Mid-trimester Genetic Amniocentesis Research Articles

Rapid diagnosis of intra-amniotic infection using nanopore-based sequencing.

Early diagnosis and treatment of intra-amniotic infection is crucial. Rapid pathogen identification allows for a definite diagnosis and enables proper management. We determined whether the 16S amplicon sequencing performed by a nanopore sequencing technique make possible rapid bacterial identification at the species level in intra-amniotic infection. Five cases of confirmed intra-amniotic infection, determined by either cultivation or 16S rDNA polymerase chain reaction (PCR) Sanger sequencing, and 10 cases of women who underwent mid-trimester genetic amniocentesis were included. DNA was extracted from amniotic fluid and PCR was performed on the full-length 16S rDNA. Nanopore sequencing was performed. The results derived from nanopore sequencing were compared with those derived from cultivation and Sanger sequencing methods. Bacteria were successfully detected from amniotic fluid using nanopore sequencing in all cases of intra-amniotic infection. Nanopore sequencing identified additional bacterial species and polymicrobial infections. All patients who underwent a mid-trimester amniocentesis had negative cultures, negative 16S PCR Sanger sequencing and nanopore sequencing. Identification of the microorganisms using nanopore sequencing technique at the bacterial species level was achieved within 5-9h from DNA extraction. This is the first study demonstrating that the nanopore sequencing technique is capable of rapid diagnosis of intra-amniotic infection using fresh amniotic fluid samples.

Protein Concentrations of Thrombospondin-1, MIP-1\u03b2, and S100A8 Suggest the Reflection of a Pregnancy Clock in Mid-Trimester Amniotic Fluid

The development of immunoassays enables more sophisticated studies of the associations between protein concentrations and pregnancy outcomes, allowing early biomarker identification that can improve neonatal outcomes. The aim of this study was to explore associations between selected mid-trimester amniotic fluid proteins and (1) overall gestational duration and (2) spontaneous preterm delivery. A prospective cohort study, including women undergoing mid-trimester transabdominal genetic amniocentesis, was performed in Gothenburg, Sweden, 2008–2016 (n = 1072). A panel of 27 proteins related to inflammation was analyzed using Meso-Scale multiplex technology. Concentrations were adjusted for gestational age at sampling, experimental factors, year of sampling, and covariates (maternal age at sampling, parity (nulliparous/multiparous), smoking at first prenatal visit, and in vitro fertilization). Cox regression analysis of the entire cohort was performed to explore possible associations between protein concentrations and gestational duration. This was followed by Cox regression analysis censored at 259 days or longer, to investigate whether associations were detectable in women with spontaneous preterm delivery (n = 47). Finally, linear regression models were performed to analyze associations between protein concentrations and gestational duration in women with spontaneous onset of labor at term (n = 784). HMG-1, IGFBP-1, IL-18, MIP-1α, MIP-1β, S100A8, and thrombospondin-1 were significantly associated with gestational duration at term, but not preterm. Increased concentrations of thrombospondin-1, MIP-1β, and S100A8, respectively, were significantly associated with decreased gestational duration after the Holm-Bonferroni correction in women with spontaneous onset of labor at term. This adds to the concept of a pregnancy clock, where our findings suggest that such a clock is also reflected in the amniotic fluid at early mid-trimester, but further research is needed to confirm this.

Metabolomic profiles of mid-trimester amniotic fluid are not associated with subsequent spontaneous preterm delivery or gestational duration at delivery

Introduction Spontaneous preterm delivery (<37 gestational weeks) has a multifactorial etiology with still incompletely identified pathways. Amniotic fluid is a biofluid with great potential for insights into the feto-maternal milieu. It is rich in metabolites, and metabolic consequences of inflammation is yet researched only to a limited extent. Metabolomic profiling provides opportunities to identify potential biomarkers of inflammatory conditioned pregnancy complications such as spontaneous preterm delivery. Objective The aim of this study was to perform metabolomic profiling of amniotic fluid from uncomplicated singleton pregnancies in the mid-trimester to identify potential biomarkers associated with spontaneous preterm delivery and gestational duration at delivery. A secondary aim was to replicate previously reported mid-trimester amniotic fluid metabolic biomarkers of spontaneous preterm delivery in asymptomatic women. Method A nested case-control study was performed within a larger cohort study of asymptomatic pregnant women undergoing mid-trimester genetic amniocentesis at 14–19 gestational weeks in Gothenburg, Sweden. Medical records were used to obtain clinical data and delivery outcome variables. Amniotic fluid samples from women with a subsequent spontaneous preterm delivery (n = 37) were matched with amniotic fluid samples from women with a subsequent spontaneous delivery at term (n = 37). Amniotic fluid samples underwent untargeted metabolomic analyses using liquid chromatography-mass spectrometry. Multivariate random forest analyses were used for data processing. A secondary targeted analysis was performed, aiming to replicate previously reported mid-trimester amniotic fluid metabolic biomarkers in women with a subsequent spontaneous preterm delivery. Results Multivariate analysis did not distinguish the samples from women with a subsequent spontaneous preterm delivery from those with a subsequent term delivery. Neither was the metabolic profile associated with gestational duration at delivery. Potential metabolic biomarker candidates were identified from four publications by two different research groups relating mid-trimester amniotic fluid metabolomes to spontaneous PTD, of which fifteen markers were included in the secondary analysis. None of these were replicated. Conclusions Metabolomic profiles of early mid-trimester amniotic fluid were not associated with spontaneous preterm delivery or gestational duration at delivery in this cohort.

Comparison of Bacterial DNA Profiles in Mid-Trimester Amniotic Fluid Samples From Preterm and Term Deliveries.

Infection and inflammation are well recognized causes of spontaneous preterm delivery (PTD) (<37 gestational weeks) and adverse infant outcomes. To date, there has been very little investigation into bacterial communities in amniotic fluid using next generation sequencing technology. In particular, it is important to characterize amniotic fluid bacterial profiles in complicated pregnancies as well as in asymptomatic women to identify predictive bacterial DNA signatures. Here, 1198 mid-trimester amniotic fluid samples from a cohort of Swedish women undergoing mid-trimester genetic amniocentesis were screened for bacterial DNA using qPCR protocols specifically designed to reduce the impacts of reagent contamination and human DNA mispriming. The majority of samples were devoid of detectable bacterial DNA; however, approximately a fifth of the cohort (19.9%) were 16S rRNA gene positive in duplicate screening. Among these, nine women had a spontaneous PTD. These nine women were matched with 18 healthy women with a delivery at term. We used PacBio SMRT technology, coupled with appropriate negative extraction and PCR controls, to sequence the full-length 16S rRNA gene in this subset of 27 women. The amniotic fluid samples contained low-abundance and low-diversity bacterial DNA profiles. Species typically associated with spontaneous PTD were absent. We were not able to identify any differences in the amniotic fluid bacterial DNA profiles of women with a subsequent spontaneous PTD compared to women who delivered at term. The findings suggest that, in a minor proportion of pregnancies, DNA from non-pathogenic bacteria may be present in the amniotic fluid far earlier than previously reported. Early detection of bacterial DNA in the amniotic fluid was, in this study, not associated with spontaneous PTD.

Interleukin-6 and C-reactive protein levels in the amniotic fluid as indicators of preterm delivery in Turkish women

Objective: The aim of this study was to determine the value of amniotic fluid interleukin-6 (IL-6) and C-reactive protein (CRP) levels in the prediction of preterm delivery in singleton pregnancies without any known risk factors for preterm delivery in Turkish women. Materials and Methods: Patients in the present perinatology department who underwent mid-trimester genetic amniocentesis due to evidence of increased risk of aneuploidy in their prenatal serum screening tests were included in the study. A sample of amniotic fluid from each patient was assessed for IL-6 and CRP. Concentrations of IL-6 and CRP in the amniotic fluid of preterm delivery and term delivery groups were compared. Results: Of 151 singleton pregnancies, 142 participants were included in the study. The participants were assigned to either the preterm or term delivery group based on pregnancy outcome. IL-6 levels in the amniotic fluid were significantly higher in the preterm delivery group, and there was a statistically significant negative correlation between IL-6 concentrations in the amniotic fluid and gestational age at delivery (correlation coefficient (CC): -18.5%, p p = 0.068). Conclusion: Measuring IL-6 in the amniotic fluid can identify women at risk of preterm delivery. Because it is not acceptable to perform amniocentesis for this screening, it is more convenient for patients in whom genetic amniocentesis is performed.

About one-half of early spontaneous preterm deliveries can be identified by a rapid matrix metalloproteinase-8 (MMP-8) bedside test at the time of mid-trimester genetic amniocentesis*

Objective: Mid-trimester amniocentesis continues to be used for the prenatal diagnosis of chromosomal anomalies and other genetic disorders. Analysis of amniotic fluid obtained at the time of mid-trimester genetic amniocentesis identifies those patients who are at risk for early spontaneous preterm delivery. This is based on a solid body of evidence that found subclinical intra-amniotic inflammation/infection to be causally linked to early spontaneous preterm birth. Although several biomarkers have been proposed to identify intra-amniotic inflammation, the accumulated data suggest that the determination of amniotic fluid matrix metalloproteinase-8 (MMP-8), or neutrophil collagenase, is a powerful predictor of spontaneous preterm delivery. MMP-8 is released by inflammatory cells in response to microbial products or “danger signals”. A rapid point-of-care test has been developed to determine MMP-8 at the bedside within 20 min, and without the requirement of laboratory equipment. The objective of this study was to determine whether an elevation of MMP-8 in the amniotic fluid, measured by a rapid point-of-care test, can identify those patients at risk for spontaneous preterm delivery after a mid-trimester genetic amniocentesis. Study design: A case–control study was designed to obtain amniotic fluid from asymptomatic singleton pregnant women who underwent mid-trimester genetic amniocentesis. An MMP-8 bedside test was performed to analyze the amniotic fluid of 64 patients with early spontaneous preterm delivery (<30 weeks) and 128 matched controls with normal pregnancy outcomes. Results: (1) The MMP-8 bedside test (Yoon's MMP-8 Check™) was positive in 42.2% (27/64) of patients with spontaneous preterm delivery but in none (0/128) of the control cases (p < 0.001); (2) the MMP-8 bedside test had a sensitivity of 42.2%, and a specificity of 100% in the prediction of spontaneous preterm delivery (<30 weeks) following a mid-trimester genetic amniocentesis; and (3) among the patients with spontaneous preterm delivery, those with a positive MMP-8 bedside test had a significantly higher rate of spontaneous delivery within 2 weeks and 4 weeks of an amniocentesis [40.7% (11/27) versus 5.4% (2/37); 63.0% (17/27) versus 24.3% (9/37)] and a shorter interval-to-delivery period than those with a negative test [interval-to-delivery: median (range), 16 d (0–95 d) versus 42 d (2–91 d); p < 0.05 for each]. Conclusion: We conclude that 42% of patients with an early spontaneous preterm delivery (< 30 weeks) could be identified by a rapid MMP-8 bedside test at the time of their mid-trimester genetic amniocentesis. The MMP-8 bedside test is a powerful predictor of early spontaneous preterm birth in asymptomatic pregnant women.

556: One third of early spontaneous preterm delivery can be identified by a rapid matrix metalloproteinase-8 (MMP-8) bedside test at the time of mid-trimester genetic amniocentesis

556: One third of early spontaneous preterm delivery can be identified by a rapid matrix metalloproteinase-8 (MMP-8) bedside test at the time of mid-trimester genetic amniocentesis

Maternal Obesity Affects Fetal Neurodevelopmental and Metabolic Gene Expression: A Pilot Study

ObjectiveOne in three pregnant women in the United States is obese. Their offspring are at increased risk for neurodevelopmental and metabolic morbidity. Underlying molecular mechanisms are poorly understood. We performed a global gene expression analysis of mid-trimester amniotic fluid cell-free fetal RNA in obese versus lean pregnant women.MethodsThis prospective pilot study included eight obese (BMI≥30) and eight lean (BMI<25) women undergoing clinically indicated mid-trimester genetic amniocentesis. Subjects were matched for gestational age and fetal sex. Fetuses with abnormal karyotype or structural anomalies were excluded. Cell-free fetal RNA was extracted from amniotic fluid and hybridized to whole genome expression arrays. Genes significantly differentially regulated in 8/8 obese-lean pairs were identified using paired t-tests with the Benjamini-Hochberg correction (false discovery rate of <0.05). Biological interpretation was performed with Ingenuity Pathway Analysis and the BioGPS gene expression atlas.ResultsIn fetuses of obese pregnant women, 205 genes were significantly differentially regulated. Apolipoprotein D, a gene highly expressed in the central nervous system and integral to lipid regulation, was the most up-regulated gene (9-fold). Apoptotic cell death was significantly down-regulated, particularly within nervous system pathways involving the cerebral cortex. Activation of the transcriptional regulators estrogen receptor, FOS, and STAT3 was predicted in fetuses of obese women, suggesting a pro-estrogenic, pro-inflammatory milieu.ConclusionMaternal obesity affects fetal neurodevelopmental and metabolic gene expression as early as the second trimester. These findings may have implications for postnatal neurodevelopmental and metabolic abnormalities described in the offspring of obese women.

Pregnancy Loss After Chorionic Villus Sampling and Genetic Amniocentesis in Twin Pregnancies

Objective To review the available evidence regarding pregnancy loss following first-trimester chorionic villus sampling (CVS) and mid-trimester genetic amniocentesis in twins. Methods We searched the MEDLINE database from January 1990 to May 2011 for randomized and cohort studies reporting on the risk of pregnancy loss after first-trimester CVS performed between 9 and 14 weeks and after genetic amniocentesis performed between 14 and 22 weeks. Where appropriate, we calculated pooled proportions and relative risks with 95% CI. Results No randomized studies were found. For CVS, nine studies fulfilled the inclusion criteria. The overall pregnancy-loss rate was 3.84% (95% CI, 2.48–5.47; n = 4). The rate of pregnancy loss before 20 weeks was 2.75% (95% CI, 1.28–4.75; n = 3) and before 28 weeks was 3.44% (95% CI, 1.67–5.81; n = 3). For amniocentesis, the overall pregnancy-loss rate was 3.07% (95% CI, 1.83–4.61; n = 4). The rate of pregnancy loss before 20 weeks was 2.25% (95% CI, 1.23–3.57; n = 2), before 24 weeks was 2.54% (95% CI, 1.43–3.96; n = 9) and before 28 weeks was 1.70% (95% CI, 0.37–3.97; n = 5). Pooled data from four case–control studies showed a higher risk (2.59% vs. 1.53%) of pregnancy loss before 24 weeks following amniocentesis (relative risk = 1.81; 95% CI, 1.02–3.19). There were no statistically significant differences in reported pregnancy loss between transabdominal and transcervical approaches, use of a single-needle system vs. a double-needle system and single uterine entry vs. double uterine entry in the CVS group. Similarly, in the amniocentesis group, there was no statistically significant difference in fetal loss between the single uterine entry vs. the double uterine entry. Conclusion In the absence of randomized studies, it is not possible to estimate accurately the excess risk following invasive procedures in twins. Currently available data show similar overall pregnancy-loss rates for both amniocentesis and CVS with the excess risk of around 1% above the background risk. Copyright © 2012 ISUOG. Published by John Wiley & Sons, Ltd.

Pregnancy loss after chorionic villus sampling and genetic amniocentesis in twin pregnancies: a systematic review

To review the available evidence regarding pregnancy loss following first-trimester chorionic villus sampling (CVS) and mid-trimester genetic amniocentesis in twins. We searched the MEDLINE database from January 1990 to May 2011 for randomized and cohort studies reporting on the risk of pregnancy loss after first-trimester CVS performed between 9 and 14 weeks and after genetic amniocentesis performed between 14 and 22 weeks. Where appropriate, we calculated pooled proportions and relative risks with 95% CI. No randomized studies were found. For CVS, nine studies fulfilled the inclusion criteria. The overall pregnancy-loss rate was 3.84% (95% CI, 2.48-5.47; n = 4). The rate of pregnancy loss before 20 weeks was 2.75% (95% CI, 1.28-4.75; n = 3) and before 28 weeks was 3.44% (95% CI, 1.67-5.81; n = 3). For amniocentesis, the overall pregnancy-loss rate was 3.07% (95% CI, 1.83-4.61; n = 4). The rate of pregnancy loss before 20 weeks was 2.25% (95% CI, 1.23-3.57; n = 2), before 24 weeks was 2.54% (95% CI, 1.43-3.96; n = 9) and before 28 weeks was 1.70% (95% CI, 0.37-3.97; n = 5). Pooled data from four case-control studies showed a higher risk (2.59% vs. 1.53%) of pregnancy loss before 24 weeks following amniocentesis (relative risk = 1.81; 95% CI, 1.02-3.19). There were no statistically significant differences in reported pregnancy loss between transabdominal and transcervical approaches, use of a single-needle system vs. a double-needle system and single uterine entry vs. double uterine entry in the CVS group. Similarly, in the amniocentesis group, there was no statistically significant difference in fetal loss between the single uterine entry vs. the double uterine entry. In the absence of randomized studies, it is not possible to estimate accurately the excess risk following invasive procedures in twins. Currently available data show similar overall pregnancy-loss rates for both amniocentesis and CVS with the excess risk of around 1% above the background risk.

Amniotic fluid interleukin-18 at mid-trimester genetic amniocentesis: relationship to intraamniotic microbial invasion and preterm delivery

Amniotic fluid interleukin-18 at mid-trimester genetic amniocentesis: relationship to intraamniotic microbial invasion and preterm delivery

Amniotic fluid interleukin-6 and tumor necrosis factor-α at mid-trimester genetic amniocentesis: Relationship to intra-amniotic microbial invasion and preterm delivery

Objective To determine the value of amniotic fluid interleukin-6 (AF IL-6) and tumor necrosis factor-α (AF TNF-α) in the diagnosis of microbial invasion of the amniotic cavity and in the prediction of preterm delivery (PTD). Study design Following amniocentesis, a sample of amniotic fluid was sent for aerobic and anaerobic bacterial cultures along with Ureaplasma urealyticum culture and it was also assessed for IL-6 and TNF-α. Results Forty-eight women who delivered preterm (<37 weeks) were matched with 96 controls. The AF IL-6 and TNF-α concentrations of women with spontaneous PTD were significantly higher than those who delivered at term (IL-6: 176.3 pg/ml [130.6–208.6] vs. 52.3 pg/ml [37.2–92.3]; TNF-α: 8.8 pg/ml [7.2–10.7] vs. 5.5 pg/ml [5.0–6.3]). AF IL-6 and TNF-α concentrations of >99.3 pg/ml and of >6.6 pg/ml respectively, had a sensitivity of 89.6% and 81.3% and a specificity of 80.3% and 79.2% for the prediction of spontaneous PTD. Moreover, AF IL-6 and TNF-α concentrations of >99.3 pg/ml and of 6.3 pg/ml respectively, had a sensitivity of 91.9% and 78.4% and a specificity of 73.8% and 70.1% for the prediction of a positive AF culture. Conclusions Elevated mid-trimester concentrations of AF IL-6, or/and of TNF-α can identify women at risk for intra-amniotic infection and for spontaneous PTD.

The efficacy of lidocaine-prilocaine cream to reduce pain in genetic amniocentesis

Objective: Evaluate whether local anesthesia by lidocaine-prilocaine cream decreases maternal pain during mid-trimester genetic amniocentesis. Material and Method: This randomized controlled study of mid-trimester genetic amniocentesis was conducted between 1 October 2006 and 30 April 2007. Pregnant women were randomized to receive lidocaine-prilocaine cream or placebo cream 30 minutes prior to amniocentesis. Patients, blinded to allocation, recorded anticipated and actual pain before and after the procedure. The visual analog score (VAS) was evaluated, using a 0-10 scale. Results: One hundred and twenty women participated in the present study. Sixty women were randomized to lidocaine-prilocaine group. The two groups were similar with respect to clinical correlations and procedure characteristics. Anticipated pain was 6.1 + 2.0 in the lidocaine-prilocaine group and 6.3 + 2.3 in the placebo group (p = 0.61). Actual pain was 2.3 + 2.2 in the lidocaine-prilocaine group and 2.9 + 2.5 in the placebo group (p = 0.16). Conclusion: Lidocaine-prilocaine cream does not decrease pain during mid-trimester genetic amniocentesis. Keywords: Pain, Amniocentesis

Mid-Trimester Genetic Amniocentesis in Twin Pregnancy and the Risk of Fetal Loss

ObjectiveTo assess the rate of fetal losses in twin pregnancies undergoing genetic mid-trimester amniocentesis. MethodsIn the first part of this investigation, a retrospective cohort study compared a group of women > 32 years old with twin pregnancies who underwent amniocentesis with a similar group unexposed to amniocentesis. Data were compiled from January 1990 to March 2004 for patients from a single institution. Pregnancies complicated by twin-to-twin transfusion syndrome, monoamniotic twins, or lethal fetal anomalies, and those treated by fetal reduction were excluded. The primary outcome was the loss of one or both fetuses prior to 24 weeks’ gestation. In the second part of the investigation, a systematic review of the literature and a meta-analysis were performed. ResultsIn the first part of the study, data were collected for 132 women exposed to amniocentesis and 248 women not exposed to amniocentesis. There was no significant difference in the rate of fetal losses between the two groups (3.0% vs. 0.8%, P = 0.10). No losses occurred within four weeks of the procedure. In the second part of the investigation, four studies, including ours, were considered for a meta-analysis of 2026 women with twin pregnancies. Compared with women unexposed to the procedure, amniocentesis in women with twin pregnancies increased the risk of fetal losses prior to 20 to 24 weeks’ gestation (odds ratio 2.42; 95% confidence intervals 1.24-4.74, P = 0.01) with an additional risk of one adverse outcome (1 or 2 fetal losses) for every 64 amniocenteses. ConclusionGenetic mid-trimester amniocentesis in twin pregnancies is associated with an increased risk of fetal loss prior to 24 weeks’ gestation.

An elevated maternal plasma, but not amniotic fluid, soluble fms-like tyrosine kinase-1 (sFlt-1) at the time of mid-trimester genetic amniocentesis is a risk factor for preeclampsia

The purpose of this study was to determine if an elevated concentration of soluble fms-like tyrosine kinase-1(sFlt-1) in maternal plasma and amniotic fluid is a risk factor for the subsequent development of preeclampsia. A case-control study was conducted to compare mid-trimester concentrations of maternal plasma and amniotic fluid sFlt-1 in patients who developed preeclampsia with those who did not. The study included 32 cases with preeclampsia (18 cases: severe preeclampsia) and 128 matched controls with normal outcomes. Patients with an abnormal fetal karyotype or major anomaly, multiple pregnancies, chronic hypertension, diabetes, and renal disease were excluded. Soluble Flt-1 concentration was measured by specific immunoassay. Nonparametric techniques were used for statistical analysis. 1) The median maternal plasma, but not amniotic fluid, sFlt-1 concentration in patients who developed preeclampsia was significantly higher than in the control cases (maternal plasma: median 730 pg/mL, range 60-3375 pg/mL vs median 441 pg/mL, range 58-1959 pg/mL, P < .05; amniotic fluid: median 10,504 pg/mL, range 5253-38,023 pg/mL vs median 10,236 pg/mL, range 4326-87,684 pg/mL, P = .65). 2) The median plasma concentration of sFlt-1 was higher in cases of severe preeclampsia than in those with mild preeclampsia without reaching statistical significance (median 762 pg/mL, range 261-3309 pg/mL vs median 334 pg/mL, range 60-3375 pg/mL; P = .07). However, there was no significant difference in the median amniotic fluid sFlt-1 concentrations between patients with severe preeclampsia and those with mild preeclampsia (P = .45). 3) An elevated maternal plasma sFlt-1 concentration (higher than 700 pg/mL) is a risk factor for the development of preeclampsia (OR 3.9, 95% CI 1.7-8.6) and severe preeclampsia (OR 7.4, 95% CI 2.5-22.1) after genetic amniocentesis. 4) The median interval from amniocentesis to the diagnosis of preeclampsia in patients with maternal plasma sFlt-1 concentrations higher than 700 pg/mL was 117 days (range 19-154 days). An elevated concentration of sFlt-1 in maternal plasma at the time of mid-trimester amniocentesis is a risk factor for the subsequent development of preeclampsia.

An elevated maternal plasma sFlt1 concentration at the time of mid-trimester genetic amniocentesis is a risk factor for preeclampsia

IN PREECLAMPTIC CASES INANC MENDILCIOGLU, SEYDA KARAVELI, GULGUN ERDOGAN, MEHMET SIMSEK, OMUR TASKIN, Akdeniz University, Obstetrics and Gynecology, Antalya, Turkey, Akdeniz University, Pathology, Antalya, Turkey OBJECTIVE: To determine whether preeclampsia is associated with an increase in placental apoptosis and differential expression of mediators of apoptosis such as fas,fas ligand, caspase-3, p53, Bax and Bcl-2. STUDY DESIGN: Placental samples from 20 women with preeclampsia and 14 normal women were analyzed using Klenow method for apoptosis. Expression of Fas, Fas ligand, caspase-3, p 53 Bcl-2, and Bax were assessed using immunohistochemistry. RESULTS: The mean percent apoptotic nuclei in trophoblasts was significantly higher for the preecampsia group than for the controls (7.79 versus 2.07; P = .015). Fas ligand expression was significantly higher in the decidua among the preeclampsia subjects compared with controls (1.35 versus 0.49; P = .036). Otherwise there was no difference in the expression of Bax , Bcl-2, p53, caspase3, fas between groups. CONCLUSION: Hypoxia triggers placental placental apoptosis which may play a important role in the pathogenesis of preeclampsia.

Rapid Detection of Group B Streptococcus and Escherichia coli in Amniotic Fluid Using Real‐Time Fluorescent PCR

OBJECTIVE: To establish reliability and validity of real-time fluorescent PCR for early detection of bacterial invasion of the amniotic cavity. METHODS: Amniotic fluid samples from 40 patients undergoing mid-trimester genetic amniocentesis were incubated for 6 h at 37 degrees C and were cultured on media specific for group B streptococcus (GBS) and E. coli. Concurrently, samples were analyzed with real-time fluorescent PCR (Roche LightCycler) using DNA primers and probes designed to detect the CAMP factor encoding cfb gene and uidA gene of GBS and E. coli, respectively. For positive control and to simulate amniotic fluid colonization, 104 cfu/ml of GBS and E. coli were inoculated on sterile amniotic fluid and incubated for 6 h. Bacterial genomic DNA for the two organisms was extracted and purified via the two-step precipitation method using a commercial kit. The real-time PCR assays were also tested against 25 non-GBS and non-E. coli bacterial species. The lower limit of detection for each pathogen was established using serial dilution of bacterial genomic DNA. RESULTS: All patient samples were negative for evidence of GBS and E. coli with both culture and real-time PCR methods. Amniotic fluid samples inoculated with GBS and E. coli were positive with real-time PCR whereas the 25 bacterial species other than GBS or E. coli tested negative with the assay. Average total sample processing time including the pre-enrichment step was 7 h 40 min. The average cost for DNA extraction and PCR testing was 8.50 dollars per test. CONCLUSION: Real-time fluorescent PCR is a valid and reliable method for detection of specific pathogens in amniotic fluid. This technique is sensitive for low inoculation levels. Real-time fluorescent PCR has potential to impact clinical management as a rapid, reliable detection method for GBS and E. coli in chorioamnionitis.

Midtrimester amniotic fluid vitamin C levels inversely correlate with interleukin-6 levels

CORRELATE WITH INTERLEUKIN-6 LEVELS EVA PRESSMAN, DEB PITTINARO, JAMES WOODS, University of Rochester, Obstetrics and Gynecology, Rochester, NY OBJECTIVE: To compare levels of vitamin C and interleukin-6 (IL-6) in pregnancies undergoing midtrimester amniocentesis for genetic indications. STUDY DESIGN: Women undergoing midtrimester genetic amniocentesis were prospectively enrolled. The initial 1 cc of amniotic fluid obtained was immediately refrigerated and processed within 24 hours for vitamin C and IL-6 analysis. Data were collected onmaternal age, gestational age, and indication for amniocentesis. Demographic data, vitamin C levels, and IL-6 levels were analyzed with Pearson’s correlation and chi-square analysis. RESULTS: Data were available for 37 patients with normal karyotypes between the gestational ages of 16 and 21 weeks (mean 18.1 weeks). Mean maternal age was 35.1 years, with 66%of patients undergoing amniocentesis due to advanced maternal age. Vitamin C levels were noted to increase with advancing gestational age (r = 0.47, P = 0.003) but did not vary with maternal age or indication for amniocentesis. IL-6 values were not affected by these factors. A negative correlation was noted between vitamin C and IL-6 (r = -0.34, P = 0.034). CONCLUSION: Elevated levels of IL-6 in midtrimester amniotic fluid indicate intra-amniotic inflammation and have been associated with adverse pregnancy outcome. The inverse correlation between IL-6 and vitamin C suggests that consumption of vitamin C may occur as part of a chronic inflammatory process. Amniotic fluid vitamin C may also serve as a defense against chronic inflammation. Evaluation of the outcomes of these pregnancies may further clarify the potential role of vitamin C in the amniotic fluid.

Reducing maternal cell contamination during midtrimester genetic amniocentesis; the effect of discarding the initial amniotic fluid collection

Reducing maternal cell contamination during midtrimester genetic amniocentesis; the effect of discarding the initial amniotic fluid collection

Does Amniotic Fluid Alpha-Fetoprotein Have Diagnostic or Prognostic Value at the Time of Second Midtrimester Genetic Amniocentesis?

In order to assess the usefulness of amniotic fluid α-fetoprotein (AFP) levels at the time of midtrimester genetic amniocentesis, 4,430 cases were retrospectively studied to compare the high, normal or low AFP values with the karyotype characteristics and fetal anatomy using ultrasound (US) scanning and confirmed by postnatal evaluation or necroscopy in the case of termination of pregnancy. All the cases presenting malformations were correctly diagnosed by US examinations. AFP levels over the 2nd standard deviation (SD) were found in 112 cases (2.52%) and below the 2nd SD in 11 cases (0.24%). The characteristics of these cases are presented and discussed. According to our results, it is concluded that routine assessment of AFP at the time of midtrimester genetic amniocentesis, if coupled with optimal US scanning, is no longer justified.