Patients

This was a single-center observational study conducted at the University Hospital Leuven in the time period between September 2007 and November 2008. We included all monochorionic diamniotic twin pregnancies diagnosed with TTTS and scheduled for an invasive prenatal procedure implying access to the recipient’s amniotic cavity (fetoscopic laser coagulation of placental vascular anastomoses, amniodrainage, or cord occlusion). Patients who underwent previous amniocentesis or amniodrainage in the index pregnancy were excluded.

TTTS was defined as the presence of oligohydramnios in the donor sac with a deepest vertical amniotic fluid pocket (DVP) of 2 cm or less, combined with polyhydramnios in the recipient sac with a DVP of 8 cm or greater prior to 20 weeks and 10 cm or greater after 20 weeks. Staging of TTTS was done according to the Quintero classification system.

Post hoc, an additional subdivision was made in stage III disease: patients with critically abnormal venous Dopplers in the recipient twin were classified as stage III-R. Those with abnormal Dopplers in the donor twin only were classified as III-D. As a control group, we recruited women with structurally normal singleton pregnancies who underwent amniocentesis for karyotyping or determination of fetal toxoplasmosis or cytomegalovirus infection.

Exclusion criteria were ultrasound signs of cardiac failure at the time of sampling, abnormal fetal karyotype, or confirmed infection. Follow-up data included the documentation of an uneventful further pregnancy and neonatal outcome.

This study was approved by the Ethics Committee on Clinical Studies of the University Hospitals Leuven. All patients gave written informed consent for the surgical procedure and sample collection for research purposes.

Ultrasound markers of fetal cardiac function

Fetal cardiac function was examined using a Voluson 730 Expert or E8 ultrasound device (GE Medical Systems, Zipf, Austria) just prior to amniotic fluid sampling in 16 controls and less than 24 hours before amniotic fluid sampling in TTTS pregnancies that underwent fetoscopic laser. Doppler measurements were obtained in the absence of fetal and maternal movements, and the Doppler sweep speed was set to maximum (10 cm/s). The angle between the sample volume and the blood flow was kept below 15°.

The left ventricular (LV) myocardial performance index (MPI) was measured by positioning the Doppler sample volume on the mitral and aortic valve in an apical 4-chamber view as described by Hernandez et al. The isovolumetric contraction time (ICT), isovolumetric relaxation time (IRT), and ejection time (ET) were determined using the valve click method. The MPI was calculated as (ICT+IRT)/ET. This index is dependent on a combination of ventricular systolic and diastolic function as well as afterload, and a higher MPI corresponds to a worse hemodynamic situation.

The right ventricular (RV) MPI cannot be obtained on a single Doppler trace because of the way the tricuspid and pulmonary valve are implanted. Therefore, the RV-MPI was calculated based on separate Doppler images depicting the duration of the time interval between 2 tricuspid inflows (TBTI) and the pulmonary outflow (PF); RV-MPI was calculated as (TBTI-PF)/PF and recorded only when the heart rate difference between tricuspid and pulmonary Doppler tracings was 5 beats/min or less. LV- and RV-MPI were transformed to z-scores based on gestational age and monochorionicity-adjusted reference ranges and classified as normal (z-score <2.0) or abnormal (z-score >2.0).

The mitral and tricuspid flow waveforms were obtained by positioning the Doppler sample volume at the tip of the atrioventricular valve leaflets in an apical 4-chamber view. The waveform was classified as normal (clearly distinct E and A wave) or abnormal (monophasic flow). The presence of atrioventricular regurgitation was assessed using color and pulsed Doppler and classified as present (holosystolic regurgitation) or absent.

The ductus venosus was assessed on a transverse or sagittal section trough the fetal abdomen. The junction between the umbilical vein and the ductus venosus was identified using color Doppler and sampled at the area of maximal aliasing. Ductus venosus waveforms were classified as normal (positive a-wave) or abnormal (absent or negative a-wave).

The umbilical vein was sampled at the level of its intraabdominal course, and its flow was classified as normal (no pulsations) or abnormal (venous pulsations).

In TTTS fetuses, fetal survival was assessed by ultrasound 48 hours after the intervention.

Biochemical markers of fetal cardiac function

For determination of biochemical markers, we used 2 mL of amniotic fluid that was visually free of bloody contamination. In all TTTS cases, a maternal plasma sample was obtained less than 1 hour before the procedure to exclude maternal biomarker changes as the source of amniotic fluid findings. Samples were centrifuged at 1200 × g , aliquoted and stored at –80°C until further processing.

For each outcome measure, all samples were analyzed in duplicate. When high variation in duplicate results was observed for a sample, it was reassayed in triplicate. Plasma and amniotic fluid cTnT levels were determined using an automated analyzer running a commercially available third-generation electrochemiluminescence assay (Modular E170; Roche Diagnostics, Vilvoorde, Belgium). Inter- and intraassay variation of this test is less than 10%. Its lowest detection level is 0.01 μg/L, and samples falling below the detection range were quantified as 0.005 μg/L (halfway between 0 and the detection limit) to allow continuous analysis.

Amniotic fluid ANP and BNP were determined using enzyme-linked immunosorbent assays (ELISA) (Phoenix Pharmaceuticals, Burlingame, CA, and Peninsula Laboratories, San Carlos, CA, respectively) following the manufacturer’s instructions. Synthetic ANP and BNP were used as positive controls. Inter- and intraassay variation of the ANP ELISA is less than 10% and less than 15%, respectively. The lowest detection level of this test is 0.13 ng/mL. Inter- and intraassay variation of the BNP ELISA is less than 5% and less than 5%, respectively. The lowest detection level of this test is 0.04 ng/mL.

Amniotic fluid total protein concentration was measured with a colorimetric bicinchoninic acid method (Pierce Biotechnology, Rockford, IL) following the manufacturer’s instructions after dilution of the samples to fall in the reference range of the test. The standard curve for this test was constructed using bovine serum albumin. The working range of this test is 20-20,000 μg/mL.

Amniotic fluid ANP, BNP, and cTnT levels were expressed as a ratio of the total protein concentration to correct for the dilution caused by the recipient’s polyuria in TTTS cases.

Data analysis

Statistical analysis was done using JMP version 7.0 (SAS Institute, Cary, NC) and Prism for Windows version 5.0 (GraphPad Software, San Diego, CA). Normality of the data was assessed using the D’Agostino Omnibus test. None of the variables was normally distributed. Therefore, data are reported as medians and interquartile range (IQR), and nonparametric tests were used to compare groups. Continuous data of 2 groups were compared using the Mann-Whitney test, whereas more than 2 groups were compared with the Kruksal-Wallis test followed by Dunn’s posttest. For nominal data, multiple groups were compared using Pearsons χ ² test. Correlations between 2 nominal parameters were assessed with Fisher exact test; for continuous data we used Spearmans correlation test. Two-sided P values less than .05 were considered statistically significant.