Objective
We sought to determine whether there is an association between bacterial load of genital mycoplasmas and histologic chorioamnionitis (HCA) in women with preterm premature rupture of membranes (PPROM).
Study Design
A total of 103 women with PPROM between 24-36 weeks of gestation were included in the study. Amniocenteses were performed, and the amounts of target genital mycoplasma DNA in amniotic fluid samples were evaluated using real-time polymerase chain reaction. The bacterial load of the genital mycoplasmas was relatively assessed using the threshold cycle value.
Results
The presence of genital mycoplasmas in amniotic fluid was found in 38% (39/103) of the women. The presence of HCA was associated with lower threshold cycle values (median 21.3, interquartile range, 16.5–28.5, vs median 29.4, interquartile range, 27.0–30.5; P = .005).
Conclusion
HCA in PPROM is associated with a higher bacterial load of genital mycoplasmas.
Genital mycoplasmas ( Ureaplasma spp and Mycoplasma hominis ) are the most frequent organisms found in the amniotic fluid (AF) of women with preterm premature rupture of membranes (PPROM). Their presence in AF is associated with higher rate of adverse pregnancy outcome with short- and long-term sequelae.
Genital mycoplasmas are among the smallest self-replicating microorganisms capable of independent growth and comparable with viruses in size. They were traditionally detected by culture methods, which required special conditions and expertise because of their delicate nature. Moreover, culture methods have fundamental flaws–they are time-consuming, and therefore, results are not available on time and cannot be used for clinical decision and management. Conventional polymerase chain reaction (PCR) is an essential tool for detection of some microbes; it dramatically improved the detection of genital mycoplasmas in comparison with culture techniques. Moreover, real-time PCR (quantitative PCR) could quantify the target DNA sequence in a sample. The amount could be expressed as an absolute or a relative value. Absolute quantification, which determines the exact amount of DNA, is more difficult and expensive because of the necessity to create reliable standards for quantification and establish the calibration curve. Moreover, these standards must be included in every real-time PCR cycle. However, it will be easier to compare data from different assays and laboratories. Relative quantification, which describes the changes in the amount of the target DNA within the same matrix by comparison to the signal from an endogenous or other reference control, is a simpler method, but the results are applicable only within the same real-time PCR assay. The inclusion of a reference control is necessary to compare among different assays.
The positive reaction in a real-time PCR is detected by accumulation of fluorescent signal, and the number of cycles required for fluorescent signal to exceed the threshold (background noise) level is called “threshold cycle” (Ct). One possible approach to the relative quantification of the target DNA in the real-time PCR is using Ct value.
Although real-time PCR for genital mycoplasmas could provide excellent methods for determining their bacterial load, the results are often interpreted only in a dichotomous manner (positive and negative). We decided to employ the Ct value as easy and inexpensive branch of relative quantification. We considered its effortless interpretation an advantage for clinicians. Therefore, use of Ct value might be helpful in clinical management of PPROM pregnancies.
Two recent studies, using different approaches of genital mycoplasma quantification, have reported the relationship between inflammatory response and the bacterial load of the U urealyticum and U parvum , respectively. With these results as basis, the quantification of the genital mycoplasmas in AF would seem to be valuable for a more accurate determination of intensity of intrauterine inflammation. Therefore, the objective of this study was to determine whether there is an association between bacterial load of genital mycoplasmas, determined by Ct value and histologic chorioamnionitis (HCA), and maternal inflammatory response, measured by maternal plasma C-reactive protein (CRP) level, as well as severe neonatal morbidity in women with PPROM.
Materials and Methods
Sample collection
A prospective cohort study was performed. Pregnant women with gestational age between 24-36 weeks, who were admitted to the Department of Obstetrics and Gynecology, University Hospital Hradec Kralove, Czech Republic, from May 2008 through November 2009 with a diagnosis of PPROM, were involved. Only women with the following criteria were enrolled: singleton pregnancy, sonographically estimated fetal weight between the 10th-90th percentiles for gestational age, and the absence of fetal structural malformations or chromosomal abnormalities. Exclusion criteria were significant vaginal bleeding and signs of fetal hypoxia.
PPROM was defined as fetal membrane rupture with leakage of AF that precedes the onset of uterine contraction by at least 2 hours at <37 weeks of gestation and was diagnosed by sterile speculum examination confirming the pooling of AF in the vagina in association with the positive test for the presence of the insulinlike growth factor-binding protein (ACTIM PROM test; Medix Biochemica, Kauniainen, Finland) in the vaginal fluid.
Ultrasound-guided transabdominal amniocenteses were performed. Approximately 5 mL of AF was aspirated, and maternal blood was collected by venipuncture; both were taken on admission before administration of corticosteroids, antibiotics, or tocolytics. The samples from the placenta, the fetal membranes, and the umbilical cord were obtained at delivery.
This study was approved by the institutional review board committee (March 19, 2008; no. 200804 SO1P), and informed consent was received from all participants.
AF analyses
DNA was isolated from the AF with the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instruction (protocol for isolation of bacterial DNA from biological fluids). Real-time PCR was performed on Rotor-Gene 6000 instrument (Qiagen) with commercial kit AmpliSens Chlamydia trachomatis / Ureaplasma / M hominis -FRT (Federal State Institution of Science, Central Research Institute of Epidemiology, Moscow, Russia) to detect DNA of C trachomatis , U parvum/urealyticum , and M hominis in common PCR tube. In real-time PCR, the amplified product was detected using fluorescent dyes. These dyes were linked to oligonucleotide probes, which bind specifically to the amplified DNA. Monitoring of the fluorescence intensities during the real-time PCR allowed the detection of accumulating product in each detection channel. Negative and positive controls were included in each amplification experiment. A control included PCR for beta-actin, a housekeeping gene, to examine the presence of inhibitors of the PCR.
PCR Ct
The amount of target DNA in the reaction, that is, in the sample, was measured on the basis of the Ct value (cycle where the instrument detects fluorescence above background noise) that is proportional to the starting template concentration. Ct is the intersection between an amplification curve and a threshold line. It is a measurement method of the concentration of target DNA in the PCR. Under ideal condition (most PCRs are close to 100% efficient), the amount of target amplicon increases at a rate of one log 10 every 3.32 cycles, which means that decreasing the bacterial load of genital mycoplasmas in the AF results in higher Ct values.
Diagnosis of HCA
Histologic examination of the placenta, the fetal membranes, and the umbilical cord was performed in all cases. Sections of tissue blocks were stained with hematoxylin and eosin. The degree of polymorphonuclear leukocyte infiltration was assessed separately in the free membranes, the chorionic plate, and the umbilical cord according to the criteria given by Salafia et al. Diagnosis of HCA was determined based on the presence of histologic grades of chorion-decidua 3-4 and/or chorionic plate 3-4 and/or umbilical cord 1-4 and/or amnion 1-4. Histopathological examination was performed by a single pathologist (H.H.) who was blinded to the clinical status of the women.
Maternal plasma CRP
The sample of maternal blood was centrifuged and supernatants were stored at −70°C until assayed. CRP concentrations were measured using an immunoturbidimetric analysis (Modular PP analyzer; Roche, Basel, Switzerland), with a method sensitivity of 0.3 mg/L.
Diagnosis of severe neonatal morbidity
For all newborns, data records regarding morbidity and mortality were reviewed. A composed entity was called “severe neonatal morbidity” and included a need for tracheal intubation; respiratory distress syndrome (RDS) defined by the presence of ≥2 of the following criteria: evidence of respiratory compromise and a persistent oxygen requirement for >24 hours, administration of exogenous surfactant, and/or radiographic evidence of hyaline membrane disease; intraventricular hemorrhage diagnosed by cranial ultrasound examination and graded I-IV by criteria defined by Papile et al ; necrotizing enterocolitis defined as a radiologic finding of either intramural gas or free intraabdominal gas; retinopathy of prematurity identified regarding retinoscopy; early-onset sepsis and late-onset sepsis (culture-proved or clinically highly suspected sepsis during the first 72 hours of life and between 4-120 days of life, respectively); bronchopulmonary dysplasia (BPD) defined as infant oxygen requirement at 28 days of age; pneumonia diagnosed regarding an abnormal chest x-ray finding; and neonatal death before hospital discharge.
Statistical analysis
The demographic and clinical characteristics were compared using unpaired t tests for continuous variables (values are presented as mean ± SD) or nonparametric Mann-Whitney U test (values are presented as median [interquartile range {IQR}]). Categorical variables were compared using Fisher’s exact test and presented as number (%). The normality of the data was tested using the D’Agostino and Pearson omnibus normality test and the Shapiro-Wilk test. Because the Ct value and maternal plasma CRP concentration were not normally distributed, a nonparametric Mann-Whitney U test was used for analyses, and variables are presented as median (IQR). Spearman rank correlation test was used for analysis of correlation between continuous variables. Differences were considered statistically significant at P < .05. A receiver operating characteristic curve was constructed to describe the relationship between the sensitivity and the false-positive rate for the different Ct values in the identification of HCA. To identify Ct value as a determinant of neonatal morbidity, a mixed linear model was performed to adjust for gestational age. All P values were from 2-sided tests, and statistical analyses were performed using GraphPad Prism 5.03 for Windows (GraphPad Software, San Diego, CA) and with SPSS 19.0 for Mac OS X (SPSS Inc, Chicago, IL).
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