Patient population

The study population consisted of fetuses whose mothers were admitted between April 2007 and September 2009 with the diagnosis of PPROM between 24 and 34 weeks of gestation and who were offered amniocentesis for the evaluation of the microbial status of the amniotic cavity. Inclusion criteria were (1) singleton gestation, (2) certain gestational age, (3) absence of chromosomal abnormality or congenital malformations detected pre- or postnatally, (4) estimated fetal weight on ultrasound >10th percentile, and (5) microbiologically proven intraamniotic infection (positive amniotic fluid culture). The diagnosis of PPROM was supported by the detection of a pool of fluid in the posterior vaginal fornix on sterile speculum examination and was confirmed by Nitrazine testing for an alkaline pH. Tocolytic agents, antibiotics, and steroids were not administered before maternal blood and amniotic fluid were collected. Calculation of gestational age was based on reliable recollection of the last menstrual period and confirmed or modified by ultrasound scans within the first 14 weeks of gestation.

The comparison group consisted of fetuses whose mothers had an uncomplicated pregnancy and were referred for fetal echocardiography. Indications for referral were related to family history of congenital heart anomaly or suspected arrhythmia that had been ruled out. We excluded fetuses of mothers with systemic diseases, such as maternal diabetes mellitus or systemic lupus erythematosus, and fetuses with chromosomal or any other congenital anomaly. Anthropometric parameters were measured in all fetuses to rule out the possibility of growth restriction. We decided to enroll at least 2 healthy control fetuses per case; we prolonged enrollment until the desired number of study subjects was achieved. The study was approved by the Institutional Review Board Committee of University of Bari. Written informed consent was obtained from all the participants.

Sonographic evaluation

The ultrasound examinations were performed with an Aloka ProSound Alpha 10 (Aloka, Tokyo, Japan), which was equipped with a phased-array duplex multifrequency transducer. All participating patients underwent a full morphologic examination of the fetal heart before quantitative assessment of right ventricular function with TDI and SRI techniques. A single investigator (E.D.N.) performed all fetal echocardiography studies. For each fetus, a high-resolution, zoomed loop of the apical 4-chamber view that incorporated at least 3 complete cardiac cycles was recorded. A narrow sector angle was used, and image depth was adjusted to allow for a maximal acquisition frame rate. Special care was taken to align the interventricular septum with the ultrasound beam (angulation, <30 degrees), and no angle correction was carried out. Digital loops were transferred to a dedicated workstation for offline analysis. Longitudinal segmental contraction and relaxation velocities of the right ventricle myocardium and strain and SR measurements were obtained by the technique described by Perles et al. Briefly, myocardial velocity profile was recorded by placement of the region of interest in the basal portion of the right free ventricular wall, adjacent to the tricuspid annulus. For strain and SR analysis, the region of interest was selected in the mid one-third of the right ventricle free wall. Through spatial derivation of the velocity data along the myocardial section of interest, Doppler-derived SR can be obtained. Further temporal integration of the SR will extract Doppler-derived strain. Two-dimensional echocardiography was used to record aortic valve closure and atrioventicular valves opening and closure, which were used for timing events during the cardiac cycle. By convention, strain is defined as positive when the distance between the points of measurements is increasing (ie, lengthening); shortening is represented by negative strain. Correspondingly, normal ventricular myocardium has a negative SR in systole and a positive SR during diastole. The following parameters were measured and averaged over 3 cycles: tissue Doppler velocities during early diastolic relaxation (E _m ) and atrial contraction (A _m ) and early diastolic SRs, as indices of right ventricle diastolic function and the peak systolic strain and SR as parameters of systolic function. E _m /A _m ratio was calculated as a surrogate measure of overall diastolic function.

Specimen collection and histologic examination

In the study group, amniocentesis was performed with a 21-gauge needle under ultrasonographic guidance with a free-hand technique. A sample of amniotic fluid was sent to the laboratory in a capped syringe immediately after collection for white blood cell count determination and cultures. Amniotic fluid was cultured for aerobic and anaerobic bacteria and for Mycoplasmas . At delivery, tissue samples were obtained from the umbilical cord and placenta. Histologic chorioamnionitis was defined as the presence of acute inflammatory changes in any of the placental tissue samples (amnion, chorion-decidua, and chorionic plate). Funisitis was diagnosed in the presence of neutrophilic infiltration in the umbilical cord wall vessels or in the Wharton’s jelly.

Statistical analysis

Statistical analysis was performed with GraphPad Prism (version 5.0c for Macintosh; GraphPad Software, San Diego CA). The t test and the Mann Whitney U test were used to compare continuous parametric and nonparametric variables, respectively. Proportions were analyzed with Fisher’s exact test. Because myocardial tissue velocity increases as a function of gestational age, the difference between the observed value and the mean value for gestational age (delta value) was computed to allow the case-controlled comparison of the E _m /A _m ratio. Expected mean values were derived by the regression equations for E _m and A _m that were provided by Perles et al.