Ivana Musilova

Intraamniotic inflammatory response to bacteria: analysis of multiple amniotic fluid proteins in women with preterm prelabor rupture of membranes.

Objective: To analyse whether intraamniotic inflammation in response to bacteria is different below and above gestational age 32 weeks in pregnancies complicated by preterm prelabor rupture of membranes (PPROM). Methods: A prospective study was performed, and 115 women with singleton pregnancies complicated by PPROM at gestational ages between 240/7 and 366/7 weeks were included in the study. Transabdominal amniocenteses were performed. Amniotic fluid was analysed using polymerase chain reactions for genital mycoplasmas and cultured for aerobic and anaerobic bacteria. The concentrations of 26 proteins in the amniotic fluid were determined simultaneously using multiplex technology. Results: Bacteria were found in the amniotic fluid of 43% (49/115) of the women. The women were stratified into two subgroups according to gestational age 32 weeks. The amniotic fluid levels of four (interleukin-6, interleukin-10, CC chemokine ligands 2, and 3) and one specific (CC chemokine ligands 2) proteins were higher in women with the presence of bacteria in the amniotic fluid below and above 32 gestational weeks, respectively. Conclusions: An intraamniotic inflammatory response to bacteria in pregnancies complicated by PPROM seems to be different below and above 32 weeks of gestation.

Prelabor rupture of membranes between 34 and 37 weeks: the intraamniotic inflammatory response and neonatal outcomes

OBJECTIVE We sought to determine the influence of microbial invasion of the amniotic cavity (MIAC) and acute histologic chorioamnionitis (HCA) on the intensity of the intraamniotic inflammatory response and neonatal morbidity in preterm prelabor rupture of membranes (PPROM) between 34-37 weeks. STUDY DESIGN This study included 99 women with singleton pregnancies complicated by PPROM between the gestational ages of 34-37 weeks. Amniocenteses were performed at the time of admission, and MIAC and amniotic fluid interleukin-6 concentrations were determined. After delivery, the placenta was evaluated for the presence of HCA. RESULTS Women with both MIAC and HCA had the highest intraamniotic inflammatory response, which was mediated by interleukin-6 concentrations (both MIAC and HCA: median 2164.0 pg/mL; HCA alone: median 654.8 pg/mL; MIAC alone: median 784.1 pg/mL; neither MIAC nor HCA: median 383.0 pg/mL; P < .0001) and the highest incidence of newborns with early-onset sepsis (P = .02). CONCLUSION Both MIAC and HCA affect the intensity of the intraamniotic inflammatory response and the incidence of early-onset sepsis following PPROM between 34-37 weeks. The intensity of the intraamniotic inflammatory response should be considered in the clinical management of PPROM between 34-37 weeks.

The microbial load with genital mycoplasmas correlates with the degree of histologic chorioamnionitis in preterm PROM

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.

The fetal inflammatory response in subgroups of women with preterm prelabor rupture of the membranes.

Abstract Objective: To evaluate the influence of microbial invasion of the amniotic cavity (MIAC) and histological chorioamnionitis (HCA) on the intensity of the fetal inflammatory response and the occurrence of fetal inflammatory response syndrome (FIRS) in preterm prelabor rupture of membranes (PPROM). Methods: One hundred and forty-nine women with singleton pregnancies complicated by PPROM between the gestational ages 24 + 0 and 36 + 6 weeks were included in the study. Blood samples were obtained by venipuncture from the umbilical cord after the delivery of the newborn. The umbilical cord blood interleukin (IL)-6 levels were evaluated using ELISA kits. The fetal inflammatory response was determined by IL-6 levels, and FIRS was defined as an umbilical cord blood IL-6 >11 pg/mL. Result: IL-6 levels and the occurrence of FIRS were higher in women complicated with both MIAC and HCA (median IL-6 35.5 pg/mL, FIRS in 68%) than in women with HCA alone (median IL-6 5.8 pg/mL, FIRS in 36%), MIAC alone (median IL-6 2.8 pg/mL, FIRS in 17%) or women without MIAC or HCA (median IL-6 4.3 pg/mL, FIRS in 29%). There were no differences in IL-6 levels or rates of FIRS among women with MIAC alone or HCA alone and women without both MIAC and HCA. Conclusion: A higher fetal inflammatory response mediated by umbilical cord blood IL-6 was identified when both MIAC and HCA were detected in pregnancies complicated by PPROM.

The association between histological chorioamnionitis, funisitis and neonatal outcome in women with preterm prelabor rupture of membranes

Abstract Objective: To determine the impact of histological chorioamnionitis (HCA) and funisitis on neonatal outcome in preterm prelabor rupture of membranes (PPROM) pregnancies. Methods: Women with PPROM between 24 + 0 to 36 + 6 weeks of gestation, admitted to the Department of Obstetrics and Gynecology at the University Hospital Hradec Kralove in the Czech Republic, between July 2008 and October 2010, were enrolled in the study (n = 231). Results: The incidence of early-onset sepsis (EOS) differed significantly in neonates born to women with and without HCA, after adjustment for gestational age (11% versus 1%, p = 0.011). The incidence of EOS in neonates was also significantly different, after adjustment for gestational age, in cases with and without funisitis (18% versus 4%, p = 0.002). The same was also found for retinopathy of prematurity (ROP) cases with and without funisitis (23% versus 4%, p = 0.014), after adjustment for gestational age. Conclusions: HCA and funisitis increase the risk of adverse perinatal outcome in PPROM pregnancies.

Intraamniotic Inflammation in Women with Preterm Prelabor Rupture of Membranes.

Objective To characterize subgroups of preterm prelabor rupture of membranes (PPROM) and short-term neonatal outcomes based on the presence and absence of intraamniotic inflammation (IAI) and/or microbial invasion of the amniotic cavity (MIAC). Methods One hundred and sixty-six Caucasian women with singleton pregnancies were included in this study. Amniotic fluid samples were obtained by transabdominal amniocentesis (n=166) and were assayed for interleukin-6 levels by a lateral flow immunoassay. The presence of Ureaplasma species, Mycoplasma hominis, Chlamydia trachomatis, and 16S rRNA was evaluated in the amniotic fluid. IAI was defined as amniotic fluid IL-6 values, measured by a point of care test, higher than 745 pg/mL. Results Microbial-associated IAI (IAI with MIAC) and sterile intraamniotic inflammation (IAI alone) were found in 21% and 4%, respectively, of women with PPROM. Women with microbial-associated IAI had higher microbial loads of Ureaplasma species in the amniotic fluid than women with MIAC alone. No differences in the short-term neonatal morbidity with respect to the presence of microbial-associated IAI, sterile IAI and MIAC alone were found after adjusting for the gestational age at delivery in women with PPROM. Conclusions Microbial-associated but not sterile intraamniotic inflammation is common in Caucasian women with PPROM. The gestational age at delivery but not the presence of inflammation affects the short-term neonatal morbidity of newborns from PPROM pregnancies.

Amniotic Fluid Cathelicidin in PPROM Pregnancies: From Proteomic Discovery to Assessing Its Potential in Inflammatory Complications Diagnosis

Background Preterm prelabor rupture of membranes (PPROM) complicated by microbial invasion of the amniotic cavity (MIAC) leading to histological chorioamnionitis (HCA) significantly impacts perinatal morbidity. Unfortunately, no well-established tool for identifying PPROM patients threatened by these disorders is available. Methodology/Principal Findings We performed an unbiased exploratory analysis of amniotic fluid proteome changes due to MIAC and HCA. From among the top five proteins that showed the most profound and significant change, we sought to confirm results concerning cathelicidin (P49913, CAMP_HUMAN), since an ELISA kit was readily available for this protein. In our exploratory proteomic study, cathelicidin showed a ∼6-fold higher concentration in PPROM patients with confirmed MIAC and HCA. We verified significantly higher levels of cathelicidin in exploratory samples (women without both MIAC and HCA: median 1.4 ng/ml; women with both conditions confirmed: median 3.6 ng/ml; p = 0.0003). A prospective replication cohort was used for independent validation and for assessment of cathelicidin potential to stratify women with MIAC leading to HCA from women in whom at least one of these conditions was ruled out. We confirmed the association of higher amniotic fluid cathelicidin levels with MIAC leading to HCA (the presence of both MIAC and HCA: median 3.1 ng/ml; other women: median 1.4 ng/ml; p<0.0001). A cathelicidin concentration of 4.0 ng/ml was found to be the best cut-off point for identifying PPROM women with both MIAC and HCA. When tested on the validation cohort, a sensitivity of 48%, a specificity of 90%, a likelihood ratio of 5.0, and an area under receiver-operating characteristic curve of 71% were achieved for identification of women with MIAC leading to HCA. Conclusions Our multi-stage study suggests cathelicidin as a candidate marker that should be considered for a panel of amniotic fluid proteins permitting identification of PPROM women with MIAC leading to HCA.

Cervical fluid IL-6 and IL-8 levels in pregnancies complicated by preterm prelabor rupture of membranes

Abstract Objective: To determine the cervical fluid interleukin (IL)-6 and IL-8 levels in pregnancies complicated by preterm prelabor rupture of membranes (PPROM) and the association of these interleukins with microbial invasion of the amniotic cavity (MIAC) and histological chorioamnionitis (HCA). Methods: Sixty women with singleton pregnancies were included in this study. Cervical fluid was sampled at the time of admission using Dacron polyester swabs, which were placed into the endocervical canal for 20 s. IL-6 and IL-8 levels were determined by ELISA. The management of PPROM was active management (except for in pregnancies <28 weeks of gestation) and occurs not later than 72 h after the rupture of membranes. Result: The women with MIAC had higher IL-6 and IL-8 levels than did the women without MIAC (IL-6: p = 0.01; IL-8: p = 0.003). There was no difference in IL-6 levels between women with and without HCA (p = 0.37). The women with HCA had higher IL-8 levels only in the crude analysis (p = 0.01) but not after adjustment for gestational age (p = 0.06). The women with both MIAC and HCA had higher levels of IL-6 and IL-8 than did the other women (IL-6: p = 0.003; IL-8: p = 0.001). IL-8 level of 2653 pg/mL was found to be the best cut-off point in the identification of PPROM pregnancies complicated by both MIAC and HCA with a likelihood ratio of 24. Conclusions: The presence of MIAC is the most important factor impacting the local cervical inflammatory response, which is determined by IL-6 and IL-8 levels in the cervical fluid. IL-8 levels seem to be a promising non-invasive marker for the prediction of pregnancies complicated by the presence of both MIAC and HCA.

Proteomic Biomarkers for Spontaneous Preterm Birth A Systematic Review of the Literature

This review aimed to identify, synthesize, and analyze the findings of studies on proteomic biomarkers for spontaneous preterm birth (PTB). Three electronic databases (Medline, Embase, and Scopus) were searched for studies in any language reporting the use of proteomic biomarkers for PTB published between January 1994 and December 2012. Retrieved citations were screened, and relevant studies were selected for full-text reading, in triplicate. The search yielded 529 citations, 51 were selected for full-text reading and 8 studies were included in the review. A total of 64 dysregulated proteins were reported. Only 14-3-3 protein sigma, annexin A5, protein S100-A8, protein S100-A12, and inter-α-trypsin inhibitor heavy chain H4 were reported in more than 1 study, but results could not be combined due to heterogeneity in type of sample and analytical platform. In conclusion, according to the existing literature, there are no specific proteomic biomarkers capable of accurately predicting PTB.

Cervical Microbiota in Women with Preterm Prelabor Rupture of Membranes

Objective To analyze the cervical microbiota in women with preterm prelabor rupture of membranes (PPROM) by pyrosequencing and to document associations between cervical microbiota, cervical inflammatory response, microbial invasion of the amniotic cavity (MIAC), histological chorioamnionitis, and intraamniotic infection (IAI). Study Design Sixty-one women with singleton pregnancies complicated by PPROM were included in the study. Specimens of cervical and amniotic fluid were collected on admission. The cervical microbiota was assessed by 16S rRNA gene sequencing by pyrosequencing. Interleukin (IL)-6 concentration in the cervical fluid and amniotic fluid was measured by ELISA and lateral flow immunoassay, respectively. Results Four bacterial community state types [CST I (Lactobacillus crispatus dominated), CST III (Lactobacillus iners dominated), CST IV-A (non-Lactobacillus bacteria dominated), and CST IV-B (Gardnerella vaginalis and Sneathia sanguinegens dominated)] were observed in the cervical microbiota of women with PPROM. Cervical fluid IL-6 concentrations differed between CSTs (CST I = 145 pg/mL, CST III = 166 pg/mL, CST IV-A = 420 pg/mL, and CST IV-B = 322 pg/mL; p = 0.004). There were also differences in the rates of MIAC, of both MIAC and histological chorioamnionitis, and of IAI among CSTs. No difference in the rate of histological chorioamnionitis was found among CSTs. Conclusions The cervical microbiota in PPROM women in this study was characterized by four CSTs. The presence of non-Lactobacillus CSTs was associated with a strong cervical inflammatory response and higher rates of MIAC, both MIAC and histological chorioamnionitis, and IAI representing a PPROM subtype with pronounced inflammation. CST I represents the dominant type of PPROM with a low rate of MIAC, IAI, and the combination of MIAC and histological chorioamnionitis.