Time Of Amniocentesis Research Articles

Inflammatory proteins in maternal plasma, cervicovaginal and amniotic fluids as predictors of intra-amniotic infection in preterm premature rupture of membranes.

ObjectiveWe aimed to assess the correlations among multiple cytokine concentrations in the maternal plasma, cervicovaginal fluid (CVF), and amniotic fluid (AF) compartments in women with preterm premature rupture of membranes (pPROM), and to develop a prediction model based on non-invasive measures, having better sensitivity and specificity for the identification of microbial invasion of amniotic cavity (MIAC).MethodThis retrospective study included 75 consecutive women with pPROM (20+0–34+0 weeks), who underwent amniocentesis. Both maternal plasma and CVF samples were collected at the time of amniocentesis. Stored AF, plasma and CVF samples were assayed for cytokine levels [interleukin (IL)-6, IL-8, monocyte chemotactic protein-1, macrophage inflammatory protein (MIP)-1α, MIP-1β] using a multiplex immunoassay kit.ResultsLevels of inflammatory proteins measured in the CVF were significantly correlated with AF proteins levels, whereas none of the proteins in plasma correlated significantly with any in the AF or CVF. Proteins levels measured in the AF and CVF were significantly higher in women with MIAC compared to those without, whereas only high levels of IL-6 in plasma were significantly associated with MIAC. By using stepwise regression analysis, a non-invasive model (using clinical factors and CVF cytokine levels) for the prediction of MIAC was developed; the area under curve of this non-invasive model was similar to that of the invasive model (using clinical factors and AF cytokines).ConclusionsThe levels of inflammatory proteins in the CVF correlated with those in the AF, whereas those in the plasma showed no correlation. A non-invasive model using clinical factors and CVF cytokine levels predicted the risk of MIAC in women with pPROM.

Plasma inflammatory and immune proteins as predictors of intra-amniotic infection and spontaneous preterm delivery in women with preterm labor: a retrospective study

BackgroundWe investigated whether various inflammatory and immune proteins in plasma predict intra-amniotic infection and imminent preterm delivery in women with preterm labor and compared their predictive ability with that of amniotic fluid (AF) interleukin (IL)-6 and serum C-reactive protein (CRP).MethodsThis retrospective cohort study included 173 consecutive women with preterm labor who underwent amniocentesis for diagnosis of infection and/or inflammation in the AF. The AF was cultured, and assayed for IL-6. CRP levels and cervical length by transvaginal ultrasound were measured at the time of amniocentesis. The stored maternal plasma was assayed for IL-6, matrix metalloproteinase (MMP)-9, and complements C3a and C5a using ELISA kits. The primary and secondary outcome criteria were positive AF cultures and spontaneous preterm delivery (SPTD) within 48 h, respectively. Univariate, multivariate, and receiver operating characteristic analysis were used for the statistical analysis.ResultsIn bivariate analyses, elevated plasma IL-6 level was significantly associated with intra-amniotic infection and imminent preterm delivery, whereas elevated plasma levels of MMP-9, C3a, and C5a were not associated with these two outcomes. On multivariate analyses, an elevated plasma IL-6 level was significantly associated with intra-amniotic infection and imminent preterm delivery after adjusting for confounders, including high serum CRP levels and short cervical length. In predicting intra-amniotic infection, the area under the curve (AUC) was significantly lower for plasma IL-6 than for AF IL-6 but was similar to that for serum CRP. Differences in the AUCs between plasma IL-6, AF IL-6, and serum CRP were not statistically significant in predicting imminent preterm delivery.ConclusionsMaternal plasma IL-6 independently predicts intra-amniotic infection in women with preterm labor; however, it has worse diagnostic performance than that of AF IL-6 and similar performance to that of serum CRP. To predict imminent preterm delivery, plasma IL-6 had an overall diagnostic performance similar to that of AF IL-6 and serum CRP. Plasma MMP-9, C3a, and C5a levels could not predict intra-amniotic infection or imminent preterm delivery.

Investigating the Role of Fetal Gene Expression in Preterm Birth

Second-trimester amniotic fluid supernatant (AFS) contains cell-free fetal RNA (cffRNA) transcripts that can provide information about fetal gene expression. In a retrospective case-control study, we measured second-trimester fetal gene expression using cffRNA extracted from AFS in women who had spontaneous preterm birth (sPTB) <34 weeks and in women who delivered >37 weeks. We extracted cffRNA from AFS of women with singletons who had second-trimester genetic amniocenteses. Twenty-one gravidas who had sPTB and 21 term controls were matched 1:1 for maternal age, fetal sex, race, gestational age (GA) at the time of amniocentesis, and medication exposure. Cell-free fetal RNA was extracted and hybridized to a customized 65-gene NanoString panel containing genes related to oxidative stress, inflammation, and hypothalamic-pituitary-adrenal (HPA) axis and included 15 housekeeping genes. Two models were run, 1 examining sPTB in relation to case/control status and 1 examining sPTB in relation to GA as a continuous variable. Among cases, the gene expression of nitric oxide synthase 1 (NOS1), D-aspartate oxidase (DDO), and Beta-2-microglobulin (B2M) was higher than controls (P value < .05; false discovery rate-corrected Q value of ≤0.10). Nitric oxide synthase 1 and DDO are genes associated with oxidative stress; B2M is a marker of the fetal inflammatory response. Fetal HPA gene expression is not associated with GA at delivery or sPTB in second-trimester AFS. Alterations of fetal gene expression related to inflammation and oxidative stress antedate clinical symptoms and may be useful for early identification of patients at risk of having an sPTB.

Frequency and clinical significance of short cervix in patients with preterm premature rupture of membranes.

ObjectiveCervical length measurement has been uggested as a useful tool for predicting intra-amniotic infection/inflammation in preterm labor, but little information is available in the setting of preterm premature rupture of membranes (pPROM). We aimed to determine whether a short cervical length is independently associated with an increased risk of intra-amniotic infection or inflammation and impending preterm delivery in women with pPROM.MethodsThis was a retrospective cohort study involving 171 consecutive singleton pregnant women with pPROM (21+0–33+6 weeks’ gestation), who underwent amniocentesis. Amniotic fluid (AF) was cultured, and assayed for interleukin (IL)-6 and IL-8. Cervical length was measured at the time of amniocentesis by transvaginal ultrasonography with an aseptic technique. Short cervical length was defined as a cervical length of ≤15 mm. Intra-amniotic infection was defined as a positive AF culture for microorganisms and intra-amniotic inflammation was defined as elevated AF concentrations of IL-6 or IL-8 (IL-6 ≥1.5 ng/mL and/or IL-8 ≥1.3 ng/mL).ResultsFifty (29.2%) women had a sonographic cervical length of ≤15mm. On univariate analysis, short cervical length was associated with an increased risk for intra-amniotic infection and/or inflammation; no other parameters studied showed a significant association. Multivariable analyses indicated that short cervical length was significantly associated with a higher risk of impending preterm delivery (within 2 days of measurement, within 7 days of measurement, and before 34 weeks), and remained significant after adjustment for potential confounders.ConclusionIn women with pPROM, short cervical length is associated with an increased risk for intra-amniotic infection/inflammation and associated with impending preterm delivery, independent of the presence of intra-amniotic infection/inflammation.

Second trimester amniotic fluid uric acid, potassium, and cysteine to methionine ratio levels as possible signs of early preeclampsia: A case report

ObjectiveThe precise etiopathogenesis of preeclampsia (PE) still remains enigmatic. In recent published work, there is a scientific trend aiming to unveil early biomarkers of PE based on amniotic fluid compositional changes before the development of clinical symptoms. Case ReportWe describe a case of an apparently clinically healthy woman, whose amniotic fluid, retrieved after amniocentesis at 222/7 gestational week, had elevated uric acid and potassium concentration, as well as cysteine to methionine ratio. At the time of amniocentesis, conventional clinical signs of PE were absent. The woman developed severe PE and intrauterine growth restriction, at the 280/7 week of gestation. ConclusionAlthough the limitation of such studies lies in the fact that amniocentesis is an invasive procedure, and thus employed only under specific indications, our scientific observations might be useful for future research towards unraveling the causes of PE.

Congenital Cytomegalovirus Infection: Prognostic Value of Maternal DNAemia at Amniocentesis.

Human Cytomegalovirus (HCMV) is the most common cause of childhood hearing loss and can lead to neurodevelopmental delay. To date, few studies have examined the correlation between maternal viremia and congenital HCMV infection. The aim of our study was to ascertain if HCMV DNA in the peripheral blood of pregnant women with primary HCMV infection at the time of amniocentesis may have a prognostic value in terms of congenital infection and neonatal symptomatic disease. We performed a prospective observational study of pregnant women referred to our maternal-fetal medicine division with suspected HCMV infection. Primary infection was diagnosed based on seroconversion for HCMV and/or HCMV immunoglobulin M-positive and low or moderate HCMV immunoglobulin G avidity. At the time of amniocentesis, maternal blood samples were collected and analyzed by means of real-time polymerase chain reaction to determine the presence of viral DNAemia. Fetuses and newborns were evaluated for the presence of congenital infection and symptomatic disease. A total of 239 pregnant women were enrolled; 32 blood samples (13.4%) were positive, and 207 (86.6%) were negative for HCMV DNA. The overall rate of transmission was 23.4%. Fifteen infected patients (26.8%) were symptomatic. Vertical transmission occurred in 14 women (43.8%) with positive and 42 (20.3%) with negative results for HCMV DNAemia (P = .006; odds ratio, 3.06; 95% confidence interval, 1.41-6.64). Symptomatic infection occurred in 6 (42.9%) infected fetuses or newborns from women with and in 9 (21.4%) from women without viral DNAemia (P = .16). Maternal viremia at amniocentesis is associated with a 3-fold greater chance of congenital infection, but it is not correlated with symptomatic disease.

Low risk of HIV transmission for women on combined antiretroviral therapy.

Care for pregnant women with HIV has evolved over the last several decades, largely owing to combination antiretroviral therapy (cARV) leading to decreased morbidity and mortality in the context of a diagnosis of HIV (Severe P, et al. NEJM 2016;363(3):257–265; INSIGHT START study group. NEJM 2015;373:795–807). This has led to steadily increasing numbers of women with HIV becoming pregnant. Historically, obstetricians were taught to avoid invasive procedures and rupture of membranes, give intravenous zidovudine in labour and recommend caesarean delivery. However, the growing body of contemporary literature refutes these management trends when a pregnant woman is on antiretroviral therapy with an undetectable viral load. In this issue of BJOG, Floridia et al. evaluate the rates of mother-to-child transmission of HIV with amniocentesis and chorionic villus sampling (CVS). This is a multicentre trial running from 2001 to 2015, containing 113 HIV-positive pregnant women who underwent invasive testing. The majority (87.6%) were on antiretroviral therapy, and none of the infants born to women on cARV therapy at the time of amniocentesis or CVS became HIV positive. Of the two cases of HIV transmission in this series, one was diagnosed with HIV late in her pregnancy after the invasive procedure, and the other was only receiving zidovudine monotherapy at the time of her procedure with an elevated viral load. Unfortunately, the authors do not provide information about the viral loads at the time of the procedures, nor the timing of infant HIV transmission (in utero versus intrapartum). While the number of HIV-positive women on cARV undergoing invasive procedures is one of the highest reported in the literature, it is underpowered to detect small increased risks of HIV transmission involved with these procedures. Importantly, one of the cases of neonatal HIV transmission occurred in a women with a negative first-trimester HIV test, who was then diagnosed with HIV later in pregnancy. This highlights the importance of prompt diagnosis and treatment of HIV, in order to reduce mother-to-child transmission. While many pregnant women with HIV are on cARV therapy, recent literature has been illustrating a disproportionate number of cases of mother-to-child transmission occurring in women with delayed diagnosis or therapy (Birkhead GS et al. Obstet Gynecol 2010;115:1247–55; Lu D, et al. Can J Public Health 2014;105:e47–52). The article by Floridia et al. provides valuable information to aid counselling pregnant women with HIV undergoing genetic testing and considering invasive procedures. We are moving into an era where pregnant women with HIV are able to weigh the risks of conditions such as aneuploidy against mother-to-child transmission of HIV, and studies such as these give physicians the ability to give evidence-based recommendations to assist in these decisions. None declared. Completed disclosure of interests form available to view online as supporting information. ■ Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Association Between Neurotrophin 4 and Long-Chain Polyunsaturated Fatty Acid Levels in Mid-Trimester Amniotic Fluid

The omega-3 long-chain polyunsaturated fatty acid (LCPUFA) docosahexaenoic acid (DHA) and the omega-6 LCPUFA arachidonic acid (AA) are essential nervous system components that increase in concentration throughout gestation. The neurotrophins, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin 3 (NT3), and neurotrophin 4 (NT4) are small basic peptides crucial for fetal brain development. The DHA supplementation during pregnancy has been suggested to enhance neural development. We evaluated whether amniotic fluid DHA and AA concentrations correlated with intra-amniotic neurotrophin levels. Amniotic fluid, obtained at 15 to 19 weeks gestation from 62 women, was tested for BDNF, NGF, NT3, and NT4 by enzyme-linked immunosorbent assay. Concentrations of DHA and AA, and saturated and monounsaturated fatty acids, were determined by gas chromatography. Associations were analyzed by the Spearman rank correlation test. Median levels of AA and DHA were 2.3% and 1.3% of the total intra-amniotic fatty acids, respectively. Median neurotrophin levels (pg/mL) were 36.7 for NT3, 26.8 for BDNF, 5.2 for NT4, and 0.8 for NGF. Intra-amniotic NT4 and BDNF levels were correlated (P = .0016), while NT3 and NGF levels were unrelated to each other or to BDNF or NT4. Only NT4 was positively correlated with amniotic fluid DHA (P < .0001) and AA (P = .0003) concentrations. There were no associations between DHA, AA, or any neurotrophin and maternal age, gestational age at time of amniocentesis, amniocentesis indication, parity, or gestational age at delivery. Elevations in intra-amniotic NT4 with increasing levels of DHA and AA suggest that these LCPUFAs may specifically influence the extent of NT4-mediated fetal brain neurogenesis.

Routine measurement of amniotic fluid alpha-fetoprotein and acetylcholinesterase: the need for a reevaluation

The objective of the study was to evaluate whether the current screening regimen of measuring amniotic fluid alpha-fetoprotein (AF-AFP) at the time of amniocentesis and reflex acetylcholinesterase testing vs ultrasound alone to detect neural tube and ventral wall defects offers improved diagnostic accuracy and cost benefit. A retrospective chart review on all patients who had amniocentesis performed at 1 center over the past 11 years was performed. Those with an elevated AF-AFP were compared with those whose AF-AFP was within normal limits. Ultrasound findings and outcomes were reviewed in all cases to assess whether neural tube defects (NTDs) or ventral wall defects (VWDs) were missed by AF-AFP or ultrasound screening. A cost-benefit analysis was then performed. Of 6232 women who underwent amniocentesis between January 2002 and December 2012, 81 had an elevated AF-AFP with or without a positive acetylcholinesterase (AChE). Of these 81 women, 13 had NTDs and 5 had VWDs. The sensitivity of the detailed ultrasound was 100% in detecting NTDs and VWDs, whereas that of the AF-AFP ranged from 22% to 77%, with the inclusion of AChE. The total expenditure for AF-AFP in our sample set (n = 6232 amniocentesis at $76.00 per AF-AFP) was $473,632, and all NTDs and VWDs were detected by ultrasound. Translated to a national laboratory (>42,447 samples/year), the cost savings in 2011 alone would be $3,225,972. Given the accuracy of high-resolution ultrasound in the detection of both NTDs and VWDs, measuring AF-AFP and AChE as a reflex-screening test is not a cost-effective approach.

Evaluation of antenatal umbilical coiling index at 16-21 weeks of gestation as a predictor of trisomy 21 and other chromosomal defects

To determine whether there is an association between sonographically assessed hyper- or hypocoiling of the umbilical cord and the presence of trisomy 21, to provide reference values for the antenatal umbilical coiling index (aUCI) at a gestational age of 16-21 weeks and to determine whether these measurements are reliable and reproducible. This was a prospective study of 737 pregnancies in which the aUCI was measured between 16 and 21 weeks of gestation by ultrasound at the time of amniocentesis. The aUCI was calculated as the reciprocal value of the mean length of one complete coil in centimeters. We created reference curves and studied the relationship with trisomy 21 and other chromosomal defects. In 30 pregnancies we studied the intra- and interobserver variation in measurements using Bland-Altman plots with associated 95% limits of agreement and intraclass correlation coefficients. aUCI was found to be non-linearly related to gestational age at 16-21 weeks and reference curves were created for the mean aUCI and the 2.3(rd) , 10(th) , 90(th) and 97.7(th) percentiles. There was no significant difference in aUCI values between the reference group (n = 714) and cases with trisomy 21 (n = 16) or other aneuploidies (n = 7) (one-way ANOVA, P = 0.716). There was good intra- and interobserver agreement in aUCI measurements. The aUCI can be measured reliably and varies according to gestational age at 16-21 weeks. The aUCI was not significantly associated with trisomy 21 or other chromosomal defects.

An elevated maternal serum C-reactive protein in the context of intra-amniotic inflammation is an indicator that the development of amnionitis, an intense fetal and AF inflammatory response are likely in patients with preterm labor: clinical implications

sObjective: We propose that an elevated maternal serum C-reactive protein (CRP) concentration in the context of intra-amniotic inflammation (IAI) is a predictor for amnionitis development, known to be the most advanced stage of maternal inflammatory response during the progression of acute histologic chorioamnionitis in preterm gestations.Methods: Study population consisted of 53 singleton gestations with IAI, who underwent amniocentesis due to preterm labor and intact membranes (PTL) and delivered preterm-neonates (<34.5 weeks) within 5 days of amniocentesis. The frequency of amnionitis and the intensity of fetal and amniotic fluid (AF) inflammatory response were examined according to the presence or absence of an elevated maternal serum CRP (≥0.7 mg/dL) at the time of amniocentesis. IAI was defined as an elevated AF matrix metalloproteinase-8 (MMP-8) (≥23 ng/mL), and fetal inflammatory response syndrome (FIRS) defined as an elevated umbilical cord plasma CRP (≥200 ng/mL).Results: (1) Patients (73.6%, 39/53) with an elevated maternal serum CRP had a significantly higher rate of amnionitis (59.0% versus 7.1%; p < 0.005), but not funisitis (46.2% versus 28.6%; p > 0.05), and higher median AF MMP-8 and umbilical cord plasma CRP concentration at birth than patients (26.4%,14/53) without that (AF MMP-8 (ng/mL): 373.1 versus 138.6: p = 0.05; umbilical cord plasma CRP (ng/mL): 363.4 versus 15.5: p < 0.05); (2) Multiple logistic regression analysis demonstrated that an elevated maternal serum CRP was a better independent predictor of amnionitis (odds ratio (OR), 12.5: 95% confidence interval (CI), 1.1–141.0; p < 0.05) than FIRS (OR, 3.6: 95% CI, 0.6–20.2; p = 0.150) and any other AF tests.Conclusions: An elevated maternal serum CRP concentration in the context of IAI is an indicator that the development of amnionitis, an intense fetal and AF inflammatory response are likely in patients with PTL.

Preeclampsia is associated with an elevation of plasma sMet concentrations in the second trimester

Objective: The aim of this study was to investigate the association between anti-angiogenic factor soluble c-Met (sMet) concentrations in maternal plasma and the risk of preeclampsia.Methods: The pregnant women included in this study (1) had subsequent preeclampsia (n = 52) and were compared to normal controls (n = 104) at the time of amniocentesis (15–20 weeks); and (2) had preeclampsia (n = 63) and were compared to normal controls (n = 112) at the time of diagnosis of preeclampsia (29–40 weeks). sMet concentrations were measured by ELISA. Non-parametric statistics were used for analysis.Results: Maternal plasma sMet concentrations were significantly higher in both women with subsequent preeclampsia (median: 1372.7 ng/ml versus 1100.5 ng/ml; p = 0.036) and women with preeclampsia (median: 1651.9 ng/ml versus 1364.7 ng/ml; p < 0.001) than in the controls. After adjusting for potential confounding factors, the risks of developing preeclampsia were as follows: adjusted odds ratio 2.5 (95% confidence interval, 1.2–5.2; p = 0.016) for second trimester sMet concentration with a cut-off value of 1223.5 ng/ml and 4.4 (95% confidence interval, 2.2–9.1; p < 0.001) for third trimester sMet concentration with a cut-off value of 1460.3 ng/ml.Conclusion: Elevated maternal plasma sMet concentrations were independently associated with the increased risk of preeclampsia.

Congenital cytomegalovirus infection: patterns of fetal brain damage

Cytomegalovirus (CMV) is the most prevalent infectious agent causing neurological dysfunction in the developing brain. This study analysed the different patterns of tissue damage, particularly in the brain, of fetuses with documented CMV infection. We studied 45 fetuses at 20-21 weeks of gestation with congenital CMV infection documented by invasive positive prenatal diagnosis. At the time of amniocentesis, abnormal ultrasound findings had been recorded for 13 of the 45 fetuses (29%). Histological and immunohistochemical characterization was performed on the placenta, brain, heart, lung, liver, kidney, and pancreas. The different degrees of brain damage were correlated with tissue viral load, inflammatory response, placental functionality, and extramedullary haematopoiesis. Even though a high CMV load was detected in all amniotic fluids, brain infection occurred in only 62% of the fetuses and with different degrees of severity. Tissues with a low viral load showed a globally weak inflammatory response, and fetuses had only mild brain damage, whereas tissues with a high CMV load showed prominent infiltration of the activated cytotoxic CD8+ T-lymphocytes responsible for immune-mediated damage. Furthermore, severe placental infection was associated with diffuse villitis and necrosis, consistent with functional impairment and possible consequent hypoxic cerebral damage. Brain injury induced by CMV congenital infection may be the result of uncontrolled viral replication, immune-mediated damage by cytotoxic CD8+ T-lymphocytes, and, in the presence of placental insufficiency, fetal hypoxia.

Phthalates and Perfluorooctanesulfonic Acid in Human Amniotic Fluid: Temporal Trends and Timing of Amniocentesis in Pregnancy

Background: Measures of prenatal environmental exposures are important, and amniotic fluid levels may directly reflect fetal exposures during hypothesized windows of vulnerability.Objectives: We aimed to detect various phthalate metabolites and perfluorooctanesulfonic acid (PFOS) in human amniotic fluid, to study temporal exposure trends, and to estimate potential associations with gestational week of amniocentesis and maternal age and parity at amniocentesis.Methods: We studied 300 randomly selected second-trimester amniotic fluid samples from a Danish pregnancy-screening biobank covering 1980 through 1996. We used only samples from male offspring pregnancies. We assayed the environmental pollutants by liquid chromatography/triple quadrupole mass spectrometry and analyzed data using generalized linear regression models.Results: We detected the di(2-ethylhexyl) phthalate (DEHP) metabolite mono(2-ethyl-5-carboxypentyl) phthalate (5cx-MEPP) at a median concentration of 0.27 ng/mL [interquartile range (IQR): 0.20–0.37 ng/mL], the diisononyl phthalate (DiNP) metabolite mono(4-methyl-7-carboxyheptyl) phthalate (7cx-MMeHP) at 0.07 ng/mL (IQR: 0.05–0.11 ng/mL), and PFOS at 1.1 ng/mL (IQR: 0.66–1.60 ng/mL). An increase of 1 calendar year was associated with 3.5% lower [95% confidence interval (CI): –4.8%, –2.1%] 5cx-MEPP levels and with 7.1% higher (95% CI: 5.3%, 9.0%) 7cx-MMeHP levels. For each later gestational week of amniocentesis, 5cx-MEPP was 9.9% higher (95% CI: 4.8%, 15.2%), 7cx-MMeHP was 8.6% higher (95: CI: 2.7%, 14.9%), and PFOS was 9.4% higher (95: CI: 3.3%, 15.9%). We observed no associations with maternal age or parity.Conclusions: Measured metabolite levels appeared to parallel decreasing DEHP exposure and increasing DiNP exposure during the study period. The environmental pollutant levels were positively associated with later gestational age at amniocentesis during pregnancy weeks 12–22.

Fetal Anterior Wall Thickness and Amniotic Fluid Insulin Levels: an Interdependence?

Excessive fetal fat as the hallmark of GDM pregnancy complications is one consequence of fetal hyperinsulinism. Noninvasive methods for fetal surveillance and measurement of fetal fat are needed. The purpose of this study was to test the hypothesis that measurements of the fetal anterior abdominal wall thickness (AAWT) in women with GDM will allow early detection of fetal hyperinsulinism. Amniocentesis was performed between 28 and 32 weeks of gestation (wks) in 220 women with GDM (diagnosed by 75g oGTT at 24 to 28 wks). Amniotic fluid insulin levels (AFIL) were determined by a commercially available radioimmunoassay. Transabdominal ultrasound provided fetal biometric measurements following standard procedures and the AAWT including fetal skin and subcutaneous tissue at the time of amniocentesis. Maternal parameters (weight, BMI, oGTT blood glucose levels and mean daily blood glucose levels) were correlated with fetal biometric data and with AFIL. There was no difference in AAWT in women with GDM and no correlation with mean AFIL. AFIL also did not correlate with any other fetal measurement or with mean oGTT blood glucose levels. AFIL only showed a correlation with maternal weight (p=0.02) and maternal BMI (p=0.01). The correlation was present for values both before pregnancy and at the time of amniocentesis. In the early third trimester, AAWT measurements do not correlate with fetal insulin levels.

Significant Performance Variation Among PCR Systems in Diagnosing Congenital Toxoplasmosis in São Paulo, Brazil: Analysis of 467 Amniotic Fluid Samples

INTRODUCTIONPerformance variation among PCR systems in detecting Toxoplasma gondii has been extensively reported and associated with target genes, primer composition, amplification parameters, treatment during pregnancy, host genetic susceptibility and genotypes of different parasites according to geographical characteristics.PATIENTSA total of 467 amniotic fluid samples from T. gondii IgM- and IgG-positive Brazilian pregnant women being treated for 1 to 6 weeks at the time of amniocentesis (gestational ages of 14 to 25 weeks).METHODSOne nested-B1-PCR and three one-round amplification systems targeted to rDNA, AF146527 and the B1 gene were employed.RESULTSOf the 467 samples, 189 (40.47%) were positive for one-round amplifications: 120 (63.49%) for the B1 gene, 24 (12.69%) for AF146527, 45 (23.80%) for both AF146527 and the B1 gene, and none for rDNA. Fifty previously negative one-round PCR samples were chosen by computer-assisted randomization analysis and re-tested (nested-B1-PCR), during which nine additional cases were detected (9/50 or 18%).DISCUSSIONThe B1 gene PCR was far more sensitive than the AF146527 PCR, and the rDNA PCR was the least effective even though the rDNA had the most repetitive sequence. Considering that the four amplification systems were equally affected by treatment, that the amplification conditions were optimized for the target genes and that most of the primers have already been reported, it is plausible that the striking differences found among PCR performances could be associated with genetic diversity in patients and/or with different Toxoplasma gondii genotypes occurring in Brazil.CONCLUSIONThe use of PCR for the diagnosis of fetal Toxoplasma infections in Brazil should be targeted to the B1 gene when only one gene can be amplified, preferably by nested amplification with primers B22/B23.

Amniotic fluid insulin-like growth factor binding protein 3 concentration as early indicator of fetal growth restriction

Objective Insulin-like growth factor-I (IGF-I) is an important regulator of fetal growth and its bioavailability depends on insulin-like growth factor binding proteins (IGFBPs). Genes coding for IGF-I and IGFBP3 are polymorphic. We hypothesized that either amniotic fluid protein concentration at the beginning of the second trimester or genotype of one of these two genes could be predictive of abnormal fetal growth. Study design Amniotic fluid samples (14–18 weeks of pregnancy) from 123 patients with appropriate for gestational age (AGA) fetuses, 39 patients with small for gestational age (SGA) fetuses and 34 patients with large for gestational age (LGA) were analyzed. Protein concentrations were evaluated by ELISA and gene polymorphisms by PCR. Results Amniotic fluid IGFBP3 concentrations were significantly higher in SGA compared to AGA group ( P = 0.030), and this was even more significant when adjusted to gestational age at the time of amniocentesis and other covariates (ANCOVA analysis: P = 0.009). Genotypic distribution of IGF-I variable number of tandem repeats (VNTR) polymorphism was significantly different in SGA compared to AGA group ( P = 0.029). 19CA/20CA genotype frequency was threefold decreased in SGA compared to AGA group and the risk of SGA occurrence of this genotype was decreased accordingly: OR = 0.289, 95%CI = 0.1–0.9, P = 0.032. Genotype distribution of IGFBP3(A-202C) polymorphism was similar in all three groups. Conclusions High IGFBP3 concentrations in amniotic fluid at the beginning of the second trimester are associated with increased risks of SGA while 19CA/20CA genotype at IGF-I VNTR polymorphism is associated with reduced risks of SGA. Neither IGFBP3 concentrations, nor IGF-I/IGFBP3 polymorphisms are associated with modified risks of LGA.

Evidence of Maternal–Fetal Transmission ofParachlamydia acanthamoebae

To the Editor: Parachlamydia acanthamoebae is a recently identified agent of pneumonia (1–3) and has been linked to adverse pregnancy outcomes, including human miscarriage and bovine abortion (4,5). Parachlamydial sequences have also been detected in human cervical smears (4) and in guinea pig inclusion conjunctivitis (6). We present direct evidence of maternal–fetal transmission of P. acanthamoebae. We tested 78 amniotic fluid samples from patients who delivered prematurely (defined as spontaneous delivery before 37 weeks of gestation) at the Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland, from 2003 to 2006. DNA was extracted by using the QIAampDNA Mini kit (QIAGEN, Hilden, Germany) and was tested by using a specific Parachlamydia real-time PCR (7). One positive sample (threshold cycle of 34.2) was confirmed by using the 16SigF-Rp2Chlam PCR, which targets a large DNA segment of the 16S rRNA gene (8). Because these 2 PCRs target different DNA segments, the positive PCR results were not due to PCR contamination with amplicons. The sequence exhibited 99.6% similarity with P. acanthamoebae strain Hall’s coccus (1) (GenBank accession no. {type:entrez-nucleotide,attrs:{text:AF366365,term_id:14030551,term_text:AF366365}}AF366365). The sample was obtained from a 29-year-old woman during her second pregnancy, at 16 weeks of gestation. Amniocentesis was performed because a first-trimester test suggested a Down syndrome risk of 1/100. At the time of amniocentesis, the patient had cough and flu-like symptoms of 3 weeks’ duration, which resolved spontaneously in a few weeks. Cytogenetic analysis showed a normal 46XX karyotype; amniotic fluid culture remained sterile. The pregnancy ended prematurely at 35 weeks and 6 days with the vaginal delivery of a 2,060-g newborn (<5th percentile). The mother and child had an uneventful hospital course. The role of Parachlamydia as the etiologic agent of premature labor and intrauterine growth retardation is likely because 1) all vaginal, placental, and urinary cultures were negative; 2) results of routine serologic tests were negative; and 3) only Parachlamydia was detected in the amniotic fluid. Intrauterine infection caused by Parachlamydia spp. may be chronic and asymptomatic until adverse pregnancy outcomes occur (4). The infection of this pregnant woman might have occurred through zoonotic contact through her work as a butcher in a rural area known for cattle breeding. Of interest, a recent study of 482 healthy Swiss men found 3 who were seropositive for Parachlamydia sp., and all 3 came from the same rural area near Lausanne (within <20-km radius) (9). Moreover, the patient owns 2 guinea pigs, potential vectors of the bacterium (6). Other modes of transmission are possible, e.g., contaminated water (free-living amebae may serve as hosts for Parachlamydia spp. and are widespread in water networks) and ingestion of undercooked meat or contaminated cow milk. A plausible pathogenic scenario for this case of possible maternal–fetal transmission of P. acanthamoebae might include bacteremia in the context of a lung infection. This could have resulted in intrauterine infection and intrauterine growth restriction.

Amniocentesis in twin pregnancies

To describe the experiences in diagnostic amniocentesis in twin pregnancies. The computerized database and medical records of pregnant women attending Maternal Fetal Medicine Unit of the hospital for diagnostic amniocentesis at 16-20 weeks gestation between January 1992 and December 2006 were retrospectively reviewed. During 15 years of experience, 7,890 amniocenteses at 16-20 weeks gestation were performed for prenatal diagnosis, including 174 procedures in 87 twin pregnancies. The mean gestational age at the time of amniocentesis was 17.13 +/- 3.35 weeks. Preterm birth rate defined as the delivery before 36 gestational weeks was 36.24%. The total fetal loss rate was 5.17%, however, the procedure related fetal loss within 2 weeks after the procedure was 1.15%. Based on our limited data and previous studies, we can counsel patients undergoing twin amniocentesis at mid-trimester that the fetal loss rate may be slightly higher than that of singleton amniocentesis.

Clinical Predictive Value of Real-Time PCR Quantification of Human Cytomegalovirus DNA in Amniotic Fluid Samples

The aim of this study was to evaluate the diagnostic reliability and prognostic significance of the quantification of cytomegalovirus (CMV) DNA in amniotic fluid (AF). We retrospectively reviewed the results for 282 amniotic fluid samples that had been tested for CMV by a quantitative real-time PCR. We observed three cases in which no CMV genomes were detected in the AF but in which the children were nevertheless congenitally infected. Hence, we conclude that a negative result by PCR for CMV in AF cannot rule out the possibility of congenital infection. No false-positive PCR results were observed. A correlation between the CMV viral load in AF and the fetal and neonatal outcomes could not be demonstrated in our study. Instead, a correlation was found between the CMV viral load and the gestational age at the time of amniocentesis.