with vertex position and singletons were selected.The neonatal and maternal outcomes were compared. Result: (1) The incidence of cord multiple cord around the neck (≥3 loops) in our hospital was 0.45%. (2) Comparison between groups: The rate of abnormal fetal movement or abnormal cardiotocography in case group was higher than those of the control group, (33.13%, 53/160) vs (8.13%, 26/320), with significant difference, P=0.000.The Umbilical Artery Systolic/diastolic (S/D) ratio of the case group was lower than that of the control group, 2.00(0.40) vs 2.14(0.40), with significant difference, P=0.000.The cesarean section rate of the case group was higher than that of the control group, (81.25%, 130/160) vs (7.50%, 24/320), and the difference was statistically significant, P=0.000.Birth Weight of the case group was lower than that of the control group, (3 143±367) g vs (3 323±349) g, with significant difference, P=0.000.(3) Comparison between subgroups: The rate of lateral incision or obstetrical forceps in the subgroup of virginal delivery among the case group (n=30) was higher than that in the control group (n=296), (30.00%, 9/30) vs (12.50%, 37/296), with significant difference, P=0.009.While, the Apgar score at 1 and 5 min of the virginal delivery case in the case group were lower than that in the control group, 10(1.25) vs 10(0) and 10(0) vs 10(0), there were both significant difference, P=0.000, 0.012, respectively.The rate of meconium-stained amniotic fluid, 1 min Apgar score of ≤7 and NICU admission were showed no significance, all P>0.05.(4) After Logistic regression, the four factors most closely associated with meconium-stained amniotic fluid in patients with multiple cord around the neck (≥3 loops), which were gestational age ≥39 weeks, Birth Weight >3 500 g, umbilical cord around the neck ≥4 loops, and trial of labor. Conclusion: (1) Multiple umbilical cord around the neck (≥3 loops) had a more positive treatment. Vaginal delivery led to lower APGAR score, but didn't increase the incidence of neonatal asphyxia.(2) Independent risk factors for meconium-stained amniotic fluid were gestational age ≥39 weeks, Birth Weight>3 500 g, umbilical cord around the neck ≥4 loops and trial of labor.

Adverse effects of maternal enterovirus infection on the pregnancy outcome: a prospective and retrospective pilot study

BackgroundEnteroviruses account for about one billion infections worldwide each year, the majority remain asymptomatic. Data on enterovirus infections during pregnancy appear to be very rare. Several cases have been reported in the literature of fetal and neonatal complications attributed to these viruses, but prospective data on these infections during pregnancy are not available.ObjectiveTo estimate the prevalence of enterovirus infections in febrile syndromes in pregnant women, and in case of in utero fetal death (IUFD).MethodsTtri-centric observational cohort study. We performed prospective inclusion for patients with fever during a four-month period. We also analyzed the amniotic fluid in patients with unexplained IUFD retrospectively during a five-year period. Investigations of enteroviruses are made by RT-PCR from routine biological samples (amniocentesis, RT-PCR in maternal blood or CSF).ResultsProspectively, 33 patients were included during the study period. We have identified 4 cases of confirmed enterovirus infection (12.4%). We have recorded a severe form of perinatal enterovirus infection involving the vital prognosis of the newborn.In the retrospective cohort of 75 IUFD cases, we had only one case of enterovirus-positive RT-PCR in amniotic fluid during 5 years, meaning a frequency of 1.3%. We did not had any positive EV case in case of early miscarriage, but the limited number of inclusions cannot help us to conclude.ConclusionEnteroviruses are probably an underestimated cause of obstetric and neonatal complications. Investigation of enterovirus by PCR should be discussed during pregnancy and peripartum in case of febrile syndrome with no obvious bacterial cause, and unexplained IUFD.

Infection-induced thrombin production: a potential novel mechanism for preterm premature rupture of membranes (PPROM)

Preterm premature rupture of membranes is a leading contributor to maternal and neonatal morbidity and death. Epidemiologic and experimental studies have demonstrated that thrombin causes fetal membrane weakening and subsequently preterm premature rupture of membranes. Although blood is suspected to be the likely source of thrombin in fetal membranes and amniotic fluid of patients with preterm premature rupture of membranes, this has not been proved. Ureaplasma parvum is emerging as a pathogen involved in prematurity, which includes preterm premature rupture of membranes; however, until now, prothrombin production that has been induced directly by bacteria in fetal membranes has not been described. This study was designed to investigate whether Ureaplasma parvum exposure can induce prothrombin production in fetal membranes cells. Primary fetal membrane cells (amnion epithelial, chorion trophoblast, and decidua stromal) or full-thickness fetal membrane tissue explants from elective, term, uncomplicated cesarean deliveries were harvested. Cells or tissue explants were infected with live Ureaplasma parvum (1×105, 1×106 or 1×107 colony-forming units per milliliter) or lipopolysaccharide (Escherichia coli J5, L-5014; Sigma Chemical Company, St. Louis, MO; 100 ng/mL or 1000 ng/mL) for 24 hours. Tissue explants were fixed for immunohistochemistry staining of thrombin/prothrombin. Fetal membrane cells were fixed for confocal immunofluorescent staining of the biomarkers of fetal membrane cell types and thrombin/prothrombin. Protein and messenger RNA were harvested from the cells and tissue explants for Western blot or quantitative reverse transcription polymerase chain reaction to quantify thrombin/prothrombin protein or messenger RNA production, respectively. Data are presented as mean values ±standard errors of mean. Data were analyzed using 1-way analysis of variance with post hoc Dunnett's test. Prothrombin production and localization were confirmed by Western blot and immunostainings in all primary fetal membrane cells and tissue explants. Immunofluorescence observations revealed a perinuclear localization of prothrombin in amnion epithelial cells. Localization of prothrombin in chorion and decidua cells was perinuclear and cytoplasmic. Prothrombin messenger RNA and protein expression in fetal membranes were increased significantly by Ureaplasma parvum, but not lipopolysaccharide, treatments in a dose-dependent manner. Specifically, Ureaplasma parvum at a dose of 1×107 colony-forming units/mL significantly increased both prothrombin messenger RNA (fold changes in amnion: 4.1±1.9; chorion: 5.7±4.2; decidua: 10.0±5.4; fetal membrane: 9.2±3.0) and protein expression (fold changes in amnion: 138.0±44.0; chorion: 139.6±15.1; decidua: 56.9±29.1; fetal membrane: 133.1±40.0) compared with untreated control subjects. Ureaplasma parvum at a dose of 1×106 colony-forming units/mL significantly up-regulated prothrombin protein expression in chorion cells (fold change: 54.9±5.3) and prothrombin messenger RNA expression in decidua cells (fold change: 4.4±1.9). Our results demonstrate that prothrombin can be produced directly by fetal membrane amnion, chorion, and decidua cells. Further, prothrombin production can be stimulated by Ureaplasma parvum exposure in fetal membranes. These findings represent a potential novel underlying mechanism of Ureaplasma parvum-induced rupture of fetal membranes.

Evidence of perturbations of the cytokine network in preterm labor

Intraamniotic inflammation/infection is the only mechanism of disease with persuasive evidence of causality for spontaneous preterm labor/delivery. Previous studies about the behavior of cytokines in preterm labor have been largely based on the analysis of the behavior of each protein independently. Emerging evidence indicates that the study of biologic networks can provide insight into the pathobiology of disease and improve biomarker discovery. The goal of this study was to characterize the inflammatory-related protein network in the amniotic fluid of patients with preterm labor. A retrospective cohort study was conducted that included women with singleton pregnancies who had spontaneous preterm labor and intact membranes (n = 135). These patients were classified according to the results of amniotic fluid culture, broad-range polymerase chain reaction coupled with electrospray ionization mass spectrometry, and amniotic fluid concentration of interleukin (IL)-6 into the following groups: (1) those without intraamniotic inflammation (n = 85), (2) those with microbial-associated intraamniotic inflammation (n = 15), and (3) those with intraamniotic inflammation without detectable bacteria (n = 35). Amniotic fluid concentrations of 33 inflammatory-related proteins were determined with the use of a multiplex bead array assay. Patients with preterm labor and intact membranes who had microbial-associated intraamniotic inflammation had a higher amniotic fluid inflammatory-related protein concentration correlation than those without intraamniotic inflammation (113 perturbed correlations). IL-1β, IL-6, macrophage inflammatory protein (MIP)-1α, and IL-1α were the most connected nodes (highest degree) in this differential correlation network (degrees of 20, 16, 12, and 12, respectively). Patients with sterile intraamniotic inflammation had correlation patterns of inflammatory-related proteins, both increased and decreased, when compared to those without intraamniotic inflammation (50 perturbed correlations). IL-1α, MIP-1α, and IL-1β were the most connected nodes in this differential correlation network (degrees of 12, 10, and 7, respectively). There were more coordinated inflammatory-related protein concentrations in the amniotic fluid of women with microbial-associated intraamniotic inflammation than in those with sterile intraamniotic inflammation (60 perturbed correlations), with IL-4 and IL-33 having the largest number of perturbed correlations (degrees of 15 and 13, respectively). We report for the first time an analysis of the inflammatory-related protein network in spontaneous preterm labor. Patients with preterm labor and microbial-associated intraamniotic inflammation had more coordinated amniotic fluid inflammatory-related proteins than either those with sterile intraamniotic inflammation or those without intraamniotic inflammation. The correlations were also stronger in patients with sterile intraamniotic inflammation than in those without intraamniotic inflammation. The findings herein could be of value in the development of biomarkers of preterm labor.

Anticardiolipin Antibody Isotype Determination in Amniotic Fluid of Pregnant Patients with Systemic Lupus Erythematosus and/or Antiphospholipid Syndrome

Objective: To determine the levels and isotypes of aCl, as well as anti beta 2 glycoprotein 1 (antiβ2-GP1) antibodies in serum and amniotic fluid of pregnant patients with SLE and/or APLS, and healthy pregnant women serving as a control group. Material and Methods: We analyzed serum and amniotic fluid of pregnant patients with SLE and/or APLS, and of healthy pregnant women through ELISA. Results were compared using a Student’s T test. Results: 6 of 13 patients (46.1%), 5 with SLE and one with primary APLS had antiphospholipid antibodies in amniotic fluid. Two patients had IgG aCl and 4 patients had antiβ2-GP1 (one of them also showing IgM) in amniotic fluid. In serum, 4 patients (30%) had antiphospholipid antibodies present (one IgG aCl and three anti β2-GP1) as opposed to none in the control group having antiphospholipid antibodies in amniotic fluid. Only one control had IgM aCl in serum. Antiβ2-GP1 in the amniotic fluid of patients showed a statistically significant value when compared to controls. Conclusion: aCl and antiβ2-GP1 may be present in the amniotic fluid of patients with and without a history of fetal loss. The presence of IgM aCl and antiβ2-GP1 in amniotic fluid suggests its localized production.

Bacteria and endotoxin in meconium-stained amniotic fluid at term: could intra-amniotic infection cause meconium passage?

Background: Meconium-stained amniotic fluid (MSAF) is a common occurrence among women in spontaneous labor at term, and has been associated with adverse outcomes in both mother and neonate. MSAF is a risk factor for microbial invasion of the amniotic cavity (MIAC) and preterm birth among women with preterm labor and intact membranes. We now report the frequency of MIAC and the presence of bacterial endotoxin in the amniotic fluid of patients with MSAF at term.Materials and methods: We conducted a cross-sectional study including women in presumed preterm labor because of uncertain dates who underwent amniocentesis, and were later determined to be at term (n = 108). Patients were allocated into two groups: (1) MSAF (n = 66) and (2) clear amniotic fluid (n = 42). The presence of bacteria was determined by microbiologic techniques, and endotoxin was detected using the Limulus amebocyte lysate (LAL) gel clot assay. Statistical analyses were performed to test for normality and bivariate comparisons.Results: Bacteria were more frequently present in patients with MSAF compared to those with clear amniotic fluid [19.6% (13/66) versus 4.7% (2/42); p < 0.05]. The microorganisms were Gram-negative rods (n = 7), Ureaplasma urealyticum (n = 4), Gram-positive rods (n = 2) and Mycoplasma hominis (n = 1). The LAL gel clot assay was positive in 46.9% (31/66) of patients with MSAF, and in 4.7% (2/42) of those with clear amniotic fluid (p < 0.001). After heat treatment, the frequency of a positive LAL gel clot assay remained higher in the MSAF group [18.1% (12/66) versus 2.3% (1/42), p < 0.05]. Median amniotic fluid IL-6 concentration (ng/mL) was higher [1.3 (0.7–1.9) versus 0.6 (0.3–1.2), p = 0.04], and median amniotic fluid glucose concentration (mg/dL) was lower [6 (0–8.9) versus 9 (7.4–12.6), p < 0.001] in the MSAF group, than in those with clear amniotic fluid.Conclusion: MSAF at term was associated with an increased incidence of MIAC. The index of suspicion for an infection-related process in postpartum women and their neonates should be increased in the presence of MSAF.

Fragmented Forms of Insulin-Like Growth Factor Binding Protein-1 in Amniotic Fluid of Patients With Preterm Labor and Intact Membranes

To determine the clinical significance of an increase in various fragmented forms of insulin-like growth factor binding protein-1 (IGFBP-1) in amniotic fluid (AF), a retrospective cohort study was conducted in 103 consecutive patients with preterm labor and intact membranes. Amniotic fluid samples were cultured for aerobic and anaerobic bacteria, and mycoplasmas, and then assayed for matrix metalloproteinase-8. Fragmented-to-intact IGFBP-1 ratios were evaluated by densitometric analysis of Western blot assays. Intact IGFBP-1 (30 kDa) and 21, 17, and 12 kDa fragments were detected in AF. Median ratios of fragmented-to-intact IGFBP-1 were higher in patients whose neonates had significant morbidity than in those whose neonates did not (P < .05), in patients spontaneously delivered within 2 and 7 days from amniocentesis than in those delivered after 2 and 7 days (P < .05), and in patients with intra-amniotic infection/inflammation than in those without (P < .001). Collectively, fragmented IGFBP-1 in AF may be indicators for adverse perinatal outcomes.

499: Fragmented forms of insulin-like growth factor binding protein-1 in amniotic fluid of patients with preterm labor and intact membranes

The purpose of this study was to examine if an increase in the various fragmented forms of insulin-like growth factor binding protein-1 (IGFBP-1) in amniotic fluid (AF) is associated with intra-amniotic infection and/or inflammation, and adverse perinatal outcomes in patients with preterm labor and intact membranes.

Unexplained fetal death is associated with increased concentrations of anti-angiogenic factors in amniotic fluid

Objective. Angiogenesis is critical for successful pregnancy. An anti-angiogenic state has been implicated in preeclampsia, fetal growth restriction and fetal death. Increased maternal plasma concentrations of the anti-angiogenic factor, soluble vascular endothelial growth factor receptor (sVEGFR)-1, have been reported in women with preeclampsia and in those with fetal death. Recent observations indicate that an excess of sVEGFR-1 and soluble endoglin (sEng) is also present in the amniotic fluid of patients with preeclampsia. The aim of this study was to determine whether fetal death is associated with changes in amniotic fluid concentrations of sVEGFR-1 and sEng, two powerful anti-angiogenic factors.Study design. This cross-sectional study included patients with fetal death (n = 35) and controls (n = 129). Fetal death was subdivided according to clinical circumstances into: (1) unexplained (n = 25); (2) preeclampsia and/or placental abruption (n = 5); and (3) chromosomal/congenital anomalies (n = 5). The control group consisted of patients with preterm labor (PTL) who delivered at term (n = 92) and women at term not in labor (n = 37). AF concentrations of sVEGFR-1 and sEng were determined by ELISA. Non-parametric statistics and logistic regression analysis were applied.Results. (1) Patients with a fetal death had higher median amniotic fluid concentrations of sVEGFR-1 and sEng than women in the control group (p < 0.001 for each); (2) these results remained significant among different subgroups of stillbirth (p < 0.05 for each); and (3) amniotic fluid concentrations of sVEGFR-1 and those of sEng above the third quartile were associated with a significant risk of unexplained preterm fetal death (adjusted OR = 10.8; 95%CI 1.3–89.2 and adjusted OR 87; 95% CI 2.3–3323, respectively).Conclusion. Patients with an unexplained fetal death at diagnosis are characterized by an increase in the amniotic fluid concentrations of sVEGFR-1 and sEng. These observations indicate that an excess of anti-angiogenic factors in the amniotic cavity is associated with unexplained fetal death especially in preterm gestations.

Detection of ureaplasmas by the polymerase chain reaction in the amniotic fluid of patients with cervical insufficiency

The purpose of this study was to determine the clinical significance of detecting microbial footprints of ureaplasmas in amniotic fluid (AF) using specific primers for the polymerase chain reaction (PCR) in patients presenting with cervical insufficiency. Amniocentesis was performed in 58 patients with acute cervical insufficiency (cervical dilatation, > or =1.5 cm) and intact membranes, and without regular contractions (gestational age, 16-29 weeks). AF was cultured for aerobic and anaerobic bacteria as well as genital mycoplasmas. Ureaplasmas (Ureaplasma urealyticum and Ureaplasma parvum) were detected by PCR using specific primers. Patients were divided into three groups according to the results of AF culture and PCR for ureaplasmas: those with a negative AF culture and a negative PCR (n=44), those with a negative AF culture and a positive PCR (n=10), and those with a positive AF culture regardless of PCR result (n=4). 1) Ureaplasmas were detected by PCR in 19.0% (11/58) of patients, by culture in 5.2% (3/58), and by culture and/or PCR in 22.4% (13/58); 2) Among the 11 patients with a positive PCR for ureaplasmas, the AF culture was negative in 91% (10/11); 3) Patients with a negative AF culture and a positive PCR for ureaplasmas had a significantly higher median AF matrix metalloproteinase-8 (MMP-8) concentration and white blood cell (WBC) count than those with a negative AF culture and a negative PCR (P<0.001 and P<0.05, respectively); 4) Patients with a positive PCR for ureaplasmas but a negative AF culture had a higher rate of spontaneous preterm birth within two weeks of amniocentesis than those with a negative AF culture and a negative PCR (P<0.05 after adjusting for gestational age at amnio-centesis); 5) Of the patients who delivered within two weeks of amniocentesis, those with a positive PCR for ureaplasmas and a negative AF culture had higher rates of histologic amnionitis and funisitis than those with a negative AF culture and a negative PCR (P<0.05 after adjusting for gestational age at amniocentesis, for each); 6) However, no significant differences in the intensity of the intra-amniotic inflammatory response and perinatal outcome were found between patients with a positive AF culture and those with a negative AF culture and a positive PCR. 1) Cultivation techniques for ureaplasmas did not detect most cases of intra-amniotic infection caused by these microorganisms (91% of cases with cervical insufficiency and microbial footprints for ureaplasmas in the amniotic cavity had a negative AF culture); 2) Patients with a negative AF culture and a positive PCR assay were at risk for intra-amniotic and fetal inflammation as well as spontaneous preterm birth.