Detection of bacteria in mid-trimester amniotic fluids by gene amplification of bacterial 16s ribosomal DNA sequences, cloning and sequence analysis

SMFM Abstracts S171 556 A SURVEY OF ASSISTED REPRODUCTIVE TECHNOLOGY (ART) BIRTHS AND IMPRINTING DISORDERS CATHY ALLEN1, ROBERT F. HARRISON1, WILLIAM REARDON2, SARAH BOWDIN3, LOUISE BRUETON3, EAMONN MAHER3, 1Human Assisted Reproduction Ireland, Dublin, Ireland, 2National Centre for Medical Genetics, Dublin, Ireland, 3University of Birmingham School of Medicine, Birmingham, United Kingdom OBJECTIVE: Genomic imprinting is an epigenetic process in which allelespecific gene expression is dependent on the parental inheritance. Although only a minority of human genes are imprinted, those that have been identified have been preferentially implicated in prenatal growth and neurodevelopment. Recently several groups have reported an increased frequency of ART births in children with Beckwith-Wiedemann syndrome (BWS) and Angelman syndrome (AS), conditions caused by imprinting disorders. However the risk of imprinting disorders in ART children is unknown. STUDY DESIGN: We undertook a systematic questionnaire-based survey of 2492 children born after ART in 2 units in Ireland and Central England. Questionnaires were sent to all patients with successful pregnancy outcomes following IVF between 1989 and 2003. Responses were assessed by a Geneticist for phenotypic signs known to be associated with an imprinting disorder. Clinical assessment was offered where the information supported a possible syndrome phenotype. Molecular analyses of blood samples were performed when phenotypic features were confirmed by physical examination. RESULTS: Response rate was 65% (n=1523 children). 70 children were identified as warranting clinical assessment; 47 were examined. 4 children had clinical features consistent with BWS. One child showed loss of methylation at KvDMR consistent with BWS. The 3 other children were negative for deletions, mutations, paternal mosaic disomy and the atypical molecular defects. One child had a number of features consistent with AS, however molecular analysis failed to detect a deletion, disomy or imprinting centre mutation in the AS region of chromosome 15. CONCLUSION: These observations suggest that the relative risk of AS and BWS may be increased in children born after ART, but the absolute risk of imprinting disorders remains small (!0.1%). We suggest that further similar surveys should be undertaken to further refine the risks of imprinting disorders after ART.

558 A VIRTUAL REALITY SYSTEM BASED ON PATIENT IMAGING DATA FOR HANDS-ON SIMULATION AND AUTOMATIC EVALUATION OF ULTRASOUND EXAMINATION AND AMNIOCENTESIS CHRISTOPHE VAYSSIERE1, CLEMENT FOREST2, OLIVIER COMAS2, DOMINIQUE CHRISTMANN3, ROMAIN FAVRE1, ISRAE¨L NISAND4, LUC SOLER2, JACQUES MARESCAUX2, 1CMCO-SIHCUS, OB/GYN, Strasbourg, France, 2IRCAD: Institut de Recherche sur le Cancer de l’Appareil Digestif, Strasbourg, France, 3CHU Hautepierre, Radiologie, Strasbourg, France, 4 CHU Hautepierre, OB/GYN, Strasbourg, France OBJECTIVE: To explore here the feasibility of creating a hands-on virtual simulator for amniocentesis based on real MRI images. STUDY DESIGN: On an up-to-date laptop, a 3D model of the patient skin is constructed and inserted into a 3D scene. A haptic force feedback device, Phantom Omni from Sensable Technologies (Woburn, MA, USA) is used to simulate the US transducer, so that the user actually feels the contact with the virtual patient. Proprietary software converts the 3D image of the patient into virtual US images according to the position of the force feedback device stylus and enables real-time navigation in all planes and directions. Using a second force feedback device, the software renders realistically the sensation of the needle penetrating the different layers (skin, uterus, placenta). RESULTS: The system has been tested successfully with a 2-mm MRI image of a fetus at 29 weeks’ gestation. By manipulating the virtual transducer, the user have to choose the best path for the needle and proceed with the insertion. The automatic assessment tool allows the evaluation of the whole procedure (success and safeness). CONCLUSION: We report the first virtual reality system for obstetric US examinations and amniocentesis that works on a virtual patient and provides an automatic evaluation. This system may represent an important step toward more effective student training and evaluation. 0002-9378/$ - see front matter doi:10.1016/j.ajog.2006.10.608

0002-9378/$ - see front matter doi:10.1016/j.ajog.2006.10.606

557 ACCURACY OF NON-INVASIVE FETAL RH CC AND RH EE GENOTYPING FROM MATERNAL BLOOD CHAD A. GROTEGUT1, JOHN P. GAUGHAN2, OSSIE GEIFMANHOLTZMAN3, 1Duke University Medical Center, Obstetrics and Gynecology, Durham, North Carolina, 2Temple University School of Medicine, Biostatistics, Philadelphia, Pennsylvania, 3Temple University School of Medicine, Obstetrics, Gynecology and Reproductive Sciences, Philadelphia, Pennsylvania OBJECTIVE: To determine the reported accuracy of non-invasive fetal Rh Cc and Rh Ee genotyping from maternal blood. STUDY DESIGN: A PubMed search of the literature describing fetal Rh Cc and Rh Ee genotyping from maternal blood was conducted. From each study, we determined the fetal Rh genotype tested, the source of the fetal DNA, gestational age at time of testing and confirmation of fetal Rh Cc and Ee type. The sensitivity, specificity, PPV and NPV of the Rh typing were calculated. RESULTS: We found 4 English-written articles reporting Rh Cc typing and 4 articles reporting Rh Ee typing from fetal DNA obtained from maternal blood. A total of 106 correct Rh Cc genotypes were documented out of 125 samples tested (84.8%). For Rh Cc typing the sensitivity, specificity, PPV and NPV were 100%, 82.6%, 80.3% and 100%, respectively. Of Rh Cc typing performed from fetal cells, 16 were correctly typed out of 28 samples tested (57.1%) and when performed from free fetal DNA in maternal plasma, 90 were correctly typed out of 97 tested (92.8%). A total of 141 correct Rh Ee genotypes were documented out of 148 tested (95.3%). For Rh Ee typing the sensitivity, specificity, PPV and NPV were 92.1%, 100%, 100% and 92.2%, respectively. Of Rh Ee typing performed from fetal cells, 34 were correctly typed out of 40 tested (85%) and when performed from free fetal DNA in maternal plasma, 107 were correctly typed out of 108 tested (99.1%). CONCLUSION: Accurate non-invasive fetal Rh Cc and Rh Ee determination using maternal blood is possible and can be applicable to the management of Rh Cc and Rh Ee alloimmunized pregnancies. Improvements of the technique and further study of the structure of the Rh Cc and Rh Ee gene may improve accuracy and allow for routine clinical use. 0002-9378/$ - see front matter doi:10.1016/j.ajog.2006.10.607

559 DETECTION OF BACTERIA IN MID-TRIMESTER AMNIOTIC FLUIDS BY GENE AMPLIFICATION OF BACTERIAL 16S RIBOSOMAL DNA SEQUENCES, CLONING AND SEQUENCE ANALYSIS CLAUDEL JEAN-PIERRE1, STEVEN WITKIN2, MARIA SCHNEIDER3, IARA LINHARES1, DANIEL SKUPSKI4, LARRY FORNEY3, 1Weill Medical College of Cornell University, Obstetrics and Gynecology, New York, New York, 2Cornell University, Obstetrics & Gynecology, New York, New York, 3University of Idaho, Biological Sciences, Moscow, Idaho, 4 Cornell University, Obstetrics and Gynecology, Flushing, New York OBJECTIVE: Mid-trimester amniotic fluids (AFs) are supposedly sterile. However, we reported that 10-15% of AFs were positive for mycoplasmas using gene amplification. We investigated whether other bacteria could be detected by amplifying the gene coding for bacterial 16S ribosomal RNA. STUDY DESIGN: AFs were collected using sterile technique and immediately transferred to a sterile tube. The bacterial 16S ribosomal RNA gene was amplified in 14 blinded AFs followed by cloning and sequence analysis. RESULTS: Ten samples were from Whites, two from Asians, one from a Black woman and one was lost to follow-up. Eleven women subsequently had a term birth and two were preterm at 29 and 36 weeks. Both preterm infants had respiratory distress syndrome. Multiple bacterial species were detected in 10 (77%) samples; four samples from term Whites were negative. The most common phylotype was most closely related to Ralstonia species, being detected in 7 (50%) of the samples. Other frequent phylotypes were Pseudomonas species (5 fluids), Flavobacterium species (4 fluids), Methylobacterium species (3 fluids), Sphingomonas species (3 fluids), and Stenotrophomonas species (3 fluids). Results from controls precluded the possibility of these being contaminants in the reagents. The AF from the 37 year old woman who delivered at 29 weeks was the only sample positive for a microbe with 89.3% homology to Burkholderia cepacia. AF from the 33 year old woman delivering at 36 weeks was the only sample positive for Streptomyces species. CONCLUSION: B. cepacia is a respiratory tract pathogen in immunosuppressed individuals that induces an intense pro-inflammatory immune response. Streptomyces species have been associated with respiratory symptoms and lung inflammation. We conclude that most mid-trimester AFs from healthy asymptomatic women are positive for bacteria that can be identified by gene amplification technology. 0002-9378/$ - see front matter doi:10.1016/j.ajog.2006.10.609