Just Accepted by The Journal of Maternal-Fetal & Neonatal Medicine Microbial load of umbilical cord blood Ureaplasma species and Mycoplasma hominis in preterm prelabor rupture of membranes Marian Kacerovsky, MD, PhD, Lenka Pliskova, MD, Ramkumar Menon, MD, PhD, Radka Kutova, PhD Ivana Musilova, MD, PhD, Jan Maly, MD, PhD and Ctirad Andrys, PhD doi: 10.3109/14767058.2014.887068
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Abstract Objective: To evaluate Ureaplasma species and Mycoplasma hominis DNA in the umbilical cord blood and its correlation with its microbial load in the amniotic fluid, as a measure of microbial burden in fetal inflammatory response and neonatal outcome in pregnancies complicated by preterm prelabor rupture of membranes (pPROM). Study design: A retrospective study of 158 women with singleton pregnancies complicated by pPROM between 240/7 and 366/7 weeks was conducted. Amniotic fluid was obtained from all women by transabdominal amniocentesis, and umbilical cord blood was obtained by venipuncture from umbilical cords immediately after the delivery of the neonates. The Ureaplasma species and Mycoplasma hominis DNA was quantitated using absolute quantification techniques. Result: Ureaplasma species and Mycoplasma hominis DNA was identified in 9% of the umbilical cord blood samples. No correlation between the amniotic fluid and umbilical cord blood microbial load of was observed. The presence of Ureaplasma species and Mycoplasma hominis DNA in the umbilical cord blood had no impact on short-term neonatal morbidity. Conclusions: A high microbial load of genital mycoplasma Ureaplasma species DNA in the umbilical cord in pregnancies complicated by pPROM is not associated with a high fetal inflammatory response and is therefore not associated with serious neonatal morbidity.
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Microbial load of umbilical cord blood Ureaplasma species and Mycoplasma hominis in preterm prelabor rupture of membranes Marian KACEROVSKY, MD, PhD1, 2, Lenka PLISKOVA, MD3, Ramkumar MENON, MD, PhD4, Radka KUTOVA, PhD3 Ivana MUSILOVA, MD, PhD2, 5, Jan MALY, MD, PhD6 and Ctirad ANDRYS, PhD7, Biomedical Research Center, University Hospital Hradec Kralove, Czech Republic. 2Department of
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1
Obstetrics and Gynecology, Charles University in Prague, Faculty of Medicine Hradec Kralove, Czech Republic. 3Institute of Clinical Biochemistry and Diagnostics, Charles University in Prague, Faculty of Medicine Hradec Kralove, University Hospital Hradec Kralove, Czech Republic 4Department of Obstetrics and Gynecology, The University of Texas Medical Branch at Galveston, USA 5Department of Obstetrics and Gynecology, University Hospital Pardubice, Czech Republic. 5Institute of Clinical Biochemistry and Diagnostics, Charles University in Prague, Faculty of Medicine Hradec Kralove, University Hospital Hradec Kralove, Czech Republic. 6Department of Pediatrics, Charles University in Prague, Faculty of Medicine Hradec Kralove, Czech Republic. 7Department of Clinical Immunology and Allergy, Charles University in Prague, Faculty of Medicine Hradec Kralove, Czech Republic. Corresponding author: Marian Kacerovsky, MD, PhD, Biomedical Research Center, University Hospital Hradec Kralove, Czech Republic, Sokolska 581, 500 05 Hradec Kralove, Czech Republic. Cellular phone: +420-777657991, Direct: +420-495832676
Email:
[email protected]
SHORT TITLE: genital mycoplasmas in umbilical cord blood
ABSTRACT
Objective: To evaluate Ureaplasma species and Mycoplasma hominis DNA in the umbilical cord blood and its correlation with its microbial load in the amniotic fluid, as a measure of microbial burden in fetal inflammatory response and neonatal outcome in pregnancies complicated by preterm prelabor rupture of membranes (pPROM).
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Study design: A retrospective study of 158 women with singleton pregnancies complicated by pPROM between 240/7 and 366/7 weeks was conducted. Amniotic fluid was obtained from all women by transabdominal amniocentesis, and umbilical cord blood was obtained by venipuncture from umbilical cords immediately after the delivery of the neonates. The Ureaplasma species and Mycoplasma hominis DNA was quantitated using absolute quantification techniques. Result: Ureaplasma species and Mycoplasma hominis DNA was identified in 9% of the umbilical cord blood samples. No correlation between the amniotic fluid and umbilical cord blood microbial load of was observed. The presence of Ureaplasma species and Mycoplasma hominis DNA in the umbilical cord blood had no impact on short-term neonatal morbidity. Conclusions: A high microbial load of genital mycoplasma Ureaplasma species DNA in the umbilical cord in pregnancies complicated by pPROM is not associated with a high fetal inflammatory response and is therefore not associated with serious neonatal morbidity. KEYWORDS: genital mycoplasmas; fetal inflammatory response; amniotic fluid.
INTRODUCTION
Preterm prelabor rupture of membranes (pPROM), defined as a leakage of the amniotic fluid before finished 37 weeks of gestation, is responsible for one-third of all preterm deliveries [1]. Ureaplasma species (Ureaplasma urealyticum and Ureaplasma parvus) and Mycoplasma hominis, the smallest self-
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replicating bacteria without a cell wall, are among the most common microorganisms associated with pPROM [2-4]. In addition to the clinical relevance of the presence of Ureaplasma species and Mycoplasma hominis in the amniotic fluid, where the intraamniotic inflammatory response appears to be microbial type-, doseand gestational age-dependent, their presence in the umbilical cord blood is indicative of a fetal inflammatory response [5-7]. Goldenberg et al. reported that the presence of Ureaplasma urealyticum and Mycoplasma hominis in umbilical cord blood was observed in 23% of preterm births with gestational ages of between 23 and 32 weeks [7]. Reports on the clinical consequences of the presence of Ureaplasma species and Mycoplasma hominis in umbilical cord blood with respect to short-term neonatal outcome are contradictory. Associations with respiratory distress syndrome, pneumonia, bronchopulmonary dysplasia and intraventricular hemorrhage have been reported by several investigators but also have not been reproduced by many others [7-10]. Therefore, the relevance of detecting Ureaplasma species and Mycoplasma hominis in umbilical cord blood warrants further investigation. Currently, little is known about the association between the microbial load of Ureaplasma species and Mycoplasma hominis in amniotic fluid and in umbilical cord blood. Moreover, no data are available about the relation between the amount of Ureaplasma species and Mycoplasma hominis in umbilical cord blood and the intensity of fetal inflammatory response. Therefore, the primary objective of this
study was to evaluate the microbial load of Ureaplasma species and Mycoplasma hominis DNA in umbilical cord blood and its association with the microbial load Ureaplasma species and Mycoplasma hominis DNA in amniotic fluid in pPROM. We also evaluated whether the fetal inflammatory response, as indicated by interleukin (IL)-6 concentration, is dependent on the microbial load of Ureaplasma species DNA. In addition, we characterized the relationship between the presence of Ureaplasma
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species and Mycoplasma hominis DNA in umbilical cord blood and selected aspects of neonatal outcomes in pPROM. MATERIAL AND METHODS The study was approved by the local institutional review board committee (March 19, 2008; no. 200804 SO1P). All women provided their written informed consent and self-identified as Caucasians. Thirty-two women in the present study group had been examined in one of our previous reports on the microbial load of Ureaplasma species and Mycoplasma hominis and intraamniotic inflammatory response in women with pPROM [6]. Sample collection Between February 2010 and February 2013, a retrospective cohort study of pregnant women at the gestational ages 240/7 and 366/7 weeks, who were admitted to the Department of Obstetrics and Gynecology, University Hospital Hradec Kralove, Czech Republic, was conducted. Pregnant women with singleton pregnancies, pPROM, and maternal age > 18 years were invited to participate in the study. Women with gestational hypertension, preeclampsia, signs of fetal growth restriction, the presence of either congenital or chromosomal fetal abnormalities, gestational or pre-gestational diabetes and signs of fetal hypoxia were excluded from the study.
Gestational ages were established by first trimester fetal biometry. In the Czech Republic, women with pPROM at less than 34 weeks of gestation are treated with corticosteroids for the induction of lung maturation (two doses of 14 mg betamethasone administered intramuscularly 24 hours apart), tocolytics for 48 hours, and antibiotics, whereas no treatment except antibiotics is initiated to delay delivery after 34 weeks. The management of pPROM in the Czech Republic is not expectant (except < 28
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gestational weeks). The induction of labor or the termination of pregnancy is initiated no later than 72 hours after rupture of the membranes, depending on the gestational age of the pregnancy, the fetal status, the maternal serum levels of C-reactive protein, and cervicovaginal streptococcus β colonization. pPROM was diagnosed by examination with a sterile speculum to verify the pooling of amniotic fluid in the vagina after confirming the presence of insulin-like growth factor-binding protein (ACTIM PROM test; MedixBiochemica, Kauniainen, Finland) in the vaginal fluid. Ultrasound-guided transabdominal amniocentesis was performed upon admission and approximately 5 mL of amniotic fluid was aspirated prior to the administration of corticosteroids, antibiotics, or tocolytics. Umbilical blood samples were obtained by venipuncture from clamped umbilical cords immediately after the delivery of the neonates and prior to the delivery of the placenta using. Detection of genital mycoplasmas DNA was isolated from the amniotic fluid and umbilical cord blood with the QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions (protocol for isolation of bacterial DNA from biological fluids). Real-time PCR was performed using a Rotor-Gene 6000 instrument (QIAGEN, Hilden, Germany) with the commercial kit AmpliSens® C. trachomatis/Ureaplasma/M. hominisFRT (Federal State Institution of Science, Central Research Institute of Epidemiology, Moscow, Russia) to detect the DNA of Ureaplasma parvum/urealyticum, Mycoplasma hominis, and Chlamydia trachomatis
in a common PCR tube. The amplified product was detected using fluorescent dyes in a real-time PCR. These dyes were linked to oligonucleotide probes, which bind specifically to the amplified DNA. The monitoring of the fluorescence intensities during the real-time PCR allowed the detection of accumulating product in each detection channel. A control included PCR for beta-actin, a housekeeping
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gene, to examine the presence of inhibitors of the PCR reaction. Quantification of genital mycoplasmas The amount of Ureaplasma species and Mycoplasma hominis DNA in copies/mL was determined by an absolute quantification technique employing an external calibration curve. Plasmid DNA (pCR4, Invitrogen) was used for the preparation of the calibration curve. The concentration of Ureaplasma species and Mycoplasma hominis DNA in copies/ L was converted into copies/mL using the following formula: concentration of Ureaplasma species/Mycoplasma hominis DNA (copies/ L) x elution volume ( L) / input volume (mL). Umbilical cord blood IL-6 levels IL-6 in the umbilical cord blood samples (1:10 dilution) was assessed by ELISA (R&D Systems Inc., Minneapolis, MN, USA). The sensitivity of the test was less than 0.70 pg/mL, and the interassay and intraassay coefficients were less than 10%. The main reason for using IL-6 to indicate the fetal inflammatory response was the fact that IL-6 umbilical cord blood levels are not expected to change as gestation progresses [11]. Diagnosis of severe neonatal morbidity Maternal and perinatal medical records were reviewed by two investigators (MK, IM) for morbidity and mortality for all newborns. For the current study, we defined “severe neonatal morbidity” as a condition composed of need for tracheal intubation, respiratory distress syndrome (defined by the presence of
two or more of the following criteria: evidence of respiratory compromise, a persistent oxygen requirement for more than 24 hours, administration of exogenous surfactant, and radiographic evidence of hyaline membrane disease), intraventricular hemorrhage (the diagnosis of which was made using cranial ultrasound examinations described by Papile et al. [12]), necrotizing enterocolitis (defined as the radiologic finding of either intramural gas or free intra-abdominal gas), retinopathy of prematurity
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(identified using retinoscopy), early and late onset sepsis [defined as any systemic bacterial infection documented by a positive blood culture and the presence of symptoms or clinically highly suspected sepsis (presence of symptoms and elevated CRP and/or affected WBC count) during the first 72 hours of life and between 4 and 120 days of life, respectively], bronchopulmonary dysplasia (defined as infant oxygen requirement at 28 days of life), pneumonia (diagnosed by an abnormal findings on chest X-rays), and neonatal death prior to hospital discharge. Statistical analysis The demographic characteristics were compared using either an unpaired t-test or the non-parametric Mann-Whitney U test for continuous variables, and the data are presented as the mean ± standard deviation (SD) and the median (range), respectively. The categorical variables were compared using the Chi-square test and are presented as percentages (%). The normality of the data was tested using the D’Agostino-Pearson omnibus normality test and the Shapiro-Wilk test. Adjusted p-values and odds ratios were obtained by binary logistic regression, with adjustment for microbial invasion of the amniotic cavity, histological chorioamnionitis, and funisitis. The differences were considered statistically significant at p < 0.05. All p-values were from two-sided tests, and all statistical analyses were performed using SPSS 19.0 for Mac OS X (SPSS Inc., Chicago, IL, USA) and with GraphPad Prism 5.03 for Mac OS X (GraphPad Software, La Jolla, CA, USA).
RESULTS A total of 193 women with singleton pregnancies complicated by pPROM between gestational ages 240/7 and 366/7 weeks were admitted to the department during the study period. Thirty-five women were excluded from the study because of different reasons: amniocentesis failures (n=19), fetal growth
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restriction (n=8), refusal to participate (n=2), structural and/or chromosomal abnormalities (n=2), severe vaginal bleeding (n=2), age under 18 years (n=1) and signs of fetal hypoxia at the time of admission (n=1). A total of 158 women with pPROM were included into the study. Among them, 18% (29/158) and 9% (15/158) had Ureaplasma species and Mycoplasma hominis DNA in their amniotic fluid and umbilical cord blood, respectively. DNA for Ureaplasma species and Mycoplasma hominis in the amniotic fluid was detected in 100% (29/29) and in 10% (3/29) of the women, respectively. The DNA for Ureaplasma species and Mycoplasma hominis in the umbilical cord blood was identified in 100% (15/15) and in 27% (4/15) of the women, respectively. The demographic data and clinical characteristics of the women with the presence of Ureaplasma species and Mycoplasma hominis DNA in their umbilical cord blood are presented in Table 1. Women with the presence of Ureaplasma species and Mycoplasma hominis DNA in their umbilical cord blood had a higher rate of microbial invasion of the amniotic cavity (p
