Time Of Amniocentesis Research Articles

DNA concentrations in amniotic fluid according to gestational age and fetal sex: data from 2573 samples

PurposeIn some cases of prenatal genetic testing, an ample amount of fetal DNA is needed, to allow for parallel testing (conducting several genetic tests simultaneously). This study investigated the association between amniotic fluid DNA concentration and various factors. We aimed to define the required amount of amniotic fluid to be extracted in amniocentesis, to allow parallel testing throughout gestational weeks.MethodsDNA concentration was analyzed from amniocentesis samples taken during the years 2016–2022. Sex association was also analyzed in postnatal whole blood samples from a separate cohort. Theoretical minimum volume of amniotic fluid needed to ensure enough DNA for chromosomal microarray analysis and exome sequencing was calculated.ResultsWe focused our analysis on 2573 samples, which were taken during weeks 17–23 and 30–35. DNA concentrations increased from weeks 17 to 21, with relatively stable concentrations thereafter. Significantly higher DNA concentrations were seen in pregnancies of female fetuses. DNA concentrations in postnatal whole blood samples did not show this association. Across most weeks, the volume needed to extract 2 µg of DNA from 95% of the samples was about 34 ml.ConclusionDNA concentrations in amniotic fluid vary according to gestational age and are higher in pregnancies of female fetuses. This should be considered when determining the volume of fluid extracted and the timing of amniocentesis, with greater volumes needed in earlier stages of pregnancy.

Predictive potential of various plasma inflammation-, angiogenesis-, and extracellular matrix remodeling-associated mediators for intra-amniotic inflammation and/or microbial invasion of the amniotic cavity in preterm labor.

To determine whether various inflammatory-, angiogenic/anti-angiogenic-, and extracellular matrix remodeling-associated proteins in plasma, alone or in combination with conventionalblood-based markers, can predict intra-amniotic inflammation and/or microbial invasion of the amniotic cavity (IAI/MIAC) in women with spontaneous preterm labor (PTL). A total of 193 singleton pregnant women with PTL (23-33weeks) were included in this retrospective cohort study. Plasma samples were obtained at the time of amniocentesis. Amniotic fluid (AF) was cultured for microorganism detection and consequent MIAC diagnosis. IL-6 levels were determined in AF and used to identify IAI (AF IL-6 ≥ 2.6ng/mL). Endostatin, haptoglobin, IGFBP-2/3, LBP, M-CSF, MMP-2/8, pentraxin 3, PlGF, S100A8/A9, and VEGFR-1 levels were assayed in plasma samples by ELISA. CRP levels and neutrophil-to-lymphocyte ratio (NLR) were measured. Plasma LBP, MMP-8, and S100A8/A9 levels, CRP levels, and NLR were significantly higher, and plasma IGFBP-2 and MMP-2 levels were significantly lower in women with IAI/MIAC than in those without this condition, whereas no baseline variables differed significantly between the two groups. Using a stepwise regression analysis, a noninvasive prediction model for IAI/MIAC was developed, which included plasma LBP, MMP-2, and MMP-8 levels (area under the curve [AUC], 0.785). The AUC for this prediction model was significantly or borderline greater than that of any single factor included in the model. IGFBP-2, LBP, MMP-2, MMP-8, and S100A8/A9 may represent valuable plasma biomarkers for predicting IAI/MIAC in women with PTL. Combination of LBP, MMP-2, and MMP-8 expression data can significantly improve the predictive potential for IAI/MIAC.

Treatment with valacyclovir during pregnancy for prevention of congenital cytomegalovirus infection: a real-life multicenter Italian observational study

Valacyclovir is the only treatment demonstrated to be effective for the prevention of vertical transmission of cytomegalovirus within a clinical randomized, placebo-controlled trial and has been reimbursed by the Italian National Health System since December 2020. This study reported the results of a real-life Italian multicenter observational study on cytomegalovirus infection in pregnancy evaluating the effect of the introduction of valacyclovir in the clinical practice for the prevention of vertical transmission of cytomegalovirus. The outcomes of women who received valacyclovir treatment and their fetuses or newborns were compared with those of a retrospective cohort observed between 2010 and 2020 who did not receive the antiviral treatment. The inclusion criterion was the diagnosis of cytomegalovirus primary infection occurring in the periconceptional period or up to 24 weeks of gestation. The primary outcome was the transmission by the time of amniocentesis. The secondary outcomes were termination of pregnancy, transmission at birth, symptomatic infection at birth, and a composite outcome (termination of pregnancy or transmission at birth). A total of 447 pregnant women from 10 centers were enrolled, 205 women treated with valacyclovir (called the valacyclovir group, including 1 twin pregnancy) and 242 women not treated with valacyclovir (called the no-valacyclovir group, including 2 twin pregnancies). Valacyclovir treatment was significantly associated with a reduction of the diagnosis of congenital cytomegalovirus infection by the time of amniocentesis (weighted odds ratio, 0.39; 90% confidence interval, 0.22-0.68; P=.005; relative reduction of 61%), termination of pregnancy (weighted odds ratio, 0.36; 90% confidence interval, 0.17-0.75; P=.0021; relative reduction of 64%), symptomatic congenital cytomegalovirus infection at birth (weighted odds ratio, 0.17; 90% confidence interval, 0.06-0.49; P=.006; relative reduction of 83%). The treatment had no significant effect on the rate of diagnosis of congenital cytomegalovirus infection at birth (weighted odds ratio, 0.85; 90% confidence interval, 0.57-1.26; P=.500), but the composite outcome (termination of pregnancy or diagnosis of congenital cytomegalovirus infection at birth) occurred more frequently in the no-valacyclovir group (weighted odds ratio, 0.62; 90% confidence interval, 0.44-0.88; P=.024). Of note, the only symptomatic newborns with congenital cytomegalovirus infection in the valacyclovir group (n=3) were among those with positive amniocentesis. Moreover, 19 women (9.3%) reported an adverse reaction to valacyclovir treatment, classified as mild in 17 cases and moderate in 2 cases. Lastly, 4 women (1.9%) presented renal toxicity with a slight increase in creatinine level, which was reversible after treatment suspension. Our real-life data confirm that valacyclovir significantly reduces the rate of congenital cytomegalovirus diagnosis at the time of amniocentesis with a good tolerability profile and show that the treatment is associated with a reduction of termination of pregnancy and symptomatic congenital cytomegalovirus infection at birth.

Reference values for interleukin-6 in the amniotic fluid of asymptomatic pregnant women.

Nowadays, proinflammatory factors are considered to play an important role in the pathophysiology of threatened preterm labor or chorioamnionitis. The aim of this study was to establish the normal reference range for interleukin-6 (IL-6) levels in the amniotic fluid and to identify factors which may alter this value. Prospective study in a tertiary-level center including asymptomatic pregnant women undergoing amniocentesis for genetic studies from October 2016 to September 2019. IL-6 measurements in amniotic fluid were performed using a fluorescence immunoassay with microfluidic technology (ELLA Proteinsimple, Bio Techne). Maternal history and pregnancy data were also recorded. This study included 140 pregnant women. Of those, women who underwent termination of pregnancy were excluded. Therefore, a total of 98 pregnancies were included in the final statistical analysis. The mean gestational age was 21.86 weeks (range: 15-38.7) at the time of amniocentesis, and 38.6 weeks (range: 30.9-41.4) at delivery. No cases of chorioamnionitis were reported. The log10 IL-6 values follow a normal distribution (W=0.990, p=0.692). The median, and the 5th, 10th, 90th, and 95th percentiles for IL-6 levels were 573, 105, 130, 1645, and 2260 pg/mL, respectively. The log10 IL-6 values were not affected by gestational age (p=0.395), maternal age (p=0.376), body mass index (p=0.551), ethnicity (p=0.467), smoking status (p=0.933), parity (p=0.557), method of conception (p=0.322), or diabetes mellitus (p=0.381). The log10 IL-6 values follow a normal distribution. IL-6 values are independent of gestational age, maternal age, body mass index, ethnicity, smoking status, parity and method of conception. Our study provides a normal reference range for IL-6 levels in the amniotic fluid that can be used in future studies. We also observed that normal IL-6 values were higher in the amniotic fluid than in serum.

The amniotic fluid proteome predicts imminent preterm delivery in asymptomatic women with a short cervix

Preterm birth, the leading cause of perinatal morbidity and mortality, is associated with increased risk of short- and long-term adverse outcomes. For women identified as at risk for preterm birth attributable to a sonographic short cervix, the determination of imminent delivery is crucial for patient management. The current study aimed to identify amniotic fluid (AF) proteins that could predict imminent delivery in asymptomatic patients with a short cervix. This retrospective cohort study included women enrolled between May 2002 and September 2015 who were diagnosed with a sonographic short cervix (< 25 mm) at 16–32 weeks of gestation. Amniocenteses were performed to exclude intra-amniotic infection; none of the women included had clinical signs of infection or labor at the time of amniocentesis. An aptamer-based multiplex platform was used to profile 1310 AF proteins, and the differential protein abundance between women who delivered within two weeks from amniocentesis, and those who did not, was determined. The analysis included adjustment for quantitative cervical length and control of the false-positive rate at 10%. The area under the receiver operating characteristic curve was calculated to determine whether protein abundance in combination with cervical length improved the prediction of imminent preterm delivery as compared to cervical length alone. Of the 1,310 proteins profiled in AF, 17 were differentially abundant in women destined to deliver within two weeks of amniocentesis independently of the cervical length (adjusted p-value < 0.10). The decreased abundance of SNAP25 and the increased abundance of GPI, PTPN11, OLR1, ENO1, GAPDH, CHI3L1, RETN, CSF3, LCN2, CXCL1, CXCL8, PGLYRP1, LDHB, IL6, MMP8, and PRTN3 were associated with an increased risk of imminent delivery (odds ratio > 1.5 for each). The sensitivity at a 10% false-positive rate for the prediction of imminent delivery by a quantitative cervical length alone was 38%, yet it increased to 79% when combined with the abundance of four AF proteins (CXCL8, SNAP25, PTPN11, and MMP8). Neutrophil-mediated immunity, neutrophil activation, granulocyte activation, myeloid leukocyte activation, and myeloid leukocyte-mediated immunity were biological processes impacted by protein dysregulation in women destined to deliver within two weeks of diagnosis. The combination of AF protein abundance and quantitative cervical length improves prediction of the timing of delivery compared to cervical length alone, among women with a sonographic short cervix.

Bile-acid levels and lung maturity test in patients with intrahepatic cholestasis of pregnancy.

Prolonged fetal exposure to maternal bile acids have been linked to fetal lung injury, but it is not known if it affects production of surfactant or fetal lung maturity tests. We set out to determine if elevated total bile acid (TBA) levels predict delayed fetal lung maturity (FLM) in patients with intrahepatic cholestasis of pregnancy (ICP). A retrospective cohort study examining patients with ICP who underwent amniocentesis between 36+0 and 37+6 between 2005 and 2014. Primary goal was to identify if a relationship exists between TBA levels and FLM defined as Lecithin:Sphingomyelin >2.5, detectable Phosphatidylglycerol, or lamellar body count of >50,000. Among the 113 patients, there was no statistically significant difference in TBA levels between women with FLM and those with immaturity (31.3 [21.6, 44.5]μmol/L and 34.9 [23.9, 62.3]μmol/L respectively; p=0.16). Logistic regression performed using predefined TBA levels of 20, 30, 40 and 50μmol/L revealed that TBA levels did not predict a negative FLM test at time of amniocentesis. TBA levels do no predict presence or absence of FLM between 36+0 and 37+6weeks of gestation. Though certain pregnancy complications can affect FLM, we could not identify a correlation between TBA levels and an accelerated or delayed lung maturation process.

Measurements of Interleukin-8 and Matrix Metalloproteinases-9 in Cervicovaginal Fluid in Women with Preterm Labor: A Direct Comparison with Amniotic Fluid.

We aimed to evaluate the correlation and agreement of interleukin (IL)-8 and matrix metalloproteinases (MMP-9) levels between cervicovaginal (CVF) and amniotic fluids (AF) in women with preterm labor (PTL) and to determine the clinical values of these proteins in CVF compared with those in AF. We designed a retrospective cohort study of 85 singleton pregnant women with PTL at 23 to 34 weeks, who underwent amniocentesis. The AF was cultured, and CVF samples were collected at the time of amniocentesis. Paired AF and CVF samples were assayed for IL-8 and MMP-9 by enzyme-linked immunoassay (ELISA) in duplicate on a single plate, using similar dilution ratios. A significant but weak correlation was found for IL-8 levels between AF and CVF (r = 0.333), while no correlation was found for MMP-9 levels between AF and CVF (r = -0.039). Intra-class correlation coefficient for the agreement of IL-8 levels between CVF and AF was 0.4335 and -0.279 for MMP-9, indicating a poor-to-fair level of agreement between the two measured values, respectively. IL-8 and MMP-9 levels in CVF were not associated with the risk of either microbial invasion of the amniotic cavity (MIAC) or spontaneous preterm delivery (SPTD) within 7 days, whereas those in AF provided good-to-excellent predictive values for these two outcomes (area under the curve [AUCs]: 0.82-0.95). AUCs for IL-8 and MMP-9 were significantly larger using AF rather than using CVF for the prediction of MIAC and SPTD. In women with PTL, IL-8 and MMP-9 levels in CVF do not precisely reflect the levels of the corresponding proteins in AF. IL-8 and MMP-9 levels in CVF had poor predictive values for the risk of MIAC and SPTD and were significantly inferior to those in AF. · IL-8 and MMP-9 levels in CVF do not precisely reflect levels of the corresponding proteins in AF.. · Diagnostic accuracy of IL-8 and MMP-9 in CVF alone is not sufficient to predict MIAC and SPTD.. · IL-8 and MMP-9 levels in AF provide good-to-excellent predictive values for these two outcomes..

Human amniotic fluid as a source of stem cells.

Human amniotic fluid collected during amniocentesis contains a heterogeneous population of differentiated and undifferentiated cells. Properties and number of these cells vary depending on the gestational age and the presence of potential fetal pathologies. The aim of this study was to analyze the effects of maternal, fetal, and environmental factors on the success rates of amniotic fluid stem cell cultures, the number of human amniotic fluid stem cells (hAFSC), their growth rates in primary cultures, and the number of cell passages. The study included 355 patients qualified for genetic amniocentesis at the Prenatal Genetic Unit, Department of Obstetrics, Gynecology and Oncologic Gynecology, Nicolaus Copernicus University Medical College in Bydgoszcz in 2011–2017. The mean age of the study participants was 34 ± 6.2 years, and mean gravidity amounted to 2.48 ± 1.4. Amniotic fluid sample volume turned out to be a highly significant (p < 0.01) predictor of culture success, and the relationship was particularly evident in women older than 40 years. Another highly significant predictor of culture success was the presence of two cell populations in the sample (p < 0.01). The likelihood of culture success correlated significantly (p < 0.05) with the season of the year at the time of amniocentesis. The number of cell passages differed significantly depending on the maternal age (p < 0.01). The number of passages also showed a highly significant relationship with the season of the year the sample was obtained (p < 0.01). Younger maternal age was identified as a determinant of high passage number (≥3), and another highly significant determinant of high passage number was the presence of two cell populations in the amniotic fluid sample (p < 0.01). Percentage of successfully established hAFSC cultures and the number of passages depended on amniotic fluid volume, the presence of two cell populations within the sample, and the season of the year. Individual characteristics of the donors, such as age and gravidity, did not exert a significant effect on the number of isolated hAFSCs and the rate of their growth. Patients’ place of residence, fetal karyotype, transportation time, and purity of the samples did not affect the success rates for primary cultures and the number of passages.

Complement Activation Fragments in Cervicovaginal Fluid Are Associated with Intra-Amniotic Infection/Inflammation and Spontaneous Preterm Birth in Women with Preterm Premature Rupture of Membranes.

We sought to determine whether the levels of complement and other inflammatory and angiogenic mediators in cervicovaginal fluid (CVF) are independently associated with intra-amniotic infection and/or inflammation (IAI) and imminent spontaneous preterm birth (SPTB, £48 hours of sampling) in women with preterm premature rupture of membranes (PPROM). This was a retrospective study consisting of 85 singleton pregnant women with PPROM at 200/7 to 336/7 weeks. Amniotic fluid (AF) obtained via amniocentesis was cultured and assayed for interleukin-6. CVF samples collected at the time of amniocentesis were assayed for complement C3a, C4a, and C5a, HSP70 (heat shock protein 70), M-CSF (macrophage colony-stimulating factor), M-CSF-R (macrophage colony-stimulating factor-receptor), S100 A8, S100 A9, thrombospondin-2, VEGF (vascular endothelial growth factor-receptor), and VEGFR-1 (vascular endothelial growth factor-receptor 1) by enzyme-linked immunosorbent assay. Multivariate logistic regression analyses revealed that elevated CVF concentrations of complement C3a, 4a, and 5a were significantly associated with an increased risk of IAI and imminent SPTB, whereas those of M-CSF were associated with IAI, but not imminent SPTB (p = 0.063), after adjustment for baseline covariates (e.g., gestational age at sampling). However, univariate, and multivariate analyses showed that the CVF concentrations of angiogenic (thrombospondin-2, VEGF, and VEGFR-1) and inflammatory (HSP70, M-CSF-R, S100 A8, and S100 A9) proteins were not associated with either IAI or imminent SPTB. In women with PPROM, elevated CVF concentrations of complement C3a, C4a, and C5a are independently related to an increased risk of IAI and imminent SPTB. These findings suggest that complement activation in CVF is significantly involved in mechanisms underlying preterm birth and in the host response to IAI in the context of PPROM. · Elevated CVF levels of C3a, 4a and 5a are associated with IAI and SPTB.. · CVF C3a, 4a and 5a have better predictability for SPTB, compared to AF WBC.. · Elevated CVF levels of M-CSF were associated with IAI, but not SPTB..

The correlation between maternal age and fetal sex chromosome aneuploidies: a 8-year single institution experience in China

BackgroundAlthough a few studies have investigated a possible association between maternal age and fetal sex chromosome aneuploidies (SCAs), most of these studies were limited to advanced maternal age (AMA) women and the results were conflicting. This study aimed to investigate the correlation between maternal age and common fetal SCAs (including 45,X, 47,XXY, 47,XXX and 47,XYY) in pregnant women of different ages that not only limited to AMA women. We retrospectively investigated a 8-year experience of prenatal diagnosis for fetal chromosome aberrations by second-trimester amniocentesis at a university teaching hospital in China. 20,409 amniotic fluid specimens collected at 19–22+6 gestational weeks were included in this study. The women were categorized into five age groups (≤ 23, 24–28, 29–33, 34–38, 39+ years) based on maternal age at the time of amniocentesis and entered as a categorical variable in all samples. The correlation between fetal SCAs and maternal age was determined using the logistic regression analysis. A chi-square test was performed to compare the incidence of fetal SCAs among age groups.ResultsA total of 179 cases of fetal SCAs were detected, and the incidence was 8.77‰ (about 1/114). The incidence of fetal SCAs increased significantly with advancing maternal age (SE, 0.014; odds ratio, 1.044; P = 0.002). Specifically, the incidence of 45,X (SE, 0.037; odds ratio, 0.916; P = 0.017) and 47,XXY (SE, 0.024; odds ratio, 1.127; P = 0.000) had significant correlation with maternal age, while the incidence of 47,XXX and 47,XYY had no correlation with maternal age (P = 0.473; P = 0.272, respectively). The incidence of fetal SCAs was also significantly different among age groups (χ2 = 10.197, P = 0.037 < 0.05), from 5.81 per 1000 fetuses at the 24–28 years to 10.92 per 1000 at the 39+ years.ConclusionsMaternal age was ascertained to be a strong risk factor for fetal SCAs.

Outcome of pregnancies with recent primary cytomegalovirus infection in first trimester treated with hyperimmunoglobulin: observational study.

To examine the efficacy of hyperimmunoglobulin (HIG) treatment in women with a recent primary cytomegalovirus (CMV) infection up to 14 weeks' gestation. This is an ongoing observational study conducted at the prenatal medicine departments of the University Hospitals of Tübingen, Bonn, Cologne and Erlangen, Germany, as well as at the Laboratory Prof. Gisela Enders and Colleagues in Stuttgart, Germany and the Institute for Medical Virology at the University of Tübingen, Tübingen, Germany. Enrolment criteria were the presence of confirmed recent primary CMV infection in the first trimester and a gestational age at first HIG administration of ≤ 14 weeks. The following inclusion criteria indicated a recent primary infection: low anti-immunoglobulin (Ig)-G levels, low anti-CMV-IgG avidity in the presence of a positive CMV-IgM test and no positive reactivity or just seroconversion anti-gB2-IgG-reactivity. HIG administration was started as soon as possible within a few days after the first visit. HIG was administered intravenously at a dose of 200 IU/kg maternal body weight and repeated every 2 weeks until about 18 weeks' gestation. The primary outcome was maternal-fetal transmission at the time of amniocentesis. Multivariate logistic regression analysis was used to determine significant covariates that could predict maternal-fetal transmission. We included 149 pregnancies (153 fetuses) that completed the treatment. Median maternal age and weight were 32.0 years and 65.0 kg, respectively. Median gestational age at the time of first referral to one of the four centers was 9.4 weeks. Median anti-CMV-IgG level, anti-CMV-IgM index and CMV-IgG avidity were 5.7 U/mL, 2.5 and 22.3%, respectively. HIG treatment was started at a median gestational age of 10.6 weeks and ended at a median of 17.9 weeks. Within this time frame, HIG was administered on average four times in each patient. Amniocentesis was carried out at a median gestational age of 20.4 weeks. In 143 (93.5%) of the 153 cases, the fetus was not infected. Maternal-fetal transmission occurred in 10 cases (6.5% (95% CI, 3.2-11.7%)). On uni- and multivariate logistic regression analysis, the level of anti-IgM index was the only factor associated significantly with maternal-fetal transmission at amniocentesis. However, only four (40.0%) of the 10 cases with maternal-fetal transmission had an anti-IgM index above 11.4, which corresponds to the 95th centile of pregnancies without transmission. HIG is a treatment option to prevent maternal-fetal transmission in pregnancy with a primary CMV infection. However, HIG treatment seems to be beneficial primarily in women with a recent primary infection in the first trimester or during the periconceptional period, and when it is administered at a biweekly dose of 200 IU/kg. © 2021 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

Rhythm of Fetoplacental 11β-Hydroxysteroid Dehydrogenase Type 2 - Fetal Protection From Morning Maternal Glucocorticoids.

Excess glucocorticoids impact fetal health. Maternal glucocorticoids peak in early morning. Fetoplacental 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) inactivates cortisol to cortisone, protecting the fetus from high glucocorticoids. However, time-specific alterations of human fetoplacental 11β-HSD2 have not been studied. We hypothesized that fetoplacental 11β-HSD2 activity shows time-specific alteration and acute affective or anxiety disorders impact fetoplacental 11β-HSD2 activity. In this observational study we investigated 78 pregnant European women undergoing amniocentesis (15.9 ± 0.9 weeks of gestation). Amniotic fluid was collected (8:00 to 16:30 hours) for analysis of fetoplacental 11β-HSD2 activity, using cortisol (F):cortisone (E) ratio in amniotic fluid, E/(E + F). Fetoplacental 11β-HSD2 rhythm and association with "acute affective or anxiety disorder" (patients with at least one of: a major depressive episode, specific phobia, panic disorder, generalized anxiety disorder, mixed anxiety and depressive disorder) and "acute anxiety disorder" (one of: panic disorder, generalized anxiety disorder, mixed anxiety, depressive disorder), assessed using Mini International Neuropsychiatric Interview, were investigated. Activity of 11β-HSD2 correlated with time of amniocentesis, peaking in the morning (r = -0.398; P < 0.001) and increased with acute affective or anxiety disorder (mean [M] = 0.70 vs M = 0.74; P = 0.037) and acute anxiety disorder (M = 0.70 vs M = 0.75; P = 0.016). These associations remained significant when controlling for confounders. 11β-HSD2 activity correlated negatively with pre-pregnancy body mass index (r = -0.225; P = 0.047). Our study indicates a time-specific alteration of fetoplacental 11β-HSD2 activity with peaking levels in the morning, demonstrating a mechanism of fetal protection from the morning maternal glucocorticoid surge.

86 Amniotic fluid microRNA profiles in twin-twin transfusion syndrome with and without severe cardiomyopathy

Twin-twin transfusion syndrome (TTTS) presents many challenges for clinicians, and the optimal means of identifying which pregnancies will benefit most from intervention is controversial. There is currently no clinically significant biomarker to detect or risk-stratify TTTS. microRNAs (miRs) are small RNAs that regulate gene expression and are biomarkers in various disease processes, including adult and pediatric heart failure. To date no studies have investigated amniotic fluid (AF) miR as a biomarker for severe TTTS. This study aims to assess whether AF miRs could be useful as biomarkers to identify pregnancies at greatest risk of severe cardiomyopathy due to TTTS. AF was collected at the time of amniocentesis or selective fetoscopic laser photocoagulation from monochorionic diamniotic twin pregnancies with TTTS at any stage and stored at -80° C. Fetal echocardiography was available for all subjects, and right ventricular Tei index of the recipient fetus ≥ 0.64 was used to define severe cardiomyopathy. RNA was extracted from the AF samples using the miRNeasy Mini Kit (Qiagen) and miR arrays were performed using the TaqMan Open Array miR panel (ThermoFisher). Student’s t-test was performed between groups and a stringent q-value of <0.005 was used to determine differentially expressed miRs. Hierarchical cluster analysis (HCA) was used to determine if these miRs properly separated the two groups. A total of 11 AF samples from pregnancies with severe cardiomyopathy were compared to 12 control TTTS samples without severe cardiomyopathy. 40 miRs were differentially expressed. HCA (Fig 1) based on these 40 miRs show a strong ability to differentiate the two groups. All differentially expressed miRs were downregulated in the severe cardiomyopathy group with the exception of upregulation of miR-208 (Fig 2), which has previously been shown to be associated with adverse outcomes in dilated cardiomyopathy. AF miRs demonstrate differential expression in TTTS with and without severe cardiomyopathy, and can be an important biomarker of disease severity in this population.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

99 Is fetal lung maturity delayed in patients with severe cholestasis of pregnancy?

With well documented higher rates of respiratory distress syndrome (RDS) in fetuses born to mothers with cholestasis, we set to determine if bile acid levels can serve as a predictor for delayed or accelerated lung maturity in patients with intrahepatic cholestasis of pregnancy. We followed 185 Hispanic patients with cholestasis of pregnancy who underwent amniocentesis at the preterm and early term periods between 2005-2014, to determine the presence or absence of fetal lung maturity, defined as Lecithin:Sphingomyelin >2.5, detectable Phosphatidylglycerol, lamellar body count of >50,000. We then compared the groups who underwent amniocentesis between 36.0-37.6 to identify the relationship between bile acid levels and fetal lung maturity. Out of the 185 patients with documented amniocentesis, 113 were performed between 36.0-37.6 weeks of gestation. When testing for lung maturity at that gestational age, there was no statistically significant difference between the positive and negative result groups with median of bile acids of 31.3 (21.6,44.5) and 34.9 (23.9,62.3) respectively (p=0.16). Logistic regression analysis was performed using predefined total bile acid cutoffs of 20 μmol/L, 30 μmol/L, 40μmol/L and 50 μmol/L. determined that bile acid levels did not correlated with a negative lung maturity test at time of amniocentesis when performed between 36.0-37.6. ROC curve using total BA was performed resulting in an AUC of 0.58 showing low clinical relevance. Bile acid levels could not serve as a predictor for presence or absence of lung maturity determined by amniocentesis (performed between 36.0-37.6 weeks of gestation). Though certain pregnancy complications have shown to impact lung maturity, we could not identify a correlation between bile acid levels and an accelerated or delayed lung maturity process. It is therefore our recommendation that late preterm and early term delivery in the setting of cholestasis be preceded by amniocentesis in order to avoid iatrogenic respiratory distress syndrome in the neonate, which has been reported in the literature.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Maternal Plasma and Amniotic Fluid LBP, Pentraxin 3, Resistin, and IGFBP-3: Biomarkers of Microbial Invasion of Amniotic Cavity and/or Intra-amniotic Inflammation in Women with Preterm Premature Rupture of Membranes.

BackgroundWe sought to determine whether lipopolysaccharide binding protein (LBP), pentraxin 3, resistin, and insulin-like growth factor binding protein (IGFBP)-3 in plasma and amniotic fluid (AF) can predict microbial invasion of the amniotic cavity (MIAC), intra-amniotic inflammation (IAI), and microbial-associated IAI in women with preterm premature rupture of membranes (PPROM).MethodsThis was a retrospective cohort study involving 168 singleton pregnant women with PPROM. AF obtained via amniocentesis was cultured and assayed for interleukin (IL)-6 to define IAI and for IL-8 to compare with AF biomarkers. Plasma samples were collected at the time of amniocentesis, and C-reactive protein (CRP) levels in serum were compared with plasma biomarkers. The stored plasma and AF samples were assayed for LBP, pentraxin 3 (PTX3), resistin, and IGFBP-3 by ELISA.ResultsMultivariate logistic regression analysis revealed that: 1) elevated plasma and AF levels of LBP were independently associated with increased risks of MIAC, IAI, and microbial-associated IAI; 2) elevated AF, but not plasma, PTX3, and resistin levels were independently associated with increased risks of MIAC, IAI, and microbial-associated IAI; 3) decreased IGFBP-3 levels in the plasma were independently associated with only IAI, whereas those in the AF were associated with only microbial-associated IAI. Among the tested biomarkers, AF PTX3 and resistin had the highest predictive performance for MIAC, IAI, and microbial-associated IAI (area under the curves [AUC] = 0.85–0.95), which is similar to the performance of AF IL-8. The AUCs of the plasma LBP and IGFBP-3 were similar to that of serum CRP with respect to IAI.ConclusionMaternal plasma LBP and IGFBP-3 are potential biomarkers for the non-invasive identification of IAI in women with PPROM, with a similar accuracy to the serum CRP level. AF LBP, PTX3, resistin, and IGFBP-3 may be involved in the intra-amniotic inflammatory responses in PPROM complicated by MIAC.

Inflammatory Proteins in the Amniotic Fluid, Plasma, and Cervicovaginal Fluid for the Prediction of Intra-Amniotic Infection/Inflammation and Imminent Preterm Birth in Preterm Labor.

This study was aimed to develop models using multiple cytokine/chemokine levels in cervicovaginal fluid (CVF) and plasma and widely used noninvasive parameters that have better accuracy for predicting intra-amniotic infection and/or inflammation (IAI) and imminent spontaneous preterm delivery (SPTD, ≤48 hours) in women with preterm labor (PTL). This was a retrospective cohort study of 95 singleton pregnant women with PTL (23-34 weeks) who underwent amniocentesis. Both CVF and plasma samples were obtained at the time of amniocentesis, and serum C-reactive protein (CRP) levels were measured. The amniotic fluid (AF), CVF, and plasma samples were assayed for interleukin (IL)-6, IL-8, IL-10, monocyte chemotactic protein-1 (MCP-1), and macrophage inflammatory protein-1β (MIP-1β) levels using a multiplex immunoassay kit. The levels of most cytokines/chemokines measured in the AF and CVF were significantly higher in the women with than in those without IAI and imminent SPTD, whereas only high-plasma IL-10 level showed a significant association with imminent SPTD. In predicting IAI, proteins in AF had significantly higher areas under the curves (AUCs) than those in CVF and plasma. However, for predicting imminent SPTD, no significant differences in the AUCs of the outcome-associated proteins were observed among the measurements in AF, CVF, and maternal plasma. By using stepwise regression analyses, noninvasive models (using protein levels in CVF and baseline clinical parameters) were developed for the prediction of IAI and imminent SPTD. The AUC of these noninvasive models were similar to those of the invasive models (using AF protein levels and baseline clinical parameters). Noninvasive models based on CVF cytokine/chemokine levels and widely used noninvasive parameters (especially CRP) act as good indicators for predicting the risk of IAI and imminent SPTD in women with PTL. Evaluation of cytokine/chemokine levels in plasma samples did not add valuable information regarding the two outcome measures in the PTL setting. · Markers in either CVF or plasma alone did not have sufficient accuracy for predicting IAI and SPTD.. · Noninvasive models using CVF cytokine and CRP act as effective tools for predicting two outcomes.. · Evaluation of cytokine level in plasma did not add valuable information regarding two outcomes..

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

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, < 34.0weeks) 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). We designed a retrospective cohort study of 145 women with PTL at 23.0-33.6weeks 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. In the multivariate analysis, elevated CVF levels of C5a and IGFBP-1 were significantly associated with IAI and SPTD at < 34weeks, 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 < 34weeks. However, univariate analysis showed no significant correlation between high CVF MMP-9 and IAI or SPTD at < 34weeks. 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 < 34weeks. These biomarkers (especially IGFBP-1) have similar or better diagnostic performance compared to AF WBCs.

Cervicovaginal Fluid Protein Microarray for Detection of Microbial Invasion of the Amniotic Cavity in Preterm Labor.

We aimed to identify cervicovaginal fluid (CVF) biomarkers that can detect microbial invasion of the amniotic cavity (MIAC) in women with preterm labor (PTL) with an antibody microarray and to develop the best combined model for detection of MIAC using these biomarkers in combination with conventional clinical variables. This retrospective cohort study included 168 singleton pregnant women with PTL (23-34weeks) who underwent amniocentesis. AF was cultured, and CVF samples were obtained at the time of amniocentesis. An antibody microarray was used to analyze the CVF proteome (n = 40). The validation of four candidate biomarkers of interest was performed by enzyme-linked immunosorbent assay (ELISA) in the final cohort (n = 168). For comparison with candidate markers, CVF IL-6 concentration was also measured. Twenty-seven molecules studied exhibited intergroup differences. Validation by ELISA confirmed significantly higher levels of CVF DKK3, M-CSF, and TIMP-1, but not of IGFBP-2, independent of gestational age, in CVF of women with MIAC. The area under the curve (AUC) of DKK3, M-CSF, and TIMP-1 from CVF was not significantly different from the AUC of IL-6 from CVF for detecting MIAC in women with PTL. By using a stepwise regression analysis, a combined detection model was developed, which included the CVF M-CSF, TIMP-1, and gestational age at sampling (AUC=0.823). An antibody microarray identified useful biomarkers (DKK3, M-CSF, and TIMP-1) in CVF for detection of MIAC, and a combined model including these biomarkers and gestational age can accurately detect MIAC in women with PTL.

49: Amniocentesis to diagnosis congenital cytomegalovirus infection following maternal primary cytomegalovirus infection in early pregnancy

Cytomegalovirus (CMV) infection is the most common congenital infection in the U.S. Our objective was to evaluate amniocentesis (AMNIO) in detecting congenital CMV infection (cCMV). Secondary analysis of a multicenter RCT designed to evaluate if CMV hyperimmune globulin (HIG) reduces cCMV in the offspring of women diagnosed with primary CMV infection prior to 24 weeks’ gestation and having no evidence of fetal infection (ultrasound findings or amniocentesis with CMV infection) at randomization. Eligible subjects were randomized to monthly infusions of HIG or placebo until delivery; those who underwent elective amniocentesis following randomization are included in this analysis. The fetus/neonate was tested for the presence of CMV at delivery or death. Comparisons were made between those with or without CMV at AMNIO, using chi-square test or Fisher’s exact test for categorical variables and the Wilcoxon rank test for continuous variables. A P-value of < 0.05 was considered significant. 399 women were randomized, and 55 (14%) women had AMNIO; 53 offspring had CMV results. Twelve AMNIOs were positive (12%) - one only by culture (8%), 7 only by PCR (58%) and four by culture and PCR (33%). The prevalence of fetal/neonatal infection was 26% (14/53). All 12 AMNIO-POS had cCMV infection confirmed at delivery, as well as 2 of the 41 AMNIO-NEG women (AMNIOs performed at 18.1 and 21 wks), for a sensitivity of 86%, specificity of 100%, positive predictive value of 100% and negative predictive value of 95%. (Table 1) Compared with AMNIO-NEG women, AMNIO-POS women had a lower avidity index at diagnosis and were more likely to undergo termination (Table 2). The gestational age (GA) at time of AMNIO was similar, as was the interval between maternal diagnosis and AMNIO. AMNIO-POS pregnancies delivered at an earlier GA and had a lower birthweight. AMNIO results are an accurate predictor of cCMV infection, particularly using PCR, even when performed at an early GA. Positive results are associated with increased termination rates and poor neonatal outcomes in continuing pregnancies.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

The earlier the gestational age, the greater the intensity of the intra-amniotic inflammatory response in women with preterm premature rupture of membranes and amniotic fluid infection by Ureaplasma species.

Objectives To determine the relationship between the intensity of the intra-amniotic inflammatory response and the gestational age at the time of diagnosis in cases with preterm premature rupture of membranes (PROM) and intra-amniotic infection caused by Ureaplasma spp. Methods A retrospective cohort study was conducted which included 71 women with preterm PROM and a positive amniotic fluid culture with Ureaplasma spp. Women with mixed intra-amniotic infections were excluded. The study population was classified into three groups according to gestational age: group 1, <26 weeks (extreme preterm PROM, n = 17); group 2, 26.0-33.9 weeks (moderate preterm PROM, n = 39); group 3, 34.0-36.9 weeks (late preterm PROM, n = 15). The intensity of the intra-amniotic and maternal inflammatory response was compared among the three groups. The intensity of the intra-amniotic inflammatory response was assessed by the concentration of amniotic fluid matrix metalloproteinase-8 (MMP-8) and white blood cell (WBC) count. The maternal inflammatory response was assessed by the concentration of C-reactive protein (CRP) and WBC count in maternal blood at the time of amniocentesis. Results (1) The median values of amniotic fluid MMP-8 concentration and WBC count were the highest in the extreme preterm PROM group and the lowest in the late preterm PROM group (P < 0.001 and P = 0.01, respectively); (2) the intensity of the maternal inflammatory response measured by maternal blood WBC count and CRP concentration was not significantly associated with gestational age at the time of diagnosis. Conclusion The earlier the gestational age at the time of PROM, the higher the intensity of the intra-amniotic inflammatory response in women with preterm PROM and intra-amniotic infection caused by Ureaplasma spp.