Understanding decidual vasculopathy and the link to preeclampsia: A review.

Preeclampsia is the archetype of a spectrum of clinical disorders related to abnormal placental development or function, characterized by placental histological lesions. Among those lesions, decidual vasculopathy is a term used to describe lesions of maternal spiral arteries, which are encountered on placental examination in about half of the women with preeclampsia. The morphological features of the lesions include perivascular lymphocytic infiltration, fibrinoid necrosis and foam cell incorporation within the vessel wall. Due to the resemblance of the latter characteristic to atherosclerosis, they are alternatively termed acute atherosis. Decidual vasculopathy correlates with worse maternal and neonatal outcomes, as well as placental pathology. In this article, we review the available literature on decidual vasculopathy and address the pitfalls in histological analysis of the lesions, including the varying definitions of the lesions and sample collection methods. We also discuss the current evidence on the etiology of the lesions and propose a novel hypothesis linking the three etiological pathways to the formation of decidual vasculopathy and, ultimately, the emergence of the heterogeneous group of placental dysfunction disorders, known as the great obstetric syndromes.

Clinical utility of non-invasive prenatal testing in pregnancies with ultrasound anomalies.

OBJECTIVE: To evaluate the application of non-invasive prenatal testing (NIPT) as an alternative to invasive diagnostic prenatal testing in pregnancies with abnormal ultrasound findings. METHODS: This was a retrospective analysis of 251 singleton and multiple pregnancies at high risk for fetal chromosomal abnormality based on findings at sonographic examination, in which NIPT was performed as a first-tier genetic test. NIPT was performed by massively parallel sequencing of cell-free DNA in maternal plasma, allowing genome-wide detection of whole-chromosome, as well as partial, autosomal aneuploidy. Sex chromosomes were not analyzed, according to the current protocol in Dutch laboratories. RESULTS: NIPT was performed at a median gestational age of 20 weeks, indicated by the presence of multiple congenital anomalies (n = 13), isolated structural anomalies (n = 57), increased nuchal translucency ≥ 3.5 mm (n = 58), soft markers (n = 73), growth restriction (n = 40) and other anomalies (n = 10). NIPT results were normal in 224 (89.2%) pregnancies, inconclusive in one (0.4%) and abnormal in 26 (10.4%). Most genetic aberrations detected by NIPT were common whole-chromosome aneuploidies: trisomy 21 (n = 13), trisomy 18 (n = 6) and trisomy 13 (n = 3). Four further NIPT results were abnormal; one was suspected of being confined placental mosaicism and one was of maternal origin. In those with normal NIPT results, sonographic follow-up or examination of the newborn indicated the need for diagnostic genetic testing in 33/224 (14.7%) pregnancies. Clinically relevant genetic aberrations were revealed in 7/224 (3.1%) cases, two of which were whole-chromosome aneuploidies: trisomy 13 and monosomy X. As sex chromosomal aberrations are not included in NIPT analysis, the latter cannot be considered a false-negative result. Other discordant findings were subchromosomal aberrations (< 20 megabases, n = 2) and monogenic aberrations (n = 3). CONCLUSIONS: NIPT should not be recommended for genetic evaluation of the etiology of ultrasound anomalies, as both resolution and sensitivity, or negative predictive value, are inferior to those of conventional karyotyping and microarray analysis. Nonetheless, some pregnant women consider NIPT to be an acceptable alternative to invasive diagnostic testing. © 2016 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of the International Society of Ultrasound in Obstetrics and Gynecology.

Three- and four-dimensional ultrasound in fetal echocardiography: an up-to-date overview.

Congenital heart diseases (CHD) are the most commonly overlooked lesions in prenatal screening programs. Real-time two-dimensional ultrasound (2DUS) is the conventionally used tool for fetal echocardiography. Although continuous improvements in the hardware and post-processing software have resulted in a good image quality even in late first trimester, 2DUS still has its limitations. Four-dimensional ultrasound with spatiotemporal image correlation (STIC) is an automated volume acquisition, recording a single three-dimensional (3D) volume throughout a complete cardiac cycle, which results in a four-dimensional (4D) volume. STIC has the potential to increase the detection rate of CHD. The aim of this study is to provide a practical overview of the possibilities and (dis)advantages of STIC. A review of literature and evaluation of the current status and clinical value of 3D/4D ultrasound in prenatal screening and diagnosis of congenital heart disease are presented.

Outcome of Multifetal Pregnancy Reduction in Women with a Dichorionic Triamniotic Triplet Pregnancy to a Singleton Pregnancy: A Retrospective Nationwide Cohort Study.

OBJECTIVE: To study the pregnancy outcomes of women with a dichorionic triamniotic triplet pregnancy that was reduced to a singleton pregnancy and to review the literature. METHODS: We performed a nationwide retrospective cohort study. We compared time to delivery and perinatal mortality in dichorionic triplet pregnancies reduced to singletons with ongoing dichorionic triplet pregnancies and primary singleton pregnancies. Additionally, we reviewed the literature on the subject. RESULTS: We studied 46 women with a reduced dichorionic triplet pregnancy and 42 women with an ongoing dichorionic triplet pregnancy. Median gestational age at delivery was 38.7 vs. 32.8 weeks, respectively (p < 0.001). Delivery <24 weeks occurred in 9 (19.6%) women with a reduced triplet pregnancy and 4 (9.5%) with an ongoing triplet pregnancy (p = 0.19). Perinatal survival rates between the reduced group and the ongoing triplet group were not significantly different. CONCLUSION: Multifetal pregnancy reduction in women with a dichorionic triplet pregnancy to a singleton pregnancy prolongs median gestational age at birth. No statistically significant association was found with miscarriage and perinatal survival rates.

Prenatal examination of the area and morphology of the atrioventricular valves using four-dimensional ultrasound in normal and abnormal hearts.

OBJECTIVE: Our aim is to evaluate the feasibility to examine the morphology and area of the atrioventricular (AV) valves in normal fetuses and fetuses with cardiac defects using spatiotemporal image correlation (STIC). METHODS: Atrioventricular valves were analyzed longitudinally in STIC volumes of 74 normal fetuses between the 15th and 36th week of pregnancy. The valve area was measured in a rendered view in diastole, the number of valve leaflets in systole. Longitudinal data analysis was performed using linear mixed models. Fifty fetuses with cardiac defects were examined. RESULTS: Examination of 355 STIC volumes of normal fetuses showed in 82.5% sufficient quality. The tricuspid valve leaflets were seen in 200 (68.3%) volumes and the mitral valve leaflets in 219 (74.7%) volumes. The tricuspid valve showed in 61.1% a round, 29.0% rectangle, and 8.9% elliptical shape and the mitral valve in 60.1% round, 28.0% rectangle, and 10.9% elliptical. Regression analysis revealed a positive relationship of the valve area with gestational age (p < 0.0001). Most heart defects with stenosis showed an area below the 5th percentile. CONCLUSION: Prenatal examination of the morphology and area of the AV valves using four-dimensional ultrasound is feasible. A rectangular valve opening is normal, which was visualized in about one third of the normal fetuses.

Pregnancy outcome after fetal reduction in women with a dichorionic twin pregnancy.

STUDY QUESTION: What are the pregnancy outcomes for women with a twin pregnancy that is reduced to a singleton pregnancy? SUMMARY ANSWER: Fetal reduction of a twin pregnancy significantly improves gestational age at birth and neonatal birthweight, however at an increased risk of pregnancy loss and preterm delivery. WHAT IS KNOWN ALREADY: Women with a multiple pregnancy are at increased risk for preterm delivery. Fetal reduction can be considered in these women. STUDY DESIGN, SIZE, AND DURATION: Retrospective cohort study of 118 women with a twin pregnancy reduced to a singleton pregnancy between 2000 and 2010. PARTICIPANTS/MATERIALS, SETTING, AND METHODS: We compared the outcome of pregnancy in consecutive women with a dichorionic twin pregnancy that was reduced to a singleton pregnancy to that of women with a dichorionic twin pregnancy that was managed expectantly and women with a primary singleton pregnancy. Reductions were performed between 10-23(6/7) weeks' gestation by intracardiac or intrathoracic injection of potassium chloride, mostly for congenital anomalies. We compared median gestational age, pregnancy loss <24 weeks, preterm delivery <32 weeks, neonatal birthweight and perinatal deaths. MAIN RESULTS AND THE ROLE OF CHANCE: We studied 118 women with a twin pregnancy that was reduced to a singleton, 818 women with an ongoing dichorionic twin pregnancy and 611 women with a primary singleton pregnancy. Loss of the entire pregnancy <24 weeks and preterm delivery occurred significantly more in the reduction group compared with the ongoing twin group (11.9 versus 3.1% <24 weeks, P< 0.001 and 18.6 versus 11.5% <32 weeks, respectively, P < 0.001). In the reduction group, the percentage of women without any surviving child was significantly higher compared with the ongoing twin and primary singleton group (14.4, 3.4 and 0.7%, respectively, P < 0.001). Median gestational age was 38.9 weeks (interquartile range (IQR) 34.7-40.3) for reduced pregnancies, 37.1 weeks (IQR 35.3-38.1) for ongoing twin pregnancies and 40.1 (IQR 39.1-40.9) for primary singletons (P < 0.001 for all comparisons). LIMITATIONS, REASONS FOR CAUTION: The main limitations of the study were its retrospective character, and the fact that indications for reduction were heterogeneous. WIDER IMPLICATIONS OF THE FINDINGS: In women with a dichorionic twin pregnancy fetal reduction increases median gestational age only at considerable risk of complete early pregnancy loss. STUDY FUNDING/COMPETING INTERESTS: The study was not funded. None of the authors has conflicts of interest.

Prevalence of hypertensive disorders in women after preeclamptic pregnancy associated with decidual vasculopathy.

OBJECTIVE: A subgroup of preeclamptic women has spiral artery lesions termed decidual vasculopathy (DV) which relate to worse clinical outcome. We aimed to determine whether a history of preeclampsia (PE) with DV is associated with adverse overall and future pregnancy outcome, including increased recurrence risk of hypertensive diseases of pregnancy. METHODS: Via posted survey women with PE and DV (DV positive) in the index pregnancy were compared to those without the lesions (DV negative) on overall and future pregnancy outcome. RESULTS: DV positive cases showed a higher incidence of chronic hypertension both preconceptionally and at time of survey, adjusted odds ratio 4.8 (2.0-11.9). The DV positive group had a higher overall incidence of pregnancies with gestational hypertension (22% vs 13%, p = 0.04), preterm birth (59% vs 45%, p = 0.02) and a lower birth weight centile (30 vs 39, p = 0.02). There was no difference in outcome of future pregnancies, irrespective of the use of prophylactic aspirin. CONCLUSION: Women with DV-associated PE have a higher overall incidence of adverse obstetric outcome and of chronic hypertension, indicating an underlying vascular pathology, putting them at risk for pregnancy and cardiovascular complications. These women constitute a target group for counseling, monitoring and possibly lifestyle or pharmacological interventions.

Differential insertion of the atrioventricular valves: measurement protocol to detect various congenital heart defects.

OBJECTIVES: The objectives of this study were to develop reference values for the distance between the atrioventricular valves, called differential insertion of the atrioventricular valves (DIAVV), in normal fetuses using four-dimensional ultrasound with spatio-temporal image correlation and to explore if DIAVV measurement can differentiate between normal hearts and hearts with cardiac defects. METHODS: The DIAVV was analysed longitudinally following a measurement protocol in 74 fetuses between 15 and 36 weeks gestational age. The DIAVV was measured in an apical four-chamber view of the heart in end-diastole. Furthermore, the DIAVV was measured in 70 fetuses with cardiac defects. RESULTS: In total, 337 normal and 70 abnormal spatio-temporal image correlation volumes were examined. Longitudinal regression analysis revealed a positive relationship of the DIAVV with gestational age and fetal biometry (p < 0.0001). The DIAVV of fetuses with double outlet right ventricle, truncus arteriosus, atrioventricular septal defects, Ebstein and tetralogy of Fallot all differed from normal fetuses (p < 0.05). CONCLUSION: Measurement of the DIAVV is a promising tool; however, a well-defined measurement protocol should be followed to accomplish the correct plane and exact moment in the cardiac cycle. This study presents new nomograms following this measurement protocol and reports an abnormal DIAVV in a wide spectrum of congenital heart disease.

Differential and linear insertion of atrioventricular valves: a useful tool?

OBJECTIVE: The differential insertion of the atrioventricular valves is the ultrasonographic representation of the more apical attachment of the tricuspid valve to the septum with respect to the mitral valve. A linear insertion is present when both valves form a linear continuum and has been suggested as a marker for atrioventricular septal defects (AVSDs). The objective of this study was to evaluate the anatomical substratum of differential and linear insertions of the atrioventricular valves in normal fetal hearts and fetal hearts with an AVSD. METHODS: The extent and position of the fibrous skeleton and attachment of the atrioventricular valves to the septum were studied in histological sections of 17 normal hearts and four hearts with an AVSD from 10 + 0 weeks' gestation to 3 days postpartum with various immunohistochemical tissue markers. In addition, spatiotemporal image correlation (STIC) volumes of 10 normal hearts and STIC volumes of eight hearts with an AVSD at 13 + 6 to 35 + 5 weeks' gestation were examined. RESULTS: The differential insertion of the atrioventricular valves was visible in normal hearts in the four-chamber plane immediately beneath the aorta, but nearer the diaphragm a linear insertion was found. In hearts with an AVSD, a linear appearance was observed in the four-chamber plane immediately beneath the aorta. Towards the diaphragm, however, first a differential insertion and, more caudally, a linear insertion was found. CONCLUSIONS: Both differential and linear insertions can be found in normal fetal hearts and fetal hearts with AVSD, depending on the plane in which the four-chamber view is visualized. Therefore, measurement of the differential insertion is likely to be useful only in experienced hands.

Alpha-fetoprotein, a fascinating protein and biomarker in neurology.

Alpha-fetoprotein (AFP) is present in fetal serum in concentrations up to 5,000,000 µg/l. After birth, AFP gene expression is turned down with a subsequent fall of the serum concentrations of this albumin-like protein to 'adult values' of circa 0.5-15 µg/l from the age of 2 years onwards. Irrespective of its assumed important functions, individuals with AFP deficiency appear fully healthy. The other way around, the presence of AFP in the circulation after the first years of life doesn't seem to harm, since individuals with 'hereditary persistence of AFP' are also without clinical abnormalities. During pregnancy, AFP (in maternal serum) has long been recognized as a marker for congenital anomalies of the fetus. Equally well known is AFP as biomarker for hepatocellular carcinoma and some other malignancies. There are at least four neurodegenerative disorders, all inherited as autosomal recessive traits and characterized by the presence of cerebellar ataxia, abnormal ocular movements, and neuropathy, for which an elevated concentration of serum AFP is an important diagnostic biomarker. The availability of a reliable biomarker is not only important during screening or diagnostic processes, but is also relevant for objective follow-up during (future) therapeutic interventions.

Age independent first trimester screening for Down syndrome: improvement in test performance.

OBJECTIVE: The aim of this study was to compare screening performance for Down syndrome of the absolute risk (AR) method to the first trimester combined test (FCT) at different maternal ages. METHODS: There was a retrospective analysis of 32,448 FCT. AR was defined as final risk divided by maternal age risk. RESULTS: The likelihood of receiving a true prediction was comparable between both methods in all age groups. With the AR method, two extra Down syndrome cases were detected in women <30 years, three cases were missed in women ≥36 years, and the likelihood of receiving a false prediction decreased overall (OR 0.82, CI 0.77-0.87; P < 0.0001), in women aged 36-40 years (0.45, CI 0.41-0.51; P < 0.0001), in women aged 41-45 years (0.18, CI 0.13-0.26; P < 0.0001) and increased in women aged ≤25 years (2.12, CI 1.52-2.96; P < 0.004). CONCLUSIONS: The AR method results in a significant decreased likelihood of receiving a false prediction with a comparable likelihood of receiving a true prediction. Thus, fewer invasive diagnostic tests will be performed. It will take away the misunderstanding about differences in screening performance for women of different ages. This might lead to a higher uptake of first trimester screening resulting in a more efficient screening policy.

Isolated abdominal wall defect with complete liver herniation without a covering or remnant membrane: an ominous sign: case report and review of literature.

Complete liver herniation in abdominal wall defects without a membrane is rare and its prognosis is not well documented. We present a case diagnosed at 12 weeks of gestation. At 27 weeks, a C-section was performed for fetal distress. The infant proved impossible to ventilate and died. In literature, 16 similar cases are described of whom 14 died in the neonatal period and two in infancy. This suggests that herniation of the complete liver in isolated abdominal wall defects without a remnant membrane is lethal and counselling should be provided accordingly.

Interobserver agreement in detailed prenatal diagnosis of congenital heart disease by telemedicine using four-dimensional ultrasound with spatiotemporal image correlation.

OBJECTIVE: To evaluate the clinical accuracy of four-dimensional (4D) echocardiography in the detailed prenatal diagnosis of congenital heart disease (CHD) in a telemedicine setting. METHODS: Ten second-trimester spatiotemporal image correlation (STIC) volumes were sent to three observers in different tertiary care centers with expertise in 4D echocardiography. The 10 volumes were selected based on the type of diagnosis to cover a wide spectrum of CHD anomalies, and also included one normal fetal heart. Observers were asked to provide the diagnosis, the postprocessing modalities used and the time spent on examination, and to give a rating of the confidence for the diagnosis on a 5-point Likert scale. They were free to consult other colleagues, including pediatric cardiologists, but were blinded to the prenatal diagnosis and the neonatal outcome. A diagnostic scoring system was used to evaluate different aspects of the heart defects. The results were compared with neonatal echocardiography or postmortem findings ('gold standard'). RESULTS: In two cases all observers correctly diagnosed all details of the volume datasets. The observer with the best performance reached perfect agreement in six cases and nearly perfect agreement in three. The volumes were most frequently studied by sectional planes and were analyzed in a median time of 11.0 (range, 2.5-30.0) min. The median confidence score was 4.0 (range, 1.0-5.0). CONCLUSIONS: In a telemedicine setting using STIC volumes, fetal cardiac anomalies can be diagnosed correctly by an expert. However, details required for adequate counseling and planning of postnatal care may be missed. STIC by telemedicine is a promising modality, although not accurate enough for exclusive use in clinical decision making regarding treatment, prognosis or termination of pregnancy.

First trimester screening for intra-uterine growth restriction and early-onset pre-eclampsia.

OBJECTIVE: To assess first trimester placental growth factor (PlGF) and pregnancy-associated plasma protein-A (PAPP-A) as screening markers for early-onset pre-eclampsia (PE) and intra-uterine growth restriction (IUGR). METHODS: PlGF concentration was retrospectively measured in first trimester serum specimens of 23 cases of early-onset PE (<34 weeks), 26 cases of IUGR (birth weight < 5th centile) and 5 controls per case. Levels were adjusted for gestational age (GA), ethnicity and smoking to obtain multiples of the expected median (MoM). Logistic regression was used to assess PlGF, PAPP-A and maternal characteristics as potential predictors of early-onset PE and IUGR. RESULTS: PlGF MoM levels were significantly lower in the early-onset PE group (P < 0.0001) compared with controls, but not in the IUGR group. PAPP-A MoM levels were significantly lower in the IUGR group (P < 0.01) compared with controls but not in the early-onset PE group. PlGF significantly improved the ability of systolic blood pressure at the first prenatal visit to predict early-onset PE [achieving a receiver-operating characteristics curve with area under the curve (AUC) of 0.8]. Combining systolic blood pressure at the first prenatal visit and PlGF did not significantly improve the predictive ability compared with PlGF alone (AUC = 0.83). CONCLUSION: Serum PlGF is an acceptable marker in first trimester screening for early-onset PE, but a poor marker in screening for IUGR. Screening performance of serum PAPP-A is poor for both early-onset PE and IUGR.

First-trimester serum marker distribution in singleton pregnancies conceived with assisted reproduction.

OBJECTIVE: To evaluate marker distribution of free beta-human chorionic gonadotrophin (fbeta-hCG) and pregnancy-associated plasma protein-A (PAPP-A) in singleton pregnancies conceived by assisted reproduction techniques (ART). METHODS: In vitro fertilization (IVF) (n = 203) and intracytoplasmic sperm injection (ICSI) (n = 192) cases from a database of 14 645 first-trimester combined tests (overall study group) were selected and matched to 1164 controls for gestational age at sample date and maternal age. RESULTS: In the IVF group and ICSI group, lnPAPP-A was lower (IVF 6.74 vs 7.08; P = 0.0001; ICSI 6.59 vs 7.07; P = 0.0001) compared with the matched controls. Lnfbeta-hCG was lower in the IVF group (3.75 vs 3.90; P = 0.005) but not significantly different in the ICSI group (3.87 vs 3.93; P = 0.27). The computed correction factors for PAPP-A and fbeta-hCG were 1.42 and 1.17 for the IVF group and 1.56 and 1.05 for the ICSI group.The false-positive rate (FPR) in the IVF and ICSI group compared with the matched controls was higher (IVF 10.3% vs 8.6% and ICSI 10.9% vs 7.5%). In the overall age-biased [maternal age significantly lower compared with all ART and control groups] study group the FPR was 6.8%. CONCLUSION: The increase in FPR in the ART groups can be explained by decreased PAPP-A values. Therefore, an adjustment in risk analysis for Down syndrome is suggested.

Reliability of fetal cardiac volumetry using spatiotemporal image correlation: assessment of in-vivo and in-vitro measurements.

OBJECTIVE: To assess the reliability of measurement of fetal cardiac ventricular volume, stroke volume, and ejection fraction with four-dimensional ultrasound using spatiotemporal image correlation (STIC). METHODS: Volume datasets were collected from two sources: 24 from fetuses over a range of gestational ages and 12 from a miniature balloon model. Datasets were analyzed by three observers, repeatedly in 12 fetal datasets and all balloon datasets. Volume calculations were obtained by manually tracing multiple parallel slices (three-dimensional (3D) slice method). Measurement error was assessed by calculating standard errors of measurement (SEM) and coefficients of variation (CV). Reliability was assessed by calculating interobserver and intraobserver intraclass correlation coefficients (ICC). RESULTS: Measurement errors of balloon volumes were small and reliability was good (SEM

First trimester maternal serum ADAM12s levels in twin pregnancies.

BACKGROUND: A disintegrin and metalloprotease 12s (ADAM12s) is a potential first trimester serum marker for fetal trisomy and adverse pregnancy outcome in singletons. In this study, ADAM12s levels in first trimester serum of uncomplicated and complicated twins were evaluated. METHODS: ADAM12s was studied in maternal serum of 215 twin pregnancies, collected between 2004 and 2008. ADAM12s was measured 'blind to outcome' using AutoDelfia (PerkinElmer, Turku, Finland). As a reference, data from 2423 singletons were used. RESULTS: The median ADAM12s level was increased in euploid twins [1.61 multiples of the median (MoM); n = 209] compared with singletons. The median ADAM12s MoM was significantly lower in monochorionic (1.36 MoM; n = 41) compared with dichorionic twins (1.67 MoM; n = 168) (Mann-Whitney U test, p = 0.005). Trisomy 21 was identified in two pregnancies. Median ADAM12s MoM in twins complicated by hypertensive disorders (1.77 MoM, n = 35) or small for gestational age fetus (1.54 MoM; n = 24) was not significantly different from uncomplicated twins (1.64 MoM; n = 134). CONCLUSION: Median ADAM12s MoM in euploid twins was increased compared with singletons. Monochorionic had significantly lower median ADAM12s MoM than dichorionic twins. Median ADAM12s MoMs were not significantly different in twins complicated by hypertensive disorders or small for gestational age fetus compared with uncomplicated twins.

Validation of volume measurements for fetal echocardiography using four-dimensional ultrasound imaging and spatiotemporal image correlation.

OBJECTIVES: To assess the accuracy and reliability of four-dimensional (4D) ultrasound imaging using spatiotemporal image correlation (STIC) employing three different techniques to measure volumes in vitro. METHODS: Customized miniature balloons attached to a pump system were used to mimic fetal cardiac chambers. After the balloon model had been immersed in a bath filled with viscous gel, 4D datasets were acquired and three methods were used for volume analysis: three dimensional (3D) slice method, Virtual Organ Computer-aided AnaLysis (VOCAL) and VOCAL combined with inversion mode. Accuracy and measurement error were measured as the difference between the volume measurements and the actual volumes. Intraobserver reliability was assessed by computing coefficients of variation (CV) and intraclass correlation (ICC). RESULTS: Measurement of 76 different volumes, ranging from 0.30 to 4.95 mL, resulted in a total of 912 measurements. The 3D slice method had a mean error of -3.3%, the inversion method underestimated the volumes with a mean error of -6.1%, and VOCAL had a mean error of -2.9%. The 3D slice method had the best agreement (95% limits of agreement (LOA), -11.2 to 4.7%), followed by VOCAL (95% LOA, -14.1 to 8.3%); the inversion mode demonstrated the worst agreement (95% LOA, -21.4 to 9.2%). All three methods were reliable with CV < 10% and ICC > 0.95. CONCLUSIONS: 4D ultrasonography with STIC is a feasible and accurate method for calculating volumes of 0.30 mL upwards. In an in-vitro model the 3D slice method proved accurate, was the least time consuming, had the best reliability and had the smallest LOA. This method may prove useful when applied to in-vivo investigations.