BILIARY ATRESIA & CHOLEDOCHAL MALFORMATION - EMBRYOLOGICAL AND ANATOMICAL CONSIDERATIONS

There are two key congenital conditions of the biliary tract which are of utmost importance to the paediatric surgeon: biliary atresia (BA)1 and choledochal malformation (CM)2. In both conditions there are important developmental components which may well be aetiological. Nevertheless, the actual mechanisms remain elusive with many unanswered questions. Still, this only reinforces the need to enhance our understanding and requires a thorough insight into the complex developmental pathway of the biliary system.

The aim of this paper is to provide an overview of the embryonic and fetal life key events and how they may interact leading to the post-natal pathology we recognise as BA and CM.

Development of the liver and bile ducts is a complex process with distinct, but interconnected steps (Fig. 1, Table 1). It is conventional to split the periods before birth into three consecutive phases based on completed weeks of gestation1:

I

Germinal phase (weeks 1-2)

II

Embryonic phase (weeks 3-8)

III

Fetal phase (weeks 9-40) [1].

The liver and biliary tract arise initially as an endodermal bud from the distal foregut within the ventral mesogastrium projecting into the mesenchyme of the septum transversum. This primordial bile duct develops into a funnel-shaped structure (pars hepatica) with a lumen evident throughout, and from about 45 days the thicker-walled gallbladder (pars cystica) also becomes apparent (Fig 2). This continues up to about the 7-8th week of gestation with the extrahepatic tract linking the GI tract with the primordial, predominantly haematopoietic, liver. From the cranial aspect of this endodermal diverticulum emerge the primordial liver cells – the hepatoblasts. Initially these are lining cuboidal cells with further proliferation resulting in a multilayered, pseudostratified endodermal structure requiring HHEX (haematopoietically expressed homeobox). These are the precursors of both hepatocytes and cholangiocytes expressing markers of both lineages: hepatocytes (α-fetoprotein (AFP), albumin (ALB), HNF4α, HHEX and cholangiocytes (HNF1β, SOX9 and CK19, a late differentiation marker). Their cellular fate is decided by signals released from adjacent mesodermal tissues.3,4

Subsequently, the hepatoblasts reduce their cell-cell contacts, delaminate and migrate into the septum transversum to become arranged in plates or “cords”, initially 3 - 4 cells thick and have an intimate relationship with the mesenchymal-derived endothelial cells lining the primitive vascular sinusoids. Proliferation and cell survival are controlled by paracrine WNT, FGF, and HGF (Hepatocyte Growth Factor) signalling from the adjacent mesodermal cells.

By this stage the liver is the dominant organ by mass within the abdominal cavity and is a large rounded mass of tissue often described by the German term – anlage (pl. anlagen)

Intrahepatic bile ducts only appear distinctly from about seven weeks gestation and their formation is organised by the infiltrating vascular network emanating from the portal vein. At seven weeks, this is infiltrating the liver anlage becoming surrounded by a layer of mesenchyme. From this a cylindrical double cell layer of darkly staining cells adjacent to the portal vein emerges termed the ductal (or limiting) plate. Actual ducts evolve from biliary epithelial cholangiocytes (BECs), a process that is characterised by remodelling and partial involution, and has four stages:

I

ductal plate stage – derived from periportal hepatoblasts, BECs begin to organise into the ductal plate - a single layered sheath of epithelial cells;

II

double layered ductal plate - a second layer of cells appear within the ductal plate;

III

"migratory stage" - tubular structures form within the double layer and the intervening ductal plate disappears or is re-absorbed;

IV

“bile duct stage” - the immature tubules are remodelled into individualised bile ducts that will form the final biliary network.5

This ductal plate evolution is co-ordinated by cell signalling pathways, the most important being NOTCH, WNT, sonic hedgehog (SHH) and TGF-β. 3,6

Persistence of the ductal plate appearance is termed a ductal plate malformation (DPM) and has been at times felt to be a prognostic histological factor in BA.7 So, it is more prevalent in BA than control liver, variously seen in 20% to 40% of BA. 8, 9, 10 Some studies suggested that the presence of DPM in liver biopsies is associated with advanced hepatic fibrosis and therefore poor prognosis after Kasai portoenterostomy10,11, nonetheless, this remains controversial.

Deranged ductal plate development may also result in an unusual spectrum of biliary tract abnormalities, often characterised by the development of multiple, small intrahepatic cysts and DPM. These include autosomal dominant polycystic kidney disease (ADPKD), recessive polycystic kidney disease (ARPKD), polycystic liver disease, Caroli disease (see later), biliary hamartomas and congenital hepatic fibrosis. 12 These are predominantly genetic in origin and frequently involve the kidney.

Bile acid synthesis starts at about 6 weeks and bile is first observed in primitive cholangioles and then transported into the fetal gut from about 12 - 14 weeks gestation implying completion of biliary continuity. This re-aligning and coalescence of extra- and intra-hepatic ducts systems at the porta hepatis is a key point in the timeline, however very little is known as to what controls it.9

The so-called "solid-phase" of biliary development, formerly a widely held belief and an obvious corollary of BA, now appears erroneous. More recent studies [9] have challenged this, stating that the developing intra- and extra-hepatic biliary tract maintains continuity throughout development, but it does remain a point of speculation at which biliary atresia begins.

Hepatic arteries develop in association with the developing bile ducts and are thought to be induced by VEGF signals from the ductal plate. The first offshoot of the hepatic artery is visible at the hepatic hilum after the 8th week. Two weeks later, the intrahepatic arterial radicals become evident in the central region of liver and subsequently extent following the intrahepatic branches of the portal system and reach the periphery at around 15th week of gestation.13 The liver continues to develop throughout the fetal stage and after birth and is characterized by maturation, hepatocyte proliferation and expansion of liver volume. The bile duct network also continues to develop at the periphery of the liver in response to Notch signalling. Until the late fetal stage, the bile ducts are all narrow. Then there is remodelling and enlargement of the ducts at the centre of the liver potentially in response to the onset of bile flow.

The pancreas is derived from the ventral and dorsal anlagen which arise from the foregut diametrically opposite each other and are distinct from about day 26 – 32. The ventral duct is an off-shoot of the bile duct and maintains this bile duct connection throughout. The dorsal anlage will give rise to the head, body and tail of the pancreas whilst the ventral bud gives rise to the uncinate process (Fig 2).

Actual fusion of pancreatic parenchyma occurs following a rotation of the ventral duct around the axis of the foregut at about 7-8 weeks gestation. This process is also accompanied by a variable degree of interconnection of the ventral and dorsal ducts. Typically, the dominant flow of pancreatic secretions from body and tail and most of the head is preferentially directed through the ventral duct (of Wirsung). The entry of the smaller dorsal duct (of Santorini) is usually more proximal in the final duodenum and reputedly drains only the uncinate process.

The final phase of pancreatic development occurs towards the end of gestation. So initially, the bile and pancreatic duct junction is outside of the wall of duodenum but during the last trimester there is gradual absorption of this junction into the wall of duodenum. The final arrangement is therefore a common chamber termed the ampulla of Vater, with both ducts emptying into it but surrounded by their own sphincter to maintain complete bile and pancreatic juice separation. Failure of this re-absorption process leads to a common (pancreato-biliary) channel that has the potential for premature mixing of exocrine secretions.

The blood supply of the liver is provided by two distinct sources: the low-pressure portal vein (PV) (responsible for ∼75% of blood flow in the adult) and the high-pressure hepatic artery (HA) (∼25%). The mixture of blood then flows through the sinusoids draining into only a single efferent system formed by coalescence of the hepatic venous channels and connecting to the vena cava and hence the right atrium. 13

The portal vein results from the confluence of the superior mesenteric and splenic veins. The development of the portal venous system is rather a complex process that occurs between the 4th and 12th week of gestation involving two distinct paired venous systems. The vitelline veins carrying blood from the gut to an evolving sub-hepatic sinusoidal plexus and the umbilical veins carrying oxygenated blood passively back from the placenta to the right side of the developing heart at the level of the sinus venosus.

During embryonic life there is major remodelling of the vitelline veins, in that three pre-hepatic venous cross-communications, resembling a “step-ladder”, sometimes in front of and then behind the embryonic intestine (Fig 3). Over time, there is selective involution with disappearance of the caudal part of the right vitelline vein, the cranial part of the left vitelline vein and the caudal-ventral anastomosis. The dorsal and cranial-ventral anastomoses persist and will eventually become the main portal vein and its right branch.13, 14, 15 Variation of this process may lead to an arrangement where the vein lies anterior with respect to the duodenum and is known as a pre-duodenal portal vein (PDPV) – a key component of the Biliary Atresia Malformation (BASM) syndrome.

The right umbilical vein disappears early in gestation, while the left bifurcates cranially, forming two new structures: one connecting with the inferior vena cava (IVC) via the ductus venosus (or Duct of Arantius). This, therefore, bypasses the liver sinusoids diverting oxygenated blood from the placenta directly to the right side of the heart; and another with the left portal vein connecting with the developing liver sinusoids. After birth, both the ductus venosus and the remaining umbilical vein involute; the former becomes the ligamentum venosum which is visible if you mobilise and retract the left lateral segment of the liver, while the latter becomes the ligamentum teres. 14

The IVC, embryologically, is a much later structure with quite an intricate ontogeny (Fig 4). The process begins with three paired venous networks: the posterior cardinal veins, subcardinal veins, and supracardinal veins. Some segments of these venous networks will anastomose, while others will regress, ultimately resulting in the predominantly right-sided IVC which is subsumed into the posterior segments of the liver. This mixed heritage can be gleaned from caval segments going caudally. Figure 4 illustrates the mixed parentage of this final version of the IVC.

pre-renal - derived from the right subcardinal vein;

renal - derived from the subcardinal – supracardinal anastomosis;

post-renal - derived from the right supracardinal vein and

hepatic - derived from the hepatic vein itself i.e. - proximal part of the right vitelline vein and hepatic sinusoids) [16].

If there is an abnormal persistence or regression of the fetal venous structures, anomalies of the IVC may result. The posterior cardinal veins initially are the main conduits for venous return before being replaced by the nascent IVC. These are still discernible in the infant as the azygous and hemiazygous veins [15], and indeed if necessary can replace the functionality of a missing or interrupted IVC, quoted to have a prevalence of 0.6% 16, a feature also frequently seen in BASM.

There is still much controversy about the timing of onset of BA relative to the time of birth. Is it truly congenital or is it acquired at some later point? One of the confounding factors is actually a key point of dogma in the understanding of BA – that it is not a single disease. Rather, it is made up of a number of “variants” which share common characteristics and may have a uniform aetiological mechanism. What such variants share with each other, however, is arguable beyond presenting with pale stools and conjugated jaundice and an obstructed biliary tract at some point in the neonatal period.

There are two syndromic variants which have BA as a consistent central component; Biliary Atresia Splenic Malformation (BASM)[17] and the Cat Eye Syndrome.18 Both were first described in patients in the Kings College Hospital series in 1993 and 2008 respectively. There also appears to be other syndromes which fall into this category where the BA is not a particular prominent or common component but is related, such as Kabuki syndrome.19 Additionally, there is a non-random association with more major anomalies such as oesophageal atresia and jejunal atresia.

BASM is much more common in European and North American series, (10-15%) 20,21, than Chinese or Japanese series. 22,23 Table 2 lists its major components with an estimate of their frequency. The main features are splenic anomalies (polysplenia, double spleen or asplenia), situs inversus, preduodenal portal vein, complete absence of the intrahepatic vena cava and cardiac anomalies. 17,20

These particular abnormalities lead us to the obvious conclusion that the features of BASM are indeed a congenital defect occurring secondary to some developmental insult during the early embryonic phase of organ development. The defect in bile duct development is perhaps the easiest to understand – at least for the surgeon. So, the typical BASM extrahepatic bile duct consists of an atrophic, often tiny, gallbladder remnant attached only to a more proximal bile duct remnant swinging back to the portal plate. Usually there is absolutely no sign of a common bile duct. Our interpretation is that the cause of this variant is failure to establish the early initial ventral out-pouching.

Determination of abdominal situs is an early event in normal development (∼20 days gestation). It achieves this (symmetry breaking) by a leftward fluid flow initiated by motile cilia followed by signal propagation by non-motile cilia. So, both types of cilia are involved in the left–right asymmetry process and functional defects in any of them can result in so-called laterality defects.24,25 Ciliary dysfunction may be a potential contributor to the genesis of the situs inversus, seen in about half of the those with BASM. There is also some evidence incriminating ciliary dysfunction even in those with the isolated form of BA. So, histological studies have shown that cholangiocyte cilia in BA are reduced in number and have a shortened length and abnormal orientation, irrespective of any laterality defects.26 Mutations in ciliopathy and laterality genes, such as Forkhead box protein A2 (FOXA2) 27, Cryptic Family 1 (CFC1) 28, nodal growth differentiation factor (NODAL), and Zic family member 3 (ZIC3) 29 have been identified at times but inconsistently. More recently, Berauer et al. using whole exome screening of a cohort of BASM patients uncovered rare biallelic variants in the polycystin 1-like 1 (PKD1L1) gene in about 10%.30 This particular gene is associated with ciliary calcium signalling and embryonic laterality determination in fish, mice, and humans, providing a plausible explanation for some of the anomalies seen.

There may be other aetiological reasons, aside from the genetic factors mentioned, that are important in BASM. Thus, we recognised maternal diabetes (particularly insulin-dependent diabetes) as being much more common in our BASM than non-BASM control cohort in 1993, confirming it in a review in 2006.17,20 This is well known as a causative factor in other somewhat eclectic congenital malformations such as transposition of the great vessels, double-outlet right ventricle, spina bifida and the caudal regression syndrome.31 The actual mechanism for this is still unclear but may be epigenetic, with an onset again during the early embryonic phase to account for the other visceral features.

A recent population-based study from Taiwan showed that mothers with type 2 diabetes have an increased risk of BA in their offspring (OR = 2.5). Now, because of the lack of granularity in the data in this type of study, we infer that this means a high incidence of the isolated form given the rarity of BASM in Asia.32 A similar, though smaller study from Texas also showed a significant association with BA with a similar OR of 2.3, especially for those with pregestational diabetes.33

Cat-eye syndrome is much less common but may present to the surgeon before appreciating that the infant actually has BA.18 One of the common features is an anorectal malformation often requiring a colostomy in the first week of life. Other associations include colobomata (defects in the iris) from which the syndrome gets its name and congenital cardiac anomalies. Genetically, these children, typically have aneuploidy of chromosome 22.18

Cystic biliary atresia (CBA) is another variant of BA which can be confidently stated to have its origins in prenatal life. Such cystic changes can be picked up during antenatal ultrasound-based screening that typically occurs from about 18-20 weeks of gestation. It is not associated with other anomalies, so it is unlikely to begin during the 1st trimester. About 25% of the cysts contains bile, so at least in these it is not unreasonable to speculate that their onset might be in the 2nd trimester during the fetal phase of the development, certainly after the initiation of bile production which we date to the 12th week of gestation.34

But what may be the cause? In 1980, Lewis Spitz reported a study in six fetal lambs whereby he ligated the common bile duct, resulting in bile-containing extrahepatic cysts reminiscent of CBA.35 Earlier, Pickett and Briggs ligated the hepatic artery in 11 fetal lambs with seven surviving to term. Each was found to have an "interruption of the common duct adjacent to the tie which was found in each case medial to the common duct".36 Perhaps the cause in CBA is ischaemic in nature, the bile duct being dependent on its arterial blood flow. There is very little circumstantial evidence in the human scenario. A postnatal study comparing ultrasound features of CBA with choledochal cysts did show that hepatic artery diameters were up to two-fold greater in the former group37 though they had little explanation for it.

Isolated BA is the largest clinical group and it is characterised, as the name suggests, by the absence of other defining characteristics. No real progress has been made in identifying its aetiological mechanism, although genetic, developmental, environmental and viral causes have all been suggested.1

To date no genes have been identified as a definitive cause of isolated BA, but the development of genome-wide association studies (GWAS) and their usage in BA research started to reveal some evidence of a genetic linkage in IBA. The initial work came from China with identification of ADD3 and XPNPEP1 mutations. 38 Other potential candidate genes responsible for the development of BA have been identified: GPC-1 39, ARF6 40, EFEMP1 41, STIP1 and REV1 42 in a European-American cohort; AMER1, INVS and OCRL in a Vietnamese cohort 43. A whole exome sequencing study identified deleterious de novo or biallelic variants in liver-expressed ciliary genes in almost one third of their BA cohort (28/89 patients) and concluded a two-fold increase in risk compared to normal.44

All the above findings might suggest that genetic factors could have a more direct role in the pathogenesis of IBA than previously thought, but better evidence needs to be revealed before a clear genetic link can be accepted.

The variation of the anatomy in biliary atresia can be staggering and underestimating it can result in severe consequences.

The porta hepatis, as its name suggests, is the gateway to the liver through which all the major neurovascular structures and biliary ducts enter or leave. It is bordered by the quadrate (Couinaud segment IV) lobe anteriorly and the caudate (Couinaud segment I) lobe posteriorly. On the left, it merges into the recessus of Rex and the umbilical fissure and is sited between Couinaud segments III and IV. The gallbladder fossa enters on the right side and below that, a little further to the right is the sulcus of Rouviere (incisura dextra) containing the posterior vascular elements (to segments VI and VII).

The usual spatial arrangement is a posteriorly-aligned portal vein dividing into right and left with the latter running in the recessus of Rex and ultimately becoming the umbilical vein, giving branches on either side to Segments II, III and IV. The common hepatic artery ascending on the left, divides into right and left branches running in front of the vein. There may also be a middle hepatic artery which dives down between the confluence of portal vein, often into the portal plate.46 (Fig. 5)

Typically, the gallbladder is atrophic in BA and essentially without a lumen. This finding alone is pathognomonic. If no bile can be aspirated from the gallbladder, the cholangiogram is deemed redundant, and the next step is to proceed to Kasai portoenterostomy. Identification and good exposure of the portal plate is a key step of this operation. In the “radical dissection” pioneered by Ryoji Ohi from Sendai and preferred at Kings College Hospital it is necessary to completely define and separate the entirety of the proximal biliary remnants from the right and left portal veins and the hepatic arteries. 46 The Rex fossa is opened to expose the junction of umbilical vein with left portal vein. Rouviere's fossa is likewise displayed. The proximal biliary remnant is transected flush with the liver capsule, starting in the gallbladder fossa and extending to the right side to incorporate the area between the anterior and posterior right vascular pedicle and to the left of the umbilical point.47. All of this denuded plate is incorporated into the Roux loop.

The Japanese classification is used to describe the intraoperative macroscopic appearance of the extrahepatic ducts and if defined by the most proximal level of bile flow obstruction.1

Type 1 (5-10%): obstruction at the level of CBD, often associated with a cyst and typically bile is found in the gallbladder

Type (1-2%): obstruction at the level of the common hepatic duct. Transection of the most proximal porta hepatis would show both right and left ducts draining bile.

Type 3 (>90%): obstruction at the level of porta hepatis, transection at this level should not show any macroscopic bile ducts

Choledochal malformations are characterised as having abnormal dilatation of parts of the biliary tract while retaining the capacity to excrete bile. Most are presumed to have a congenital origin. Figure 6 illustrates the current Kings College Hospital classification48, which is derived from Todani's work 49. The commonest forms (∼80%) consist of extrahepatic dilatation with either a cystic (Type 1C) or fusiform (Type 1F) morphology. When either have a dilated intrahepatic component then these are described as Type 4, either C or F depending on morphology of the extrahepatic component. Type 2 is rare and best described as a diverticulum of the common bile duct while Type 3 is a localised dilatation of the intramural part of the distal bile duct. Purely intrahepatic cysts are described as Type 5 and while there are some isolated examples most lesions are multiple and fall within the Caroli spectrum with liver fibrosis and are associated with renal fibrosis and cyst formation (see ductal plate malformation above).

Around 25% of Type 1C malformations are detected antenatally on the screening maternal ultrasound at around 18-20 weeks gestation. Postnatally most actually do excrete bile with resolution of physiological jaundice. At operation they will have a tenuous, almost thread-like distal common bile duct within the head of the pancreas joining its duct to form a common channel. Intracystic pressure, if measured, is invariably high and the level of amylase in bile low, implying the absence of any significant pancreatic reflux. We speculate that it is this atrophic distal common duct that is driving the dilatation but the origins of this are unclear. 50

Fusiform choledochal malformations do appear different from their cystic brethren. There is an association in Type 1F lesions with duodenal atresia (unpublished observation) where the insertion of CBD into the duodenum may well be abnormal. These tend to present later on in childhood and might also be related in some way to injury during the surgical repair. The majority of Type 1F lesions present later, outside of infancy although they are usually smaller in diameter, and here the common channel appears to play a more significant role. They usually exhibit free reflux of pancreatic juice and therefore high bile amylase values. Furthermore, about 25% of these will present with acute pancreatitis, thought to be due to the reflux of bile into the pancreatic duct. So, is the common channel the key to their aetiology? It seems unlikely that abnormal reflux of pancreatic juice into the bile duct causes dilatation – though this is the basis of Babbitt's hypothesis. 51 Animal models inducing reflux of pancreatic juice into the biliary tract cause modest biliary dilatation only 52 and we have shown that the mucosal damage in clinical choledochal malformation is related to the effects of elevated pressure rather than pancreatic reflux. 50

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