Fetal Lambs Research Articles

Multiomics analysis reveals the presence of a microbiome in the gut of fetal lambs

ObjectiveMicrobial exposure is critical to neonatal and infant development, growth and immunity. However, whether a microbiome is present in the fetal gut prior to birth remains debated. In this study,...

Conception during the lactation and rearing period affects the ewe-lamb bond at birth

Conceiving during the early postpartum period, when ewes are investing energy resources nursing an offspring is energetically demanding. Moreover, it requires artificial weaning during ewe gestation, which induces a strong stress response. All this together can impact fetal growth and lambs’ mortality rate. The aim of the study was to determine if conceiving during the lactation and rearing period affects lambs’ weight and body temperature, and ewe and lamb behaviors after birth. Ewes were induced to ovulate during October (spring) and lambed in autumn. When they conceived, they either remained lactating while nursing their current offspring (LAC, n = 10) or had their last parturition the previous year (CON, n = 49). Lambs born from LAC ewes were lighter than those born from CON ewes (4.13 ± 0.35 kg vs 4.77 ± 0.26; P = 0.02, respectively). More LAC ewes lambed lying down than CON ewes (100 % vs 42.9 %; P = 0.03). At birth, lambs born from LAC ewes made more attempts to stand up (11.4 ± 2.2 vs 6.1 ± 1.1, P = 0.043), and had a longer latency before first suckling (79.4 ± 11.2 s vs 45.6 ± 5.9 s; P = 0.012) than lambs born from CON ewes. At the end of a separation-reunion test performed 24–36 h after birth, LAC ewes were further away from their lambs than CON ewes (5.1 ± 1.0 m vs 2.95 ± 0.54 m; P = 0.045). Lambs born from LAC took longer than those born from CON ewes to meet their mother (24.9 s vs 8.5 ± 3.5 s ± 7.7; P = 0.059), and suckle (142.5 ± 22.8 s vs 86.3 ± 10.3 s; P = 0.029). Overall, conceiving during the lactation and rearing period affected the establishment of the ewe-lamb bond. More LAC ewes lambed lying down, their lambs were lighter and less vigorous after birth. LAC ewes were less interested in their lamb, both at parturition and during the separation-reunion test. LAC ewes were less disposed to invest in their lamb, both at parturition as well as during a separation-reunion test performed the day after birth. On the other hand, although lambs from LAC ewes were less vigorous, they tended to require more attention during the short separation test. These results highlight the need of research to develop strategies aiming to improve the welfare of ewes and lambs in accelerated lambing systems.

82 Neuroprotection is improved by watertightness of spina bifida repair in the fetal lamb

We investigated the neurofunctional consequences of the watertightness of prenatal spina bifida (SB)-repair in the fetal lamb model. The degree to which cerebrospinal fluid leakage (CSF) is prevented by the neurosurgical technique may translate to the way fetal SB-repair is done clinically. Neurological outcomes of watertight and non-watertight multilayer repairs were compared to those in unrepaired lambs. 75-day fetal lambs (term=145 days) underwent standardized induction of lumbar SB. At 100 days, they were assigned to one of the three groups. At 1-2 days after term delivery, we assessed hindbrain herniation (HH)-reversal on magnetic resonance imaging as primary outcome measure. Secondary proxies of neuroprotection were CSF-leakage at the repair site; hindlimb function based on joint movement score, locomotor grade, Motor and Somatosensory Evoked Potentials; neuroprotection (i.e. score of 4/4 encompassing live birth, HH-reversal, absent CSF-leakage, joint score ≥9/15), and brain and spinal cord histology. Compared to unrepaired lambs, watertight repair was neuroprotective in 5/7 lambs, as evidenced by a 100% HH-reversal, lower CSF-leakage rate (14%), better hindlimb motor function, higher neuronal density in the hippocampus and corpus callosum, and larger area of astroglial cells at the lesion epicenter (Table 1). Conversely, non-watertight SB-repair did not achieve the same level of neuroprotection (1/7), i.e. 86% HH-reversal rate, no motor function improvement, high CSF-leakage rate (43%), low brain neuron count and astroglial cell area. These findings demonstrate that a watertight fetal SB-repair improves brain and spinal cord structure and function more. This has important clinical implication and neurosurgical technique that achieves watertightness should be adopted.

Placental gas exchange during amniotic carbon dioxide insufflation in sheep.

Insufflation of the amniotic cavity with carbon dioxide (CO2 ) is used clinically to improve visibility during complex fetoscopic surgery. Insufflation with heated, humidified CO2 has recently been shown to reduce fetal hypercapnia and acidosis in sheep, compared with use of cold and dry CO2 , but the underlying mechanisms are unclear. The aim of this study was to investigate whether differences in placental CO2 and oxygen (O2 ) exchange during insufflation with heated and humidified vs cold and dry CO2 could explain these findings. Thirteen fetal lambs at 105 days of gestation (term, 146 days) were exteriorized partially, via a midline laparotomy and hysterotomy, and instrumented with an umbilical artery catheter, an umbilical vein catheter and a common umbilical vein flow probe. Arterial and venous catheters and flow probes were also inserted into the maternal uterine circulation. Six ewes were insufflated with cold, dry CO2 (22°C; 0-5% humidity) and seven with heated, humidified CO2 (40°C; 95-100% humidity) at 15 mmHg for 180 min. Blood-flow recordings and paired arterial and venous blood gases were sampled from uterine and umbilical vessels. Rates of placental CO2 and O2 exchange were calculated. After 180 min of insufflation, fetal survival was 33% (2/6) using cold, dry CO2 and 71% (5/7) using heated, humidified CO2 . By 120 min, fetuses insufflated with heated, humidified CO2 had lower arterial CO2 levels and higher arterial pH compared to those insufflated with cold, dry gas. Insufflation decreased significantly placental gas exchange in both groups, as measured by rates of both (i) fetal CO2 clearance and O2 uptake and (ii) maternal O2 delivery and CO2 uptake from the fetal compartment. Lower arterial CO2 and higher pH levels in fetuses insufflated with heated and humidified, compared to cold and dry, CO2 could not be explained by differences in placental gas exchange. Instead, heated and humidified insufflation appeared to reduce fetal CO2 absorption from the uterus, supporting its use in preference to cold, dry CO2 . © 2019 International Society of Ultrasound in Obstetrics and Gynecology.

Contrast-Enhanced Brain Ultrasound Perfusion Metrics in the EXTra-Uterine Environment for Neonatal Development (EXTEND): Correlation With Hemodynamic Parameters.

Contrast-enhanced ultrasound (CEUS) can provide quantitative perfusion metrics and may be useful to detect cerebral pathology in neonates and premature infants, particularly in extrauterine environments. The effect of hemodynamics on cerebral perfusion metrics is unknown, which limits the clinical application of this technology. We aimed to determine associations between systemic hemodynamics and concurrently measured brain perfusion parameters in an animal model of extrauterine support. Nine fetal lambs were transferred to an extrauterine support device. Lumason® ultrasound contrast (0.1-0.3 ml) was administered via the umbilical vein and 90-second cine clips were obtained. Time-intensity-curves (TICs) were generated and time-dependent and area-under-curve (AUC) parameters were derived. Associations between brain perfusion metrics and hemodynamics including heart rate (HR) and mean arterial pressure (MAP) were evaluated by multilevel linear mixed-effects models. Eighty-six ultrasound examinations were performed and 72 examinations were quantifiable. Time-dependent measurements were independent of all hemodynamic parameters (all p ≥.05). Oxygen delivery and mean blood flow were correlated with AUC measurements (all p ≤.01). Physiologic HR and MAP were not correlated with any measurements (all p ≥.05). Detected aberrations in time-dependent CEUS measurements are not correlated with hemodynamic parameters and are thought to reflect the changes in cerebral blood flow, thus providing a promising tool for evaluation of brain perfusion. CEUS brain perfusion parameters are not correlated with physiologic HR and MAP, but AUC-dependent measurements are correlated with oxygen delivery and blood flow, suggesting that CEUS offers additional value over standard monitoring. Overall, these findings enhance the applicability of this technology.

Reducing lung liquid volume in fetal lambs decreases ventricular constraint.

This study evaluated whether an increased left ventricular (LV) pump function accompanying reduction of lung liquid volume in fetal lambs was related to increased LV preload, augmented LV contractility, or both. Eleven anesthetized preterm fetal lambs (gestation 128 ± 2 days) were instrumented with (1) an LV micromanometer-conductance catheter to obtain LV end-diastolic volume (EDV) and end-diastolic pressure (EDP), the maximal rate of rise of LV pressure (dP/dtmax), LV output, LV stroke work, and LV end-systolic elastance (Ees), a relatively load-independent measure of contractility; (2) an endotracheal tube to measure mean tracheal pressure and to reduce lung liquid volume. LV transmural pressure was calculated as LV EDP minus tracheal pressure. Reducing lung liquid volume by 16 ± 4 ml kg-1 (1) augmented LV output (by 16%, P = 0.001) and stroke work (29%, P < 0.001), (2) increased LV EDV (12%, P < 0.001), (3) increased LV transmural pressure (2.2 mmHg, P < 0.001), (4) did not change LV dP/dtmax normalized for EDV (P > 0.7), and (5) decreased LV Ees (20%, P < 0.01). These findings suggest a rise in LV pump function evident after reduction of lung liquid volume in fetal lambs was related to increased LV preload secondary to lessening of external LV constraint, without any associated rise in LV contractility. This study has shown that reducing the volume of liquid filling the fetal lungs lessens the degree of external constraint on the heart. This lesser constraint permits a rise in left ventricular dimensions and thus greater cardiac filling that leads to increased left ventricular pumping performance. This study has defined a mechanism whereby a reduction in lung liquid volume results in enhanced pumping performance of the fetal heart. These findings suggest that a reduction in lung liquid volume which occurs during the birth transition contributes to increases in left ventricular dimensions and pumping performance known to occur with birth.

Continuous chest compressions with asynchronous ventilations increase carotid blood flow in the perinatal asphyxiated lamb model.

Background:The neonatal resuscitation program (NRP) recommends interrupted chest compressions (CC) with ventilation in the severely bradycardic neonate. The conventional 3:1 compression-to-ventilation (C:V) resuscitation provides 90 CC/min, significantly lower than the intrinsic newborn heart rate (120–160 beats/min). Continuous CC with asynchronous ventilation (CCCaV) may improve success of return of spontaneous circulation (ROSC).Methods:Twenty-two near-term fetal lambs were randomized to interrupted 3:1 C:V (90 CC + 30 breaths/min) or CCCaV (120 CC + 30 breaths/min). Asphyxiation was induced by cord occlusion. After five min of asystole, resuscitation began following NRP guidelines. The first dose of epinephrine was given at 6 minutes. Invasive arterial blood pressure and left carotid blood flow were continuously measured. Serial arterial blood gases were collected.Results:Baseline characteristics between groups were similar. Rate of and time to ROSC was similar between groups. CCCaV was associated with a higher PaO2 (22±5.3 vs. 15±3.5 mmHg, p<0.01), greater left carotid blood flow (7.5±3.1 vs. 4.3±2.6 ml/kg/min, p<0.01) and oxygen delivery (0.40±0.15 vs. 0.13±0.07 mL O2/kg/min, p <0.01) compared to 3:1 C:V.Conclusion:In a perinatal asphyxiated cardiac arrest lamb model, CCCaV showed greater carotid blood flow and cerebral oxygen delivery compared to 3:1 C:V resuscitation.

HIF-1\u03b1 promotes cellular growth in lymphatic endothelial cells exposed to chronically elevated pulmonary lymph flow

Normal growth and development of lymphatic structures depends on mechanical forces created by accumulating interstitial fluid. However, prolonged exposure to pathologic mechanical stimuli generated by chronically elevated lymph flow results in lymphatic dysfunction. The mechanisms that transduce these mechanical forces are not fully understood. Our objective was to investigate molecular mechanisms that alter the growth and metabolism of isolated lymphatic endothelial cells (LECs) exposed to prolonged pathologically elevated lymph flow in vivo within the anatomic and physiologic context of a large animal model of congenital heart disease with increased pulmonary blood flow using in vitro approaches. To this end, late gestation fetal lambs underwent in utero placement of an aortopulmonary graft (shunt). Four weeks after birth, LECs were isolated and cultured from control and shunt lambs. Redox status and proliferation were quantified, and transcriptional profiling and metabolomic analyses were performed. Shunt LECs exhibited hyperproliferative growth driven by increased levels of Hypoxia Inducible Factor 1α (HIF-1α), along with upregulated expression of known HIF-1α target genes in response to mechanical stimuli and shear stress. Compared to control LECs, shunt LECs exhibited abnormal metabolism including abnormalities of glycolysis, the TCA cycle and aerobic respiration. In conclusion, LECs from lambs exposed in vivo to chronically increased pulmonary lymph flow are hyperproliferative, have enhanced expression of HIF-1α and its target genes, and demonstrate altered central carbon metabolism in vitro. Importantly, these findings suggest provocative therapeutic targets for patients with lymphatic abnormalities.

Safety and efficacy of smart tracheal occlusion device in diaphragmatic hernia lamb model.

To investigate the efficacy and safety of the 'smart' tracheal occlusion (Smart-TO) device in fetal lambs with diaphragmatic hernia (DH). DH was created in fetal lambs on gestational day 70 (term, 145 days). Fetuses were allocated to either pregnancy continuation until term (DH group) or fetoscopic endoluminal tracheal occlusion (TO), performed using the Smart-TO balloon on gestational day 97 (DH + TO group). On gestational day 116, the presence of the balloon was confirmed on ultrasound, then the ewe was walked around a 3.0-Tesla magnetic resonance scanner for balloon deflation, which was confirmed by ultrasound immediately afterwards. At term, euthanasia was performed and the fetus retrieved. Efficacy of occlusion was assessed by the lung-to-body-weight ratio (LBWR) and lung morphometry. Safety parameters included tracheal side effects assessed by morphometry and balloon location after deflation. The unoccluded DH lambs served as a comparator. Six fetuses were included in the DH group and seven in the DH + TO group. All balloons deflated successfully and were expelled spontaneously from the airways. In the DH + TO group, in comparison to controls, the LBWR at birth was significantly higher (1.90 (interquartile range (IQR), 1.43-2.55) vs 1.07 (IQR, 0.93-1.46); P = 0.005), while on lung morphometry, the alveolar size was significantly increased (mean linear intercept, 47.5 (IQR, 45.6-48.1) vs 41.9 (IQR, 38.8-46.1) μm; P = 0.03); whereas airway complexity was lower (mean terminal bronchiolar density, 1.56 (IQR, 1.0-1.81) vs 2.23 (IQR, 2.14-2.40) br/mm2 ; P = 0.005). Tracheal changes on histology were minimal in both groups, but more noticeable in fetal lambs that underwent TO than in unoccluded lambs (tracheal score, 2 (IQR, 1-3) vs 0 (0-1); P = 0.03). In fetal lambs with DH, TO using the Smart-TO balloon is effective and safe. Occlusion can be reversed non-invasively and the deflated intact balloon expelled spontaneously from the fetal upper airways. © 2020 International Society of Ultrasound in Obstetrics and Gynecology.

Immortalization of primary sheep embryo kidney cells.

Sheep primary epithelial cells are short-lived in cell culture systems. For long-term in vitro studies, primary cells need to be immortalized. This study aims to establish and characterize T immortalized sheep embryo kidney cells (TISEKC). In this study, we used fetal lamb kidneys to derive primary cultures of epithelial cells. We subsequently immortalized these cells using the large T SV40 antigen to generate crude TISEKC and isolate TISEKC clones. Among numerous clones of immortalized cells, the selected TISEKC-5 maintained active division and cell growth over 20 passages but lacked expression of the oncogenic large T SV40 antigen. Morphologically, TISEKC-5 maintained their epithelial aspect similar to the parental primary epithelial cells. However, their growth properties showed quite different patterns. Crude TISEKC, as well as the clones of TISEKC proliferated highly in culture compared to the parental primary cells. In the early passages, immortalized cells showed heterogeneous polyploidy but in the late passages the karyotype of immortalized cells became progressively stable, identical to that of the primary cells, because the TISEKC-5 cell line has lost the large SV40 T antigen expression, this cell line is a valuable tool for veterinary sciences and biotechnological productions.

Ovine abortion and stillbirth investigations in Australia.

Fetal loss and lamb mortality between mid-pregnancy and weaning are important economic and welfare issues for the Australian sheep industry. The aim of this study was to determine common causes of ovine abortion and stillbirths based on submissions to veterinary laboratories and identify factors that impact the determination of an aetiological diagnosis. Data for 529 investigations on abortion or stillbirth between 2000 and 2018 were retrieved from four state veterinary laboratories in Western Australia, South Australia, Victoria and Tasmania. An aetiological diagnosis was made for 57% of investigations. Investigations that included placental tissue samples were more than twice as likely to have an aetiological diagnosis compared to investigations without placenta (P = 0.017, 95% confidence interval 1.1, 4.5). Of the investigations where an aetiological diagnosis was made, 81% involved infectious abortion, with Campylobacter spp. (32%), Listeria spp. (25%) and Toxoplasma gondii (9%) being the three most common abortigenic pathogens implicated. The remaining 19% of investigations with an aetiological diagnosis included a wide range of infectious and non-infectious diseases. Diagnoses made varied year to year and between states. No evidence of exotic abortigenic pathogens were reported. Veterinary practitioners can improve the probability of an aetiological diagnosis by emphasising to farmers the importance of collecting any aborted material, especially placenta, and appropriate storage of the tissues until they can be submitted to the laboratory. Some diseases that cause abortion in Australian sheep have zoonotic potential, and veterinary practitioners play an important role in educating clients about appropriate hygiene when handling pregnant and lambing ewes or any aborted material.

Indirect ELISA (iELISA) standardization for the diagnosis of bovine enzootic leukosis

ABSTRACT: Enzootic bovine leukosis (EBL) is an infectious disease caused by bovine leukemia virus (BLV) that affects cattle worldwide. Agar gel immunodiffusion (AGID) was the reference test for EBL diagnosis for many years, but enzyme-linked immunosorbent assay (ELISA) showed higher sensitivity, was faster to perform, and resulted in an objective reading. However, the importation of ELISA kits is lengthy and expensive, and currently, no AGID kits are available in Brazil. The aim of this work was to standardize an indirect ELISA (iELISA) for EBL diagnosis using BLV antigens produced in Tadarida brasiliensis lung (Tb1Lu) cells, which are Bovine viral diarrhea virus (BVDV) free, unlike fetal lamb kidney (FLK) cells, currently used for this purpose. Following standardization, iELISA results were compared with those obtained by AGID and the commercial Chekit Leucose-Serum ELISA. Compared to AGID, iELISA had 94,44% sensitivity, 75.68% specificity, 79.10% positive predictive value (PPV) and 93.30% negative predictive value (NPV), with 84% concordance and a Kappa index of 0.699. Compared to the Chekit Leucose-Serum ELISA, iELISA showed 92.60% sensitivity, 87.09% specificity, 90.27% PPV and 90,00% NPV, with 90.27% concordance and a Kappa index of 0.801. Taking into account the high agreement with the traditional tests and the absence of non-specific reactions with BVDV, the developed assay could be used as diagnostic method to control EBL in Brazil.

Conventional revolution: the ethical implications of the natural progress of neonatal intensive care to artificial wombs

Research teams have used extra-uterine systems (Biobags) to support premature fetal lambs and to bring them to maturation in a way not previously possible. The researchers have called attention to...

Surviving Lambs with Myelomeningocele Repaired in utero with Placental Mesenchymal Stromal Cells for 6 Months: A Pilot Study

Background: Fetal repair of myelomeningocele (MMC) with placental mesenchymal stromal cells (PMSCs) rescues ambulation in the ovine model up to 48 h postnatally. Outcomes past 48 h are unknown as MMC lambs have not been survived past this timepoint. Objective: We aimed to survive lambs for 6 months following the fetal repair of MMC with PMSCs. Methods: Fetal MMC lambs were repaired with PMSCs. Lambs received either no additional treatment or postnatal bracing and physical therapy (B/PT). Motor function was assessed with the sheep locomotor rating (SLR). Lambs with an SLR of 15 at birth were survived for 6 months or until a decline in SLR less than 15, whichever came first. All lambs underwent a perimortem MRI. Results: The lambs with no postnatal treatment (n = 2) had SLR declines to 7 and 13 at 29 and 65 days, respectively, and were euthanized. These lambs had a spinal angulation of 57° and 47°, respectively. The B/PT lamb (n = 1) survived for 6 months with a sustained SLR of 15 and a lumbar angulation of 42°. Conclusion: Postnatal physical therapy and bracing counteracted the inherent morbidity of the absent paraspinal muscles in the ovine MMC model allowing for survival and maintenance of rescued motor function of the prenatally treated lamb up to 6 months.

Hypoxic modulation of fetal vascular MLCK abundance, localization, and function.

Changes in vascular contractility are among the most important physiological effects of acute and chronic fetal hypoxia. Given the essential role of myosin light-chain kinase (MLCK) in smooth muscle contractility and its heterogeneous distribution, this study explores the hypothesis that subcellular changes in MLCK distribution contribute to hypoxic modulation of fetal carotid artery contractility. Relative to common carotid arteries from normoxic term fetal lambs (FN), carotids from fetal lambs gestated at high altitude (3,802 m) (FH) exhibited depressed contractility without changes in MLCK mRNA or protein abundance. Patterns of confocal colocalization of MLCK with α-actin and 20-kDa regulatory myosin light chain (MLC20) enabled calculation of subcellular MLCK fractions: 1) colocalized with the contractile apparatus, 2) colocalized with α-actin distant from the contractile apparatus, and 3) not colocalized with α-actin. Chronic hypoxia did not affect MLCK abundance in the contractile fraction, despite a concurrent decrease in contractility. Organ culture for 72 h under 1% O2 decreased total MLCK abundance in FN and FH carotid arteries, but decreased the contractile MLCK abundance only in FH carotid arteries. Correspondingly, culture under 1% O2 depressed contractility more in FH than FN carotid arteries. In addition, hypoxia appeared to attenuate ubiquitin-independent proteasomal degradation of MLCK, as reported for other proteins. In aggregate, these results demonstrate that the combination of chronic hypoxia followed by hypoxic culture can induce MLCK translocation among at least three subcellular fractions with possible influences on contractility, indicating that changes in MLCK distribution are a significant component of fetal vascular responses to hypoxia.

Physiologic-Based Cord Clamping Maintains Core Temperature vs. Immediate Cord Clamping in Near-Term Lambs.

Background: Physiologic-based cord clamping (PBCC) involves deferring umbilical cord clamping until after lung aeration. It is unclear if infant is at risk of becoming hypothermic during PBCC.Objectives: To test if PBCC would maintain core temperature more effectively than immediate cord clamping (ICC).Design: At 0.93 gestation, fetal lambs were surgically exteriorized and instrumented from pregnant ewes under general anesthesia. Prior to the start of the experiment, lambs were thoroughly dried, placed on hot water bottles, and core temperature was continuously monitored using a rectal thermometer. PBCC lambs (n = 21), received intermittent positive pressure ventilation (iPPV) for ≥5 min prior to umbilical cord clamping. In ICC lambs (n = 23), iPPV commenced within 60 s after umbilical cord clamping. iPPV was provided with heated/humidified gas. Lambs were moved under a radiant warmer after umbilical cord clamping. Additional warmth was provided using a plastic overlay, hairdryer, and extra water bottles, as needed. Two-way mixed and repeated measures one-way ANOVAs were used to compare temperature changes between and within a single group, respectively, over time.Results: Basal fetal parameters including core temperature were similar between groups. ICC lambs had a significant reduction in temperature compared to PBCC lambs (p < 0.001), evident by 1 min (p = 0.002). ICC lambs decreased temperature by 0.51°C (± 0.42) and 0.79°C (± 0.55) at 5 and 10 min respectively (p < 0.001). In PBCC lambs, temperature did not significantly change before or after umbilical cord clamping (p = 0.4 and p = 0.3, respectively).Conclusions: PBCC stabilized core temperature at delivery better than ICC in term lambs. Hypothermia may not be a significant risk during PBCC.

Development of an in situ hybridization assay using an AS1 probe for detection of bovine leukemia virus in BLV-induced lymphoma tissues.

Enzootic bovine leukosis (EBL) is a malignant B cell lymphoma caused by infection with bovine leukemia virus (BLV). Histopathological examination is commonly used for diagnosis of the disease, but observation of lymphoma alone does not confirm EBL because cattle may be affected by sporadic forms of lymphoma that are not associated with BLV. Detection of BLV in tumor cells can be definitive evidence of EBL, but currently, there is no technique available for such a purpose. In this study, we focused on a viral non-coding RNA, AS1, and developed a novel in situ hybridization assay for the detection of BLV from formalin-fixed paraffin-embedded (FFPE) tissues. RNA-seq analysis revealed that all examined B lymphocytes derived from clinical EBL abundantly expressed AS1 RNA, indicating a possible target for detection. The in situ hybridization assay using an AS1 probe clearly detected AS1 RNA in fetal lamb kidney cells persistently infected with BLV. The utility of this assay in clinical samples was assessed using three EBL-derived lymph node specimens and one BLV-negative specimen, and AS1 RNA was detected specifically in the EBL-derived tissues. These results suggest that AS1 RNA is a useful target for the detection of BLV from FFPE specimens of tumor tissues. This technique is expected to become a powerful tool for EBL diagnosis.

OC02.03: The “Smart” tracheal occlusion device is safe and effective in fetal lambs with diaphragmatic hernia

OC02.03: The “Smart” tracheal occlusion device is safe and effective in fetal lambs with diaphragmatic hernia

New device permitting non-invasive reversal of fetal endoscopic tracheal occlusion: ex-vivo and in-vivo study.

One of the drawbacks of fetal endoscopic tracheal occlusion (FETO) for congenital diaphragmatic hernia is the need for a second invasive intervention to re-establish airway patency. The 'Smart-TO' device is a new balloon for FETO that deflates spontaneously when placed in a strong magnetic field, therefore overcoming the need for a second procedure. The safety and efficacy of this device have not yet been demonstrated. The aim of this study was to investigate the reversibility, local side effects and occlusiveness of the Smart-TO balloon, both in a simulated in-utero environment and in the fetal lamb model. First, the reversibility of tracheal occlusion by the Smart-TO balloon was tested in a high-fidelity simulator. Following videoscopic tracheoscopic balloon insertion, the fetal mannequin was placed within a 1-L water-filled balloon to mimic the amniotic cavity. This was held by an operator in front of their abdomen, and different fetal and maternal positions were simulated to mimic the most common clinical scenarios. Following exposure to the magnetic field generated by a 1.5-T magnetic resonance (MR) machine, deflation of the Smart-TO balloon was assessed by tracheoscopy. In cases of failed deflation, the mannequin was reinserted into a water-filled balloon for additional MR exposure, up to a maximum of three times. Secondly, reversibility, occlusiveness and local effects of the Smart-TO balloon were tested in vivo in fetal lambs. Tracheal occlusion was performed in fetal lambs on gestational day 95 (term, 145 days), either using the balloon currently used in clinical practice (Goldbal2) (n = 5) or the Smart-TO balloon (n = 5). On gestational day 116, the presence of the balloon was assessed by tracheoscopy. Deflation was performed by puncture (Goldbal2) or MR exposure (Smart-TO). Six unoccluded fetal lambs served as controls. Following euthanasia, the lung-to-body-weight ratio (LBWR), lung morphometry and tracheal circumference were assessed. Local tracheal changes were measured using a hierarchical histologic scoring system. Ex vivo, Smart-TO balloon deflation occurred after a single MR exposure in 100% of cases in a maternal standing position with the mannequin at a height of 95 cm (n = 32), 55 cm (n = 8) or 125 cm (n = 8), as well as when the maternal position was 'lying on a stretcher' (n = 8). Three out of eight (37.5%) balloons failed to deflate at first exposure when the maternal position was 'sitting in a wheelchair'. Of these, two balloons deflated after a second MR exposure, but one balloon remained inflated after a third exposure. In vivo, all Smart-TO balloons deflated successfully. The LBWR in fetal lambs with tracheal occlusion by a Smart-TO balloon was significantly higher than that in unoccluded controls, and was comparable with that in the Goldbal2 group. There were no differences in lung morphometry and tracheal circumference between the two balloon types. Tracheal histology showed minimal changes for both balloons. In a simulated in-utero environment, the Smart-TO balloon was effectively deflated by exposure of the fetus in different positions to the magnetic field of a 1.5-T MR system. There was only one failure, which occurred when the mother was sitting in a wheelchair. In healthy fetal lambs, the Smart-TO balloon is as occlusive as the clinical standard Goldbal2 system and has only limited local side effects. © 2020 International Society of Ultrasound in Obstetrics and Gynecology.

Quantitative contrast-enhanced ultrasound of the brain on twin fetal lambs maintained by the extrauterine environment for neonatal development (EXTEND): initial experience.

With the development of an artificial environment to support the extremely premature infant, advanced imaging techniques tested in this extrauterine system might be beneficial to evaluate the fetal brain. We evaluated the feasibility of (a) performing contrast-enhanced ultrasound (CEUS) and (b) quantifying normal and decreased brain perfusion in fetal lambs maintained on the extrauterine environment for neonatal development (EXTEND) system. Twin premature fetal lambs (102days of gestational age) were transferred to the EXTEND system. Twin B was subjected to sub-physiological flows (152mL/kg/min) and oxygen delivery (15.9mL/kg/min), while Twin A was maintained at physiological levels. We administered Lumason contrast agent into the oxygenator circuit and performed serial CEUS examinations. We quantified perfusion parameters and generated parametric maps. We also recorded hemodynamic parameters, serum blood analysis, and measurements across the oxygenator. Postmortem MRIs were performed. No significant changes in hemodynamic variables were attributable to CEUS examinations. On gray-scale images, Twin B demonstrated ventriculomegaly and progressive parenchymal volume loss culminating in hydranencephaly. By CEUS, Twin B demonstrated decreased peak enhancement and decreased overall parenchymal perfusion when compared to Twin A by perfusion parameters and parametric maps. Changes in perfusion parameters were detected immediately following blood transfusion. Postmortem MRI confirmed ultrasonographic findings in Twin B. In this preliminary experience, we show that CEUS of the brain is feasible in fetal lambs maintained on the EXTEND system and that changes in perfusion can be quantified, which is promising for the application of CEUS in this extrauterine system supporting the premature infant.