Pilot studies

A series of pilot studies leading to our final device were performed that identified potential obstacles and allowed sequential design modifications. All pilot studies utilized the pumpless arterial–venous (AV) circuit described below. The primary design modifications were related to the fluid environment and our approach to vascular access. A consistent observation throughout our pilot studies was the haemodynamic stability and efficient gas exchange achieved by the pumpless AV circuit over a wide range of circuit flows. The initial series of experiments were performed in late gestational lambs (125–140 days of gestation; term=145 days) and utilized an open fluid bath with continuous recirculation of an electrolyte solution (designed to mimic amniotic fluid) through micropore filters. To avoid the potential for umbilical venous spasm we utilized the carotid artery (CA) and jugular vein (JV) for vascular access (Table 1, open CA/JV). These studies lasted 23 to 108 h but were limited by sepsis and cannula-related complications. This led to our second design that included a semi-closed fluid bath with continuous exchange of amniotic fluid, rather than recirculation (Table 1, semi-closed CA/JV). With the improved incubator, five experimental animals with CA/JV cannulation (ranging in age from 120 to 125 days of gestation) were maintained on the system for 346.6±93.5 h, a marked improvement over the original design. Importantly, one animal was maintained on the circuit for 288 h (120–132 days of gestation) and was successfully weaned to spontaneous respiration, with long-term survival confirming that animals can be transitioned to normal postnatal life after prolonged extra-uterine support. However, sepsis remained limiting in 3 of 5 lambs resulting in design of a closed fluid circuit (the Biobag). With introduction of the Biobag, pilot studies were performed with the aim to apply our system to earlier gestational fetuses. From the perspective of lung development, lambs at 100 −115 days of gestation are in the mid to late canalicular phase of lung development18, which is the biological equivalent of the 22–24-week gestation premature human infant19. However, in 110-day gestational age (GA) lambs with CA/JV cannulation, diminishing circuit flows and progressive oedema developed within the first few days. Low circuit flows were due to reduced perfusion pressures across the oxygenator owing to lower mean arterial pressure in earlier GA lambs combined with elevated right-sided venous pressures in the 110-day GA CA/JV animals relative to published in utero controls (inferior vena cava pressures 9.6±2 mm Hg versus 4±2 mm Hg, respectively)20,21,22. To offload the right atrium, we opted to utilize the umbilical vein (UV) for venous inflow to mimic normal fetal umbilical venous return and improve streaming of oxygenated blood across the foramen ovale20,21. To avoid umbilical venous spasm23,24,25, we advanced the umbilical venous cannula to a position with the tip just inside the abdominal fascia. CA/UV cannulation resulted in the stable support of five 106–113-day GA lambs for 13 to 26 days in the Biobag (Table 1, Biobag, CA/UV). All five lambs demonstrated long-term haemodynamic stability and stable circuit flows and oxygenation parameters (CA/UV group in Fig. 2). However, flow to the oxygenator in CA/UV lambs was well below the normal physiologic flow to the placenta (70–120 versus 150–200 ml kg−1 min−1)26,27,28, primarily due to the inherently small-caliber carotid artery. The limiting problem of carotid vascular inflow led to the development of our final device to achieve physiologic fetal support (Fig. 1 and Supplementary Movie 1). The three components of the system are described below.

Table 1 Overview of experimental animals. Full size table

Figure 1: UA/UV Biobag system design. (a) Circuit and system components consisting of a pumpless, low-resistance oxygenator circuit, a closed fluid environment with continuous fluid exchange and an umbilical vascular interface. (b) Representative lamb cannulated at 107 days of gestation and on day 4 of support. (c) The same lamb on day 28 of support illustrating somatic growth and maturation. Full size image

Figure 2: Haemodynamic and oxygen parameters in CA/UV lambs versus UA/UV lambs. CA/UV lambs data represented by grey circles (n=5) and UA/UV lambs as black circles (n=8). (a) Heart rate. (b) Mean pre-membrane oxygenator pressure calculated as 1/3 systolic+2/3 diastolic. (c) Body weight-adjusted circuit flow. (d) Post-membrane oxygen saturation (sat). (e) Haemoglobin. (f) Oxygen delivery. Data are presented as mean±s.e.m. Statistically significant difference between groups in (a–f) is denoted by *P<0.05 (analysis of variance (ANOVA)). Full size image

A pumpless arteriovenous circuit

From the inception of the study, we reasoned that a pumpless circuit—in which blood flow is driven exclusively by the fetal heart—combined with a very low resistance oxygenator would most closely mimic the normal fetal/placental circulation. In most of our studies we utilized a small-volume, near-zero-resistance oxygenator and short segments of circuit tubing to minimize surface area and priming volumes. This system is comparable to the volume of the placenta itself—the reported placental blood volume of the sheep is 23.1 to 48.1 ml per kg29, and most of the studies in this report utilized the Quadrox-ID Pediatric oxygenator (Maquet Quadrox-ID Pediatric Oxygenator: Maquet Cardiopulmonary AG, Rastatt, Germany) that has a priming volume of 81 ml. Recently, with smaller lambs (0.5 to 1 kg) we have utilized a modified Quadrox Neonatal oxygenator (Maquet Quadrox-I Neonatal and Pediatric Oxygenator: Maquet Cardiopulmonary AG) with a priming volume of 38 ml. Thus, our circuit priming volumes for 1 to 3 kg lambs are within the normal placental blood volume range. Throughout the development of our device, with the exception of our earlier gestational CA/JV animals described above, all animals demonstrated complete haemodynamic stability, without need for vasopressors or evidence of progressive acidosis or circulatory failure.

A closed sterile fluid environment

To further address issues of sterility, size adaptability and efficiencies of space and fluid volume, a ‘Biobag’ design was developed—a single-use, completely closed system that minimizes amniotic fluid volumes and can be customized to more closely replicate the size and shape of the uterus. The Biobag consists of polyethylene film that is translucent, sonolucent and flexible to permit monitoring, scanning and manipulation of the fetus as necessary. An open, sealable side was incorporated to facilitate insertion of the fetus at the time of cannulation, and various water-tight ports were designed to accommodate cannulas, temperature probes and sterile suction tubing. After cannulation, the Biobag is sealed and transferred to a mobile support platform that incorporates temperature and pressure regulation, padding and the fluid reservoirs and fluid exchange circuitry. The development of the Biobag essentially solved the problem of gross fluid contamination, and has eliminated pneumonia on lung pathology. Throughout the subsequent experiments, low-level amniotic fluid contamination was observed only in circumstances where Biobag re-entry was required. When this occurred, contamination could be cleared by increasing the fluid exchange rate and injecting antibiotics into the bag fluid on a daily basis.

Umbilical vascular access

To more closely approximate flow dynamics in utero, carotid cannulation was abandoned in favour of double umbilical artery and single umbilical vein cannulation (abbreviated UA/UV) cannulation. We developed a technique for umbilical cord vessel cannulation that maintains a length of native umbilical cord (5–10 cm) between the cannula tips and the abdominal wall, to minimize decannulation events and the risk of mechanical obstruction (Fig. 1b,c). The umbilical and venous cannulas are only 2 cm long, most of which is used for securing the cannulas, and therefore the interface is functionally end to end. Umbilical cord spasm was mitigated by a combination of topical papaverine administration, atraumatic operative technique and maintaining warmth and physiologic oxygen saturation of the umbilical venous inflow on initiation of circuit flow. The Biobag was modified to accommodate exclusively umbilical cannulas (Fig. 1b,c and Supplementary Movie 1).

Physiologic extracorporeal support of the fetus

The combination of the pumpless oxygenator circuit, the closed fluid circuit and Biobag and umbilical cord access constitute our device. We have run 8 lambs with maintenance of stable levels of circuit flow equivalent to the normal flow to the placenta. We have run 5 fetuses from 105 to 108 days of gestation for 25–28 days, and 3 fetuses from 115 to 120 days of gestation for 20–28 days (Table 1, Biobag UA/UV). The longest runs were terminated at 28 days due to animal protocol limitations rather than any instability, suggesting that support of these early gestational animals could be maintained beyond 4 weeks. Haemodynamic and circuit flow parameters for all eight UA/UV lambs are summarized in Fig. 2 and compared directly with CA/UV lambs. The UA/UV lambs demonstrated levels of circuit flow comparable to what is considered normal placental flow (Fig. 2c, 150–250 ml kg−1 min−1) throughout the duration of their runs. With physiologic circuit flows in UA/UV animals, we were able to maintain lower post-membrane saturation (Fig. 2d) with normal fetal oxygen delivery (Fig. 2f) and reduced transfusion requirement (Fig. 2e) relative to CA/UV lambs. To prevent excessive oxygen delivery, we typically lowered initial sweep-gas oxygen concentration to 11–14% by blending nitrogen with room air.

Normal oxygen delivery is required for physiologic metabolic support, substrate utilization and growth and development. Figure 3a shows the calculated parameters related to oxygen delivery for our UA/UV lambs on both the 5 earlier gestation lambs (105 to 108 days of gestation) and the 3 later gestation lambs (115 to 120 days of gestation). The values for both groups are comparable to the previously published normal values for fetal lambs30. During studies, efforts were made to maintain normal fetal oxygen tension and carbon dioxide exchange while providing normal oxygen delivery (Fig. 3b). In CA/UV lambs a progressive fall in haemoglobin (Hgb) levels was noted, ultimately requiring transfusion with a large volume of adult blood (∼40 ml kg−1 per week) to maintain O 2 delivery. We reasoned that this was likely due to impaired erythropoietin production from the fetal liver due to supraphysiologic O 2 content of the post-membrane UV blood31. Subsequent experiments with the first three UA/UV animals demonstrated reduced transfusion requirement (∼10 ml kg−1 per week), likely due to lower PaO 2 in UV blood. In the last five UA/UV animals we administered daily erythropoietin that slowed the progression of anaemia and nearly eliminated (∼6 ml kg−1 per week) or, in the last 3 animals, completely eliminated the need for blood transfusion (Fig. 3c). Fetuses had normal pH values (Fig. 3d) and lactate levels (Fig. 3e) throughout the studies.

Figure 3: Oxygen parameters in UA/UV lambs. Data from n=8 lambs. (a) Haemodynamic and laboratory parameters. (b) Pre-membrane P a CO 2 and P a O 2 . (c) Haemoglobin levels with and without erythropoietin (EPO). (d) Pre-membrane pH. (e) Plasma lactate. † In utero control values in (a) derived from measured data in ref. 18. Data in (b–e) are presented as mean±s.e.m. Statistically significant difference between groups in (c) denoted by *P<0.05 (analysis of variance (ANOVA)). Full size image

Daily echocardiography confirmed physiologic cardiac outputs and maintenance of the fetal cardiac circulation throughout the UA/UV runs, with near-normal ductus arteriosus flows (Fig. 4a–c), patency and flow through the ductus venosus and right to left shunting through the foramen ovale (Supplementary Movies 2, 3, 4)32. Cardiac contractility was excellent and chamber and vena caval size could be used as an indicator of general volume status, allowing adjustments in fluid administration. An experienced fetal cardiologist (J.R.) reviewed all echocardiographic data and agreed that cardiac function appeared grossly normal in all respects.

Figure 4: Echocardiographic parameters in UA/UV lambs. Data from n=8 lambs. (a) Echocardiographic parameters. (b) Combined cardiac output. (c) Ductus arteriosus (DA) flow to right ventricular output ratio. Data in (b,c) are presented as mean±s.e.m. *In utero microsphere data in (a) derived from measured data in ref. 20. †Lamb 3 noted to have Tetralogy of Fallot (TOF) with restricted pulmonary flow before cannulation was excluded from mean value calculations (‡) due to TOF physiology. Full size image

Growth and organ maturation

The nutrition provided via the circuit was based on substrate uptake of late-gestation fetal lambs33,34,35, and hence consisted predominantly of carbohydrate and amino acid, with trace lipid. Our strategy in CA/UV and UA/UV lambs was to titrate dextrose and amino acid administration to levels of plasma glucose (<40 mg dl−1) and blood urea nitrogen (<30 mg dl−1) to avoid an osmotic diuresis and/or hyperosmolar state. Substrate tolerance generally correlated with oxygen delivery, and at relatively higher oxygen delivery, UA/UV lambs tolerated physiologic levels of substrate delivery (Fig. 5a) and demonstrated growth comparable to breed-matched controls (Fig. 5b,c). From a qualitative perspective, there was obvious growth and maturation with prolonged runs. Animals opened their eyes, became more active, had apparently normal breathing and swallowing movements, grew wool and clearly occupied a greater proportion of space within the bags (Fig. 1b,c). The addition of insulin infusions in the last two lambs further improved substrate utilization and allowed administration of higher caloric loads with further enhancement of fetal growth.

Figure 5: Nutrition and growth in UA/UV lambs. Data from n=8 lambs. (a) Nutritional substrate and laboratory parameters; data are presented for individual animals and as group averages (mean± s.e.m.). (b) Control growth curve using UA/UV and control lamb weights at hysterotomy (n=19). Solid line represents exponential best fit. (c) UA/UV growth curve. Solid line represents exponential best fit of UA/UV non-insulin lamb weights at hysterotomy and at end of studies. Dashed line represents control growth curve. †In utero control values in (a) derived from measured data in refs 36, 37, 59. *P value in (c) refers to between-group analysis of variance (ANOVA; statistical significance defined as P<0.05). Full size image

As a surrogate for organ maturation in our system, we assessed lung maturation in UA/UV lambs by detailed morphometric analysis (Fig. 6a–e), histologic assessment (Fig. 6f–i), surfactant protein B analysis (Fig. 6j,k) and analysis of function after birth (Fig. 6m). Lambs at 106 −113 days of gestation are in the mid to late canalicular phase of lung development18, which is the biological equivalent of the 23–24-week gestation premature human infant19. Morphometric analysis demonstrated progression from the canalicular to saccular stages of lung development in parallel with age-matched normal control lambs (Fig. 6a–e). From a functional perspective, lambs were easily ventilated after removal from the circuit, and nearly comparable to 141-day GA control lambs delivered by caesarean section and immediately ventilated (Fig. 6m). Other metabolic parameters reflective of organ function and nutritional status were surprisingly stable despite the known maternal contributions to hepatic and renal function (Table 2). Bilirubin levels and liver function tests showed only very mild elevation or remained within the normal range.

Figure 6: Structural and biochemical lung development and early neonatal pulmonary function in UA/UV lambs. (a–e) Morphology of control ((f), 113d GA and (g),139d GA) and experimental lambs ((h), 132d GA, CA/UV lamb 3; (i), 144d GA, UA/UV lamb 5) following 19 and 28 days on circuit, respectively (scale bars, 50 μm). Ongoing alveolar formation on circuit is evidenced by increased density and height of secondary septae (arrowheads). Density of surfactant protein-B-positive alveolar cells ((j), arrowheads in k,l) and neonatal pulmonary function were not different from that of age-matched controls (m). *P value in (j) refers to difference between groups (Student’s unpaired t-test, statistical significance defined as P<0.05). †Control group in (j) includes near-term lambs only (n=4, mean GA 141 days). §Mean control values in (m) derived from measured data in ref. 24. ‡Near-term control lambs in (m) delivered by caesarean section and ventilated in the same manner as experimental animals. Data in (j,m) are presented as mean±s.e.m. Full size image

Table 2 Metabolic, haematologic and fluid parameters in UA/UV lambs cannulated at GA 105–111 days and GA 115–120 days. Full size table

Brain growth and development were also assessed in UA/UV animals. Brains were grossly normal appearing after runs with no difference in brain-to-body weight ratios in experimental animals versus age-matched in utero controls (Fig. 7a). To assess brain maturation we analysed gyral thickness that also demonstrated no difference from controls (Fig. 7b). Biparietal diameter has been reported as a surrogate for brain growth36 and the growth curve for lambs maintained on our system was similar to the expected curve determined from weight-based calculations (Fig. 7c)37. As the effects of exogenous insulin on brain growth are unknown, animals receiving insulin were excluded from brain growth assessments (n=2). On whole-brain sectioning we observed no evidence of haemorrhage or infarct in any of the UA/UV animal brains (n=5). Finally, to assess ischaemic injury and global brain integrity on the surviving lamb, postnatal T1-, T2- and diffusion-weighted magnetic resonance imaging sequences were performed at 6 months of age (Fig. 7d). Despite utilization of the CA in this animal, there was no evidence of ischaemia or structural defects. To confirm these findings at a histologic level we performed routine haematoxylin and eosin staining on critical brain regions (Fig. 8a) and, as white matter is particularly sensitive to ischaemic injury, we assessed critical regions of the brain by densitometry of myelin-stained sections38,39,40 in UA/UV animals. There was no difference in myelin density in any of the brains analysed (Fig. 8b–d). In addition, neuropathologists from two separate institutions were unable to identify any histologic lesions associated with ischaemia, infarction or demyelination when blinded to experimental and control tissues.

Figure 7: Neurologic development and maturation in experimental lambs. (a) Post-mortem brain-to-body weight ratio of experimental (Exp.) and control (Ctrl.) animals; animals that received insulin are excluded. (b) Gyral width on haematoxylin and eosin (H&E) stained sections by region as compared with 140-day gestation controls. (c) Biparietal diameter in experimental (Exp.) versus predicted (equation derived from ref. 26) animals. (d) Postnatal T2-weighted coronal magnetic resonance imaging (MRI) at 6 months of age. Scale bar in (d) is 1 cm. Data in (a–c) are presented as mean±s.e.m. P values in (a–c) refer to difference between control and experimental groups (Student’s t-test for each region/time point, with statistical significance defined as P<0.05). BPD, bronchopulmonary dysplasia. Full size image

Figure 8: Histologic and other parameters of brain development and maturation. (a) Representative haematoxylin and eosin (H&E)-stained sections of post-therapy cerebrum/cerebellum and their respective cortices displaying normal brain parenchyma and absence of injury. (b) Representative Luxol fast blue myelin stain and digital pixel identification depicting myelin density (orange). (c) Percent positive pixels identified in the selected regions. (d) Maximum positivity (density of myelin) in the selected regions. (e) Ocular EMG activities in instrumented in utero control (Ctrl.) and experimental (Exp.) fetal lambs at 120 days (upper tracing) and 134 and 139 days, respectively (lower tracing). White area of bar represents activity and black areas quiescence. (f) Correlation of middle cerebral artery pulsatility index (MCA PI) and fetal oxygen delivery; solid line represents linear best fit. Data in (c,d) are presented as mean±s.e.m. The scale bars in (a,b) from left to right are 4 mm, 300 μm, 1.5 cm, 150 μm, 4 mm and 2 mm, respectively. P values in (c,d,f) refer to difference between control and experimental groups (Student’s t-test in c,d and Pearson’s correlation coefficient in f, with statistical significance defined as P<0.05). Full size image

From a gross functional level, the UA/UV animals demonstrated normal or increased movement, sleep/wake cycles, intermittent breathing and swallowing and generally appeared comfortable and nondistressed. Fetal breathing movements were noted regularly throughout the incubation period. As one measure of neurologic development, we compared ocular electromyography (EMG) in two chronically catheterized fetal lambs41, with two lambs maintained in the system over the same range of gestation. A developmental progression from fragmented to consolidated sleep/wake cycles between the two gestational ages is apparent in both in utero and experimental animals (representative data shown in Fig. 8e). Finally, although brain perfusion was not directly assessed, the middle cerebral artery pulsatility index was routinely measured and correlated with oxygen delivery (Fig. 8f), consistent with normal autoregulation of cerebral blood flow. Taken together with our observation of normal cardiac outputs, oxygen delivery and fetal circulatory pathways, we feel our limited data to this point is encouraging with respect to cerebral perfusion and brain development. It is important to note however that there are important differences between fetal lamb brain maturation and human brain maturation, most important of which is the earlier maturation of the germinal matrix in the lamb (70 days)42. Thus, the risk of intracranial haemorrhage cannot be assessed in the ovine model43. In addition, long-term neurologic follow-up is difficult in our model, due to difficulties with survival of premature lambs and to the limitations in assessment of lamb neurologic function. Thus, any conclusions regarding neurologic development must be qualified.