Neonatal Cholestasis: Clinical Guidelines

List of Abbreviations

ALT – ALanine Aminotransferase

AST – ASpartate Aminotransferase

aPTT – activated Partial Thromboplastin Time

BA – Biliary Atresia

GGT – γ-Glutamyl Transferase

BA – Bile Acids

INR – International Normalized Ratio

PUFA – PolyUnsaturated Fatty Acids

PFIC – Progressive Familial Intrahepatic Cholestasis

PT – Prothrombin Time

AGS – Alagille Syndrome

MCT – Medium-Chain Triglycerides

LT – Liver Transplantation

ALP – Alkaline Phosphatase

ELISA – Enzyme-linked immunosorbent assay

Ultrasound – Ultrasound Investigation

Terms and Definitions

Cholestasis syndrome is a decrease or complete cessation of bile flow due to impaired bile formation and/or excretion, leading to elevated bile components in the blood and their deficiency in the intestine.

Cytolysis syndrome is an elevated serum level of transaminases (alanine aminotransferase, aspartate aminotransferase) associated with compromised hepatocyte membrane integrity.

Hepatocellular insufficiency is impaired liver function manifested by decreased albumin, fibrinogen, cholinesterase, cholesterol, glucose, prothrombin index or prolonged prothrombin time, and an increased international normalized ratio.

Posterior embryotoxon is a congenital opacity of the peripheral cornea in the form of a milky-white ring separated from the limbus by a narrow strip of clear tissue.

Butterfly vertebra is a sagittal defect of the vertebral body caused by impaired fusion of two lateral chondrification centers during embryogenesis (on radiographs, the vertebra resembles a "flying butterfly").

Liver fibrosis is excessive proliferation of connective tissue in portal tracts with septum formation.

Liver cirrhosis is structural remodeling of liver tissue with the formation of regenerative nodules and impaired blood circulation within them.

Portal hypertension is a persistent increase in pressure within the portal venous system caused by impaired blood flow of various etiologies localized in the portal vessels, hepatic veins, and inferior vena cava.

Hypoplasia of the intrahepatic bile ducts is a decrease in the number and/or size of interlobular bile ducts.

1. Brief information on the disease or condition

1.1 Definition of the disease or condition

Neonatal cholestasis is an impairment of the excretory function of the hepatobiliary system during the neonatal period, leading to elevated bile components in the blood and their deficiency in the intestine [1–3].

Synonyms: neonatal hyperbilirubinemia due to impaired bilirubin excretion; direct neonatal hyperbilirubinemia; bile plug syndrome.

1.2 Etiology and pathogenesis of the disease or condition (group of diseases or conditions)

The morphofunctional characteristics of the liver and bile ducts in newborns are marked by a high rate of bile acid synthesis and immaturity of their enterohepatic circulation, predisposing to impaired bile flow. Newborns exhibit a discrepancy between the increased production of bilirubin, bile acids, and other bile components and a limited capacity for their elimination from the body. Normally, on days 4–7 of life, significantly more bile acids are produced than in adults. Neonatal bile acids are termed "atypical" due to the presence of additional hydroxyl groups on their surface, which reduce their toxicity index while simultaneously and significantly decreasing their cholekinetic activity. Relative immaturity of the liver enzyme systems responsible for capturing bile components from the blood, their intracellular transport, and excretion into the intrahepatic biliary system, increased permeability of intercellular junctions, low excretory activity of the biliary system, and increased reabsorption of bile components in the intestine contribute to slow bile elimination and elevated levels of bile acids and other bile components in the blood. In newborns, this is manifested by a relative hepatomegaly (up to 2 cm below the costal margin), an increase in the direct bilirubin fraction up to 15–20% of the total, higher blood levels of enzymes such as ALP, GGT, and bile acids, and may be considered during this period as a transitional state—physiological neonatal cholestasis. The complete establishment of bile formation mechanisms and enterohepatic circulation is completed after one month of age, reaching adult levels by 3–6 months of life [4–6].

Causes of neonatal cholestasis may include:

– Extrahepatic perinatal pathology.

– Liver and biliary tract diseases.

The main diseases manifesting with neonatal cholestasis are presented in Table 1.

Table 1. Liver and bile duct diseases manifesting with cholestasis syndrome in newborns.

Relevant current clinical guidelines describe in detail such diseases as galactosemia [7], tyrosinemia [8], Niemann-Pick disease type C [9], cystic fibrosis [10], and hypothyroidism [11].

Causes of cholestasis syndrome in the neonatal period can be either transient conditions associated with severe perinatal pathology and infections manifesting as hepatitis, or a number of congenital liver diseases resulting from developmental anomalies of the biliary system, impaired synthesis and/or transport of bile components, and metabolic disorders. Some of the most common and/or effectively treatable diseases are presented below.

1. Neonatal cholestasis due to extrahepatic perinatal pathology – an impairment of the excretory function of the hepatobiliary system caused by a combination of pathological and iatrogenic factors of the perinatal period, as well as morphofunctional immaturity of the liver. Among extrahepatic causes of neonatal cholestasis, the leading role belongs to conditions accompanied by hypoxia or ischemia of the hepatobiliary system, gastrointestinal tract (GIT) hypoperfusion, persistent hypoglycemia, metabolic acidosis, and congestive heart failure. Impairment of the hepatobiliary excretory function may be caused by elevated bilirubin levels in hemolytic disease of the newborn (HDN) due to significant alterations in the colloidal properties of bile, increased viscosity, and in some cases due to the direct toxic effect of bilirubin on hepatocyte membranes and cellular mitochondria. Systemic and localized bacterial infections play an important role by triggering the synthesis and excretion of a complex cascade of inflammatory mediators by Kupffer cells, as well as hepatocytes and sinusoidal endothelial cells, which directly affect bile formation and excretion. Therapeutic measures provided to newborns in neonatal intensive care units (NICUs) involve potentially hepatotoxic drugs and parenteral nutrition (PN) solutions. These also contribute to disrupting the functional state of the hepatobiliary system. Cholestasis is more frequently observed in preterm infants under the simultaneous action of multiple pathological and iatrogenic factors affecting liver function and bile duct status. These changes are based on various destructive alterations of the biliary ducts, impaired permeability of hepatocyte membranes and intercellular junctions (reversible in most cases). The development of neonatal cholestasis against the background of extrahepatic perinatal pathology is characteristic of the neonatal period, as it is precisely at this age that the morphofunctional immaturity of the hepatobiliary system exists as a result of the combined action of pathological and iatrogenic factors [1,4,12–14].

2. Diseases of the liver and biliary tract.

Depending on the level of the hepatobiliary lesion, diseases are conventionally divided into those presenting with extrahepatic and intrahepatic cholestasis.

Causes of extrahepatic cholestasis in newborns may include:

• Biliary atresia (BA).

• Choledochal cyst.

• "Bile plugs" or bile duct stones.

• Compression of the common bile duct.

2.1 Biliary Atresia (BA) is a progressive obliteration of the extrahepatic bile ducts, beginning during intrauterine development, with gradual involvement of the intrahepatic biliary system and the development of biliary cirrhosis. Currently, the etiology remains unclear. Theories under discussion include developmental malformation, viral, genetic, and others. The lack of patency in the biliary tract leads to the accumulation of bile components in the hepatobiliary system and their increased entry into the blood. Bile does not enter the intestine, and as a result, the digestion and absorption of fats and fat-soluble vitamins are impaired. The immune system plays a certain role in the pathogenesis of BA. Cytochemical analysis of liver biopsies reveals cellular markers of inflammation, including CD14-positive macrophages that trigger a cascade of immunological reactions. The expression of type I intracellular adhesion molecules promotes the formation of leukocyte antigens around the bile ducts, which in turn triggers a cytotoxic "lymphocyte attack." The inflammation of the bile duct epithelial cells is accompanied by the active production of a growth factor that stimulates the transcription of type I collagen, which underlies periductal fibrosis. Four types of the disease are distinguished: type I (3%) – atresia of the common bile duct only; type II (6%) – a cyst at the porta hepatis connected to the intrahepatic bile ducts; type III (19%) – atresia of the left and right hepatic ducts, while the gallbladder, cystic duct, and common bile duct remain patent; type IV (72%) – atresia of the entire extrahepatic system [15,16].

2.2 Choledochal Cyst is a congenital dilatation of the bile duct that, in 2–5% of cases, causes complete obstruction of the biliary ducts and can be a cause of extrahepatic cholestasis. Primary choledochal cysts are caused by initial thinning or absence of the muscular wall and its replacement by connective tissue. Secondary cysts are caused by a malformation forming during the regression stage of solid embryogenesis (at 3–7 weeks). Resulting kinks, stenoses, or valves of the terminal common bile duct lead to its dilation and wall thinning. Isolated dilatations of the common bile duct alone, without involvement of the cystic duct and gallbladder wall, are characteristic. An abnormal connection of the cyst with the pancreatic duct is also possible; it is believed that the reflux of pancreatic enzymes into the common bile duct contributes to cyst development. When a choledochal cyst is combined with biliary system obstruction, the pathogenesis does not differ from that of BA. In other cases, bile stasis in the dilated bile duct leads to altered colloidal properties, increased viscosity, and incomplete bile outflow. Prolonged retention of bile in the ducts predisposes to infectious complications with the development of cholangitis.

2.3. Neonatal Hepatitis (Congenital Hepatitis, Fetal Hepatitis) – liver pathology of infectious origin in the perinatal and postnatal periods. Viruses (cytomegalovirus, rubella virus, herpes virus, Coxsackie and others, hepatitis B and C viruses), bacteria (Listeria, Mycobacterium tuberculosis and others), protozoa (Toxoplasma), as well as Treponema pallidum and others, can cause hepatitis. Hepatitis developing against the background of neonatal sepsis is most often caused by the same pathogens as the sepsis itself. A high risk of fetal and neonatal hepatitis B infection exists under the following conditions: development of acute hepatitis B in the mother in the third trimester of pregnancy; maternal HB_SAg carriage combined with a positive HB_EAg and absence of anti-HB_E; detection of hepatitis B DNA by PCR; high titer of anti-HB_cAg. The pathogenesis is based on a specific inflammatory response to infection with microorganisms tropic to hepatocytes. Cytomegalovirus-induced neonatal cholestasis is detailed in the clinical guidelines "Congenital Cytomegalovirus Infection" [17].

2.4 Alagille Syndrome (Syndromic Intrahepatic Bile Duct Hypoplasia, Arteriohepatic Dysplasia) is a syndromic form of pathology comprising a combination of at least three of five major features: chronic cholestasis based on congenital intrahepatic bile duct hypoplasia, cardiovascular defects, vertebral anomalies, eye defects, and characteristic craniofacial features [18]. Alagille syndrome has an autosomal dominant inheritance pattern. The gene defect is associated with a partial deletion of the short arm of chromosome 20 [20p11-12], where the Jagged1 (JAG1) gene is localized. Mutations in another gene, called NOTCH2, are identified less frequently. The liver changes in Alagille syndrome are based on congenital intrahepatic bile duct hypoplasia, the severity of which can vary widely and determine both the time of onset of the first clinical symptoms and the prognosis of the disease. Hypoplasia of the intrahepatic bile ducts hinders bile outflow, leading to the accumulation of its components in the hepatobiliary system and their increased entry into the blood. On the other hand, insufficient bile flow into the intestine leads to impaired absorption of fats and fat-soluble vitamins.

2.5. Nonsyndromic Intrahepatic Bile Duct Hypoplasia is diagnosed in the absence of anomalies and/or malformations characteristic of Alagille syndrome, pathogenic variants in the JAG1 and NOTCH2 genes, and upon detection of hypoplasia during histological examination of a liver biopsy. The etiology of this disease is not established; it is hypothesized that a combination of various teratogenic factors affects the initiation and formation of intrahepatic bile ducts. It should also be noted that this histological sign can be a manifestation of other conditions, including infectious diseases (rubella, CMV, and syphilis), metabolic disorders (alpha-1 antitrypsin deficiency, cystic fibrosis), endocrine disorders (hypopituitarism), and chromosomal diseases (trisomy 18 and 21). Therefore, when intrahepatic bile duct hypoplasia is detected, attention should be paid to clinical and laboratory features characteristic of these disorders, and additional studies should be performed. The exclusion of these diseases serves as the basis for diagnosing nonsyndromic (idiopathic) intrahepatic bile duct hypoplasia.

2.6. Progressive Familial Intrahepatic Cholestasis (PFIC) is a class of chronic cholestatic liver diseases caused by a genetically determined disruption of the hepatocyte canalicular membrane structure, leading to impaired excretion of bile acids and/or other bile components. Currently, up to 12 types of PFIC are distinguished (Table 2) [19–27]. However, the 5 best-studied types are: type 1 (Byler disease), type 2 (Byler syndrome), type 3 (MDR3 gene deficiency), type 4 (TJP2 gene deficiency), and type 5 (farnesoid X receptor deficiency).

Table 2. PFIC Types

Impaired excretion of bile acids and/or other bile components leads to their accumulation in hepatocytes. Upon reaching a certain concentration in hepatocytes, bile components enter the blood, exerting a toxic effect. The absence or deficiency of bile acids in the intestine leads to impaired absorption of fats and fat-soluble vitamins.

2.7 Inborn errors of bile acid synthesis due to enzymopathy – a group of rare hereditary disorders caused by a deficiency of an enzyme involved in the synthesis of primary bile acids. Depending on the deficiency of a specific enzyme system, 9 disorders are distinguished, two of which have been described to manifest in the neonatal period (Table 3).

Table 3 Congenital defects of bile acid synthesis manifesting with neonatal cholestasis

Deficiency of an enzyme involved in bile acid synthesis leads to the accumulation of intermediate products of their synthesis. These compounds are not excreted into the intestine and accumulate in hepatocytes. Upon reaching a certain concentration in hepatocytes, these intermediate products enter the bloodstream, exerting a toxic effect primarily on the hepatocytes themselves. The absence of bile acids in the intestine leads to impaired absorption processes, including the malabsorption of fat-soluble vitamins.

1.3 Epidemiology of the Disease or Condition

The prevalence of cholestasis syndrome depends on the etiology. The epidemiology of certain conditions is listed in Table 4 [6]. The total incidence is approximately 1 in 2,500 newborns [21,28].

Table 4. Prevalence of certain congenital liver diseases and metabolic disorders accompanied by neonatal cholestasis.

1.4 Features of Coding the Disease or Condition

P58.2 Neonatal jaundice due to infection

P58.4 Neonatal jaundice due to drugs and toxins transmitted from mother or given to newborn

P59.1 Bile plug syndrome

P59.2 Neonatal jaundice from other and unspecified hepatic cell damage

Subsequently, when the cause of neonatal cholestasis is clarified, the disease coding is transformed into the corresponding code of the identified nosology.

1.5 Classification of the disease or condition

Currently, there is no classification of cholestasis syndrome in newborns.

1.6 Clinical presentation of the disease or condition

Clinical manifestations of transient cholestasis syndrome include greenish-tinged jaundice, hepatomegaly, dark yellow urine, and episodes of stool acholia, which develop against the background of perinatal pathology. As the child's general condition improves and the underlying disease resolves, cholestasis gradually decreases in most cases. However, residual manifestations may persist for a long time, up to 6–8 months of life.

Neonatal hepatitis is in most cases a manifestation of a generalized infection, although in more than 50% of cases of congenital infection, liver changes are reactive in nature. Clinical manifestations of hepatitis include impaired general condition, signs of an infectious process, jaundice, hepatosplenomegaly, hemorrhagic syndrome, and other features of the TORCH syndrome (Toxoplasmosis, Other infections [including syphilis, hepatitis, HIV], Rubella, Cytomegalovirus, and Herpes simplex virus). Most newborns infected with hepatitis B or C virus develop asymptomatic lifelong carriage or primary chronic hepatitis.

Patients with biliary atresia are in most cases born full-term with anthropometric parameters within the normal physiological range. Jaundice appears on the 2nd–3rd day of life, i.e., within the timeframe typical for physiological jaundice. Approximately 2/3 of patients exhibit a "lucent interval"—a decrease in the intensity of jaundice by the end of the 1st–2nd week of life, followed by its gradual increase and the appearance of a greenish skin tint by the end of the 1st month. Stool acholia is the earliest and most constant clinical sign of the disease. Its appearance is often preceded by the passage of meconium. When assessing stool color, it should also be remembered that when using certain therapeutic formulas with added medium-chain triglycerides, various shades of gray can be the equivalent of pale/acholic stools. Biliary atresia is characterized by the absence of hepatomegaly at birth, followed by an increase in liver size and a change in its consistency from elastic to firm during the first two months of life. By 1 month of age, hemorrhagic syndrome may develop (bleeding from the mucous membranes of the gastrointestinal tract, umbilical stump, intracranial hemorrhage). By 1–2 months of age, weight loss generally develops, the severity of which depends on the type of infant feeding. The most pronounced deficit is observed with breastfeeding or standard formula intended for healthy newborns. When using therapeutic, high-calorie formulas with partially hydrolyzed components, weight deficits may be absent or minimal. Without surgical treatment, signs of portal hypertension appear as early as 5–6 months of life, which subsequently progress rapidly and indicate the development of biliary cirrhosis [15,29].

Clinical manifestations of a common bile duct cyst can be observed with impaired bile duct patency or the development of infectious complications (cholangitis). In the former case, clinical and laboratory findings do not differ from those of biliary atresia. When cholangitis develops, a combination of extrahepatic cholestasis and inflammatory changes is characteristic. The clinical picture includes fever, worsening of the general condition, and other symptoms [30].

In Alagille syndrome, cholestasis appears in the neonatal period, less commonly during the first months of life. There is greenish-tinged jaundice, hepatomegaly, intermittent stool acholia, and dark urine. Later, after 4–6 months of life, a decrease or disappearance of jaundice is observed, along with normalization of stool and urine color. However, pruritus appears, which subsequently intensifies and becomes the leading clinical symptom of the disease, whereas other manifestations remain intermittent [31–34].

Clinical manifestations of progressive familial intrahepatic cholestasis (PFIC) include jaundice and intermittent stool acholia, with early onset of pruritus within the first months of life being characteristic. Most types of PFIC are characterized by low GGT activity [26,27,35]. The main characteristics of various PFIC types are presented in Table 5.

Table 5. Main characteristics of progressive familial intrahepatic cholestasis

In disorders of bile acid synthesis, the onset of initial symptoms occurs in the neonatal period, less commonly during the first months of life. Jaundice, hepatomegaly, dark urine, and intermittent stool acholia are noted. A dissociation is characteristic between low serum GGT and cholesterol levels and elevated levels of other cholestasis markers, along with a significant increase in ALT and AST. Impaired protein-synthetic function of the liver is also characteristic (decreased albumin, cholinesterase, fibrinogen, prothrombin index, and elevated INR and other parameters). Morphological examination of liver biopsy specimens reveals signs of predominantly intrahepatic cholestasis, giant cell transformation of hepatocytes, and disorganization of hepatic lobules. Children also show delayed physical development and conditions associated with fat-soluble vitamin deficiency. Hemorrhagic syndrome may develop.

2. Diagnostics, medical indications and contraindications for diagnostic methods

Criteria for establishing the diagnosis/condition:

Cholestasis syndrome, regardless of etiology, can be diagnosed based on an increase in direct bilirubin by more than 20% of the total bilirubin level or above 17 µmol/L in infants in the first 7 days of life, combined with elevated activities of alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT), cholesterol, and bile acids.

The diagnosis of "neonatal cholestasis" as a complication of severe extrahepatic pathology is established upon identifying predisposing factors and ruling out primary diseases of the liver and bile ducts.

2.1 Complaints and Anamnesis

Comments: parents may report jaundice, pale stools, poor weight and height gain, and regurgitation in the child.

Comments: when taking the medical history, it is necessary to pay attention to a burdened family history (consanguineous marriage, similar cases of the disease in siblings); presence of relatives with liver pathology, renal failure, as well as cases of death from liver failure in the family.

Comments: in patients with cholestasis syndrome at onset, the timing of the appearance of jaundice, acholic stools, hepatomegaly, and splenomegaly should be noted. History-taking reveals pathological conditions of the perinatal period and therapeutic interventions contributing to impaired excretory liver function.

2.2 Physical Examination

Comments: during the examination, the color of the skin and sclera, the size of the liver and spleen, and the color of stool and urine are evaluated. It is necessary to measure the liver along three lines: the right anterior axillary line, the right midclavicular line, and the midline. The spleen is palpated along the left anterior axillary line. In the case of a common bile duct cyst, a space-occupying lesion can be determined in the right hypochondrium upon abdominal palpation.

2.3 Laboratory Diagnostics

Comments: in newborns with severe perinatal pathology, infectious liver damage, as well as the development of hypersplenism, anemia, thrombocytopenia, etc., are observed.

Comments: an increase in cholestasis markers—direct bilirubin level, alkaline phosphatase, gamma-glutamyltransferase, cholesterol, and bile acids—is pathognomonic for cholestasis syndrome. It is important to note that the indication for examining a newborn to rule out biliary atresia and other cholestatic liver diseases is an increase in direct bilirubin above 17 µmol/L in newborns up to two weeks of age, regardless of the total bilirubin level, and in patients older than two weeks of age, a direct bilirubin level > 17 µmol/L with a total bilirubin level of less than 85 µmol/L or greater than 20% of the total bilirubin level when the total bilirubin level is greater than 85 µmol/L. Most types of progressive familial intrahepatic cholestasis (PFIC), as well as congenital bile acid synthesis disorders, are characterized by a dissociation between a low gamma-glutamyltransferase level and an elevation of other cholestasis markers (direct bilirubin, alkaline phosphatase).

In transient neonatal cholestasis, a delayed (relative to cholestasis) increase in cytolysis enzymes by less than 6–8 times is often noted. The ALT/AST ratio is less than 1. Indicators reflecting the synthetic function of the liver (albumin, cholinesterase) generally do not change.

In biliary atresia (BA), the degree of increase in cholestasis markers dynamically progresses from a minimal elevation during the first 2–3 weeks of life to a significant increase by 2–3 months; the activity of cytolysis enzymes (ALT, AST) in BA increases moderately and, as a rule, with a delay. In most cases, during the first 2–3 weeks after birth, these indicators remain within normal limits and then gradually increase; the levels of albumin and cholinesterase, which reflect the synthetic function of the liver, do not change in the early stages of the disease (during the first 3–4 months of life).

Infectious neonatal hepatitis is characterized, in addition to elevated cholestasis markers, by an 8- to 10-fold or greater increase in ALT and AST levels, an ALT/AST ratio greater than 1, as well as impaired synthetic function of the liver, manifested by decreased albumin, cholinesterase, fibrinogen, prothrombin index (PTI), etc. Creatine kinase activity is measured for differential diagnosis with conditions presenting with cytolysis syndrome.

Blood urea and creatinine testing is performed to rule out renal impairment, which may accompany inherited liver diseases such as Alagille syndrome, where renal developmental anomalies frequently lead to renal failure.

Comments: vitamin K-dependent coagulopathy may manifest as a decreased prothrombin index (PTI) (prolonged prothrombin time (PT)) and/or an elevated international normalized ratio (INR) due to impaired intestinal absorption of vitamin K. These changes, along with a decrease in fibrinogen, may occur with the development of hepatocellular insufficiency.

Comments: the test is performed if the facility has the appropriate technical capability.

Comments: the panels may include tests for free triiodothyronine (fT3) in the blood, free thyroxine (fT4) in blood serum, and thyroid-stimulating hormone (TSH) in the blood.

• In newborns with hypoglycemia combined with cholestasis syndrome, it is recommended to perform a comprehensive assay of tropic hormones (blood thyroid-stimulating hormone (TSH), blood adrenocorticotropic hormone, and blood growth hormone) to rule out hypopituitarism [40–44].

LeRR CLoE 4Level of recommendation confidence C (level of evidence – 4)

• All newborns with cholestasis syndrome are recommended to undergo antibody testing for potential hepatitis pathogens using enzyme-linked immunosorbent assay (ELISA) (determination of antibodies to Cytomegalovirus in blood, antibodies to Coxsacki virus in blood, antibodies to Hepatitis С virus in blood, antibodies to Herpes simplex virus in blood, antibodies to Toxoplasma gondii in blood, antibodies to Treponema pallidum in blood to determine the etiology of hepatitis [45–47] [48,49] [98,99].

GCR CLEL 5Grade of Recommendation C (Level of Evidence – 5)

• All newborns with cholestasis syndrome are recommended to undergo PCR blood testing to identify potential pathogens of neonatal hepatitis (quantitative determination of Epstein - Barr virus DNA by PCR in peripheral and cord blood, quantitative determination of Cytomegalovirus DNA by PCR in peripheral and cord blood, quantitative determination of Hepatitis С virus RNA in blood by PCR, determination of Hepatitis С virus antigen in blood, quantitative determination of Hepatitis В virus DNA in blood by PCR, determination of HbsAg of Hepatitis В virus in blood, qualitative determination of Rubella virus RNA by PCR in peripheral and cord blood, quantitative determination of Herpes simplex virus types 1, 2 DNA by PCR in blood, quantitative determination of Toxoplasma gondii DNA by PCR in peripheral and cord blood) [45–47] [48,49,98,99].

LeRR CLoE 4Level of recommendation confidence C (level of evidence – 4)

Comments: the absence of pathological abnormalities according to neonatal screening data does not rule out the presence of metabolic disorders in the child and, in progressive cholestasis syndrome, requires re-testing due to the likelihood of false-negative results. The study is performed if the facility has the appropriate capability.

Comments: the study is performed if the facility has the appropriate capability. The spectrum of organic amino acids in urine should include the determination of bile acid content.

• In all newborns with cholestasis syndrome of unclear etiology, it is recommended to perform a set of studies to evaluate cholestatic syndrome (molecular genetic testing, determination of pathogenic variants in genes responsible for the development of inherited liver diseases by targeted high-throughput sequencing) in order to exclude inherited liver diseases [21,59,61].

GCR CLEL 5Grade of Recommendation C (Level of Evidence – 5)

Comment: if there are signs highly suggestive of any inherited liver disease, targeted gene testing using Sanger sequencing is recommended to detect the pathogenic variant. In the absence of a characteristic clinical presentation, it is advisable to perform an expanded molecular genetic study using high-throughput sequencing of genes associated with hereditary diseases accompanied by cholestasis, or whole-exome sequencing. The study is performed if the facility has the appropriate capability.

Comments: the study is performed if the facility has the appropriate capability.

2.4 Instrumental Diagnostics

Comments: in transient neonatal cholestasis, ultrasound (US) shows non-specific changes such as varying degrees of hepatomegaly and a mild increase in the echogenicity of its parenchyma.

Changes characteristic of biliary atresia (BA) include: the gallbladder is not visualized on an empty stomach or is detected as a hyperechoic "cord"; gallbladder (GB) size is significant: its length is less than 1.5 cm, as well as the presence of the "GB-ghost" triad: length less than 1.9 cm, irregular contour, and absence of a smooth, regular echogenic GB mucosa; no response to feeding throughout the entire interval between feeds; the "triangular cord" sign is detected in the majority of patients with BA, with sensitivity, specificity, and accuracy for BA of 85%, 100%, and 95%, respectively. This is a hyperechoic elongated cord located above the portal vein trunk. The presence of a triangular cord and a fibrous plate thickness greater than 3.4 mm increases the probability of BA. Up to 1–1.5 months of age, the "triangular cord" may not be prominent or may be completely absent in 30–40% of patients with BA, and after 2–2.5 months it is difficult to distinguish from periportal fibrosis, which can also occur in other liver diseases, complicating the diagnosis of BA. Doppler markers of BA: diameter of the right hepatic artery greater than 1.5 mm and the ratio of the right hepatic artery to the portal vein greater than 0.45; the mean diameter of the hepatic artery in patients with BA is significantly larger than in patients with other liver diseases. Additional criteria include dilatation of intrahepatic bile ducts, less frequently cysts in the porta hepatis; in 7–15% of children with BA, splenic fragmentation with fibrous strands in the parenchyma or multiple accessory spleens (polysplenia) are detected.

An ultrasound sign of a bile duct cyst is a cystic lesion in the projection of the common bile duct.

However, in most other inherited liver diseases, characteristic ultrasound signs may be absent.

Comments: in patients with cholestasis syndrome, renal structural features may be one of the extrahepatic manifestations of a number of inherited liver diseases, such as Alagille syndrome, which is characterized by increased echogenicity of the renal cortical parenchyma, reduced differentiation of renal tissue into cortex and medulla due to a decrease in the number of pyramids in a cross-section to 4 or less, as well as structural abnormalities of the pyramids in the form of an elongated shape and various cystic changes in the pyramids and parenchyma [71].

Comments: congenital heart defects may be one of the extrahepatic manifestations of a number of inherited liver diseases; in particular, stenosis of the pulmonary artery and its branches is a major clinical sign of Alagille syndrome.

Comments: vertebral anomalies, particularly "butterfly" vertebrae, are observed in patients with Alagille syndrome.

2.5 Other Procedures

Comments: posterior embryotoxon may be detected in 78–89% of patients with Alagille syndrome.

• In all patients with neonatal cholestasis and suspected biliary atresia, it is recommended to have an initial consultation (examination) with a pediatric surgeon to determine indications for surgical treatment [3,75,76].

GCR CLEL 5Grade of Recommendation C (Level of Evidence – 5)

Comments: if biliary atresia is suspected, consultation with a pediatric surgeon is recommended to address the issue of performing the Kasai portoenterostomy.

• In all patients with neonatal cholestasis of unclear etiology, it is recommended to have an initial consultation (examination) with a geneticist to determine the scope of molecular genetic diagnostic methods [77].

GCR CLEL 5Grade of Recommendation C (Level of Evidence – 5)

3. Treatment, including drug and non-drug therapies, diet therapy, and pain management

3.1 Conservative Treatment

3.1.1. Medical Nutrition

Without adequate bile flow, bile acids cannot be normally secreted into the intestinal lumen. Bile acid salts are an essential component of micelle formation in the lumen of the small intestine. Micelles allow the absorption of insoluble molecules, including most lipids and fat-soluble vitamins. In cholestasis syndrome, bile salt secretion is reduced, leading to inefficient fat absorption. Malabsorption due to cholestasis can lead to growth retardation, weight loss, and deficiencies in fat-soluble vitamins, including vitamin A, vitamin D, vitamin E, and vitamin K [78–81].

• Newborns with cholestasis syndrome are recommended to be fed a specialized infant formula for dietary (therapeutic) use with an increased content of medium-chain triglycerides to prevent lipid metabolism disorders and fat-soluble vitamin deficiencies [82,83].

GDR CLE 5Grade of recommendation C (level of evidence – 5)

Comments: in conditions of fat malabsorption associated with cholestatic syndrome, medium-chain triglycerides are an important component of medical nutrition. Medium-chain triglycerides (MCTs) have greater water solubility and are absorbed in the stomach and small intestine without the involvement of bile acids. The optimal proportion of total lipids as MCTs ranges from 30 to 50%. Higher dietary MCT content (>80%) should be avoided without adequate supplementation of polyunsaturated fatty acids (PUFAs), as this may lead to essential fatty acid (EFA) deficiency. PUFAs are also precursors of eicosanoids, which improve immune function, reduce systemic inflammation, and participate in platelet aggregation. Deficiency of PUFAs and oligounsaturated fatty acids (OUFAs) can result from low intake, impaired fat digestion/malabsorption, and ineffective elongation of OUFA precursors. Deficiency can also be iatrogenic if the diet is very high in MCTs and low in long-chain triglycerides (LCTs).

The development of cholestasis caused by extrahepatic perinatal pathology is most frequently observed in premature infants, including extremely premature infants; therefore, the following factors must be considered when selecting medical nutrition:

• severity and duration of cholestasis;

• gestational and postnatal age of the child;

• impaired digestion and absorption of other components, including proteins and carbohydrates.

In children with severe perinatal pathology, impaired absorption of proteins and carbohydrates may be observed, which, along with fat malabsorption syndrome due to cholestasis, serves as an indication for the use of appropriate medical nutrition. In this case, it is advisable to use a lactose-free formula based on protein hydrolysate containing 50% medium-chain triglycerides. In premature infants, and especially extremely premature infants, the prescription of formulas intended for feeding premature infants (pre-formulas) enriched with medium-chain triglycerides is indicated. It should be noted that there are no contraindications to the use of breast milk. At the same time, exclusive breastfeeding cannot meet the child's demand for essential nutrients and, above all, the lipid component; therefore, it is advisable to combine breast milk with a therapeutic formula under the control of weight dynamics.

3.1.2. Pharmacotherapy

In newborns with transient cholestasis, general therapeutic approaches include: adequate treatment of the underlying disease, exclusion or restriction of potentially hepatotoxic drugs and blood products, early initiation of enteral nutrition, and choleretic therapy. When choosing choleretic therapy, it is necessary to take into account the morphofunctional features of the hepatobiliary system in newborns, which include a high rate of primary bile acid synthesis and immaturity of excretory mechanisms.

Comments: Ursodeoxycholic acid** (ATC code A05AA02) is a tertiary bile acid. Its proposed mechanisms of action include enhancing bile excretion and altering the bile acid composition to be more hydrophilic and less toxic.

Comments: deficiency of fat-soluble vitamins due to steatorrhea is common in chronic cholestasis. These include vitamins A, D, E, and K. Most patients with cholestatic syndrome require supplementation with one or more of these fat-soluble vitamins. Vitamin E deficiency can lead to neurological symptoms, including peripheral neuropathy, cerebellar ataxia, peripheral axonal degeneration, and posterior column neuronopathy. Vitamin A deficiency can lead to ocular diseases, including night blindness, corneal dryness or ulceration, retinopathy, and ultimately blindness. Xerophthalmia and keratomalacia due to vitamin A deficiency have been described in a child with biliary atresia (BA). Vitamin K is essential for the synthesis of several blood clotting factors, including factors II, VII, IX, and X. Vitamin K preparations (ATC code B02BA) are usually administered orally, although patients with severe cholestasis may require parenteral administration. Simultaneous administration of water-soluble vitamin E (ATC code A11HA) with vitamin D and its analogs (ATC code A11CC) may increase vitamin D absorption [79,85].

Fat-soluble vitamins are prescribed when cholestasis lasts for more than 10 days, in the following dosages:

Cholecalciferol** (ATC code A11CC) orally 1000–2500 IU/day for a duration of at least 1 month. Dose adjustment is performed under the control of blood 1,25-OH vitamin D levels.

#Retinol** (ATC code A11CA) orally 3000–10000 IU/day [4].

Vitamin E (ATC code A11HA) orally 50 mg/day if body weight is greater than 1 kg, 25 mg/day if body weight is less than 1 kg.

Vitamin K group (ATC code B02BA) [4,12,86–93]:

#Phytomenadione (ATC code B02BA01) 1% emulsion. The prophylactic dosing regimen ranges from 1 mg/kg once a week in children with a body weight of less than 5 kg to 2.5–5.0 mg 1 to 3 times a week or 10 mg once every 2 weeks in children with a body weight of more than 5 kg. The dose and frequency of prophylaxis are selected individually depending on the coagulogram results and in severe cases may reach daily administration. In the presence of cholestasis or malabsorption syndrome, parenteral administration of vitamin K group preparations (ATC code B02BA) is advisable. In the absence of #phytomenadione (ATC code B02BA01), it is permissible to use #menadione sodium bisulfite** (ATC code B02BA02) 1% solution. It must be borne in mind that its action will begin 8–24 hours after administration.

3.2 Surgical Treatment

Surgical treatment of cholestatic syndrome in the neonatal period is not performed except in cases where the diagnosis of biliary atresia is highly probable, in which the Kasai hepatic portoenterostomy procedure is performed. Currently, a mini-laparotomy approach or laparoscopic intervention is used.

Liver transplantation is not performed in the neonatal period.

Treatment of a choledochal cyst is exclusively surgical—complete excision of the cyst with the formation of a biliodigestive anastomosis using an isolated Roux-en-Y jejunal loop [30].

3.3 Other Treatment

Not applicable.

4. Medical Rehabilitation and Sanatorium-Resort Treatment

Carried out by specialized professionals (pediatrician, gastroenterologist, ophthalmologist, pediatric surgeon, etc.) depending on the concomitant pathology and complications.

5. Prevention and Dispensary Observation

• For children with cholestatic syndrome due to hereditary liver diseases, dispensary follow-up (examination, consultation) by a pediatrician is recommended for the prevention of disease complications [1,36].

GDR CLE 5Grade of recommendation C (level of evidence – 5)

Comments: after discharge from the hospital, dispensary monitoring is recommended: physical examination by a specialist (pediatrician, gastroenterologist, pediatric surgeon, etc.) who observes the patient, assessing the manifestations of the disease every 1–3 months during the first year of life, and then every 3–6 months in the second year of life. At each specialist visit, disease activity is monitored: complete (clinical) blood count and general (clinical) urinalysis (proteinuria), general biochemical blood test (investigation of total blood protein levels, blood albumin levels, total blood bilirubin levels, free (unconjugated) blood bilirubin levels, bound (conjugated) blood bilirubin levels, blood triglyceride levels, blood cholesterol levels; determination of blood creatine kinase activity, aspartate aminotransferase activity, alanine aminotransferase activity, gamma-glutamyl transferase activity, alkaline phosphatase activity), preliminary hemostasis testing with determination of prothrombin index, fibrinogen levels, prothrombin (thromboplastin) time in blood, activated partial thromboplastin time, thrombin time in blood, international normalized ratio (INR).

• For children with cholestatic syndrome due to hereditary liver diseases, a repeat consultation (examination) by a geneticist is recommended for medical genetic counseling [77,94].

GDR CLE 5Grade of recommendation C (level of evidence – 5)

Comments: a geneticist consultation is recommended for parents after a diagnosis is established in a child, in order to provide genetic counseling regarding the genetic risk of having children with hereditary disorders in this family.

• Children with cholestasis syndrome are recommended to receive vaccination to prevent infectious diseases [95].

GCR CLEL 5Grade of Recommendation C (Level of Evidence – 5)

Comments: newborns with neonatal cholestasis are recommended to be vaccinated in accordance with the National Immunization Schedule, provided there are no contraindications from other organs and systems. To prevent viral hepatitis B, all newborns are administered hepatitis B vaccine** (ATX code J07BC01) within the first 24 hours of life, followed by doses at 1 and 6 months of age. If the mother is an HBsAg carrier, an additional vaccination is administered at 12 months of age.

6. Organization of Medical Care

Indications for hospitalization in a medical organization:

1) Severe, unstable condition of the child.

2) Need for correction of hypoalbuminemia.

Indications for patient discharge from a medical organization

1) Stable, satisfactory condition of the child.

2) Stabilization of laboratory parameters, absence of hypoalbuminemia.

3) Absence of contraindications to discharge.

If a malformation of the biliary tract is suspected, the patient is referred to a specialized surgical department for additional diagnostic methods and inpatient surgical treatment.

7. Additional Information

Outcomes of cholestasis syndrome depend on the etiology of the disease.

Criteria for Evaluating the Quality of Medical Care

References

Appendix A1. Working Group Composition

1. Marina B. Albegova – PhD in Medical Sciences, Head of the Consultative and Pediatric Department, Institute of Neonatology and Pediatrics, National Medical Research Center for Obstetrics, Gynecology and Perinatology named after Academician V.I. Kulakov of the Russian Ministry of Health.

2. Madlena E. Bagaeva – Senior Research Scientist, Department of Pediatric Gastroenterology, Hepatology and Dietotherapy, Federal Research Center of Nutrition and Biotechnology, Associate Professor, Department of Gastroenterology and Dietology, Pirogov Russian National Research Medical University of the Russian Ministry of Health.

3. Ekaterina N. Balashova – PhD in Medical Sciences, Leading Research Scientist, Prof. A.G. Antonov ICU, Institute of Neonatology and Pediatrics, National Medical Research Center for Obstetrics, Gynecology and Perinatology named after Academician V.I. Kulakov of the Russian Ministry of Health, Associate Professor, Department of Neonatology, Board Member of the Russian Society of Neonatologists.

4. Nikolay N. Volodin – Academician of the Russian Academy of Sciences, Professor, MD; President of the Russian Association of Perinatal Medicine Specialists, President of the Association of Specialists in Perinatal Medicine, Healthcare Provision for Pregnant Women, Fetuses, Newborns, and Children under 3 Years of Age (ASPM+).

5. Marina S. Gothie – Pediatrician, Consultative and Pediatric Department, Institute of Neonatology and Pediatrics, National Medical Research Center for Obstetrics, Gynecology and Perinatology named after Academician V.I. Kulakov of the Russian Ministry of Health.

6. Dmitry N. Degtyarev – MD, Professor, Deputy Director for Research, National Medical Research Center for Obstetrics, Gynecology and Perinatology named after Academician V.I. Kulakov of the Russian Ministry of Health, Head of the Department of Neonatology, Pediatric Faculty, I.M. Sechenov First Moscow State Medical University of the Russian Ministry of Health (Sechenov University), Board Member of the Russian Society of Neonatologists.

7. Anna V. Degtyareva – MD, PhD, Professor, Head of the Department of Pediatrics, Institute of Neonatology and Pediatrics, National Medical Research Center for Obstetrics, Gynecology and Perinatology named after Academician V.I. Kulakov of the Ministry of Health of Russia; Professor, Department of Neonatology, Pediatric Faculty, I.M. Sechenov First Moscow State Medical University of the Ministry of Health of Russia (Sechenov University); Member of the Council of the Russian Society of Neonatologists.

8. Alina A. Dokshukina – Medical Geneticist, Consultative and Pediatric Department, Institute of Neonatology and Pediatrics, National Medical Research Center for Obstetrics, Gynecology and Perinatology named after Academician V.I. Kulakov of the Ministry of Health of Russia.

9. Svetlana I. Zhdanova – PhD, Assistant Professor, Department of Hospital Pediatrics, Kazan State Medical University of the Ministry of Health of Russia; Neonatologist, Newborn Department, Republican Clinical Hospital of the Ministry of Health of the Republic of Tatarstan (Kazan); Specialist, Department of Nutritional Technologies, Institute of Neonatology and Pediatrics, National Medical Research Center for Obstetrics, Gynecology and Perinatology named after Academician V.I. Kulakov of the Ministry of Health of Russia.

10. Ekaterina Yu. Zakharova – MD, PhD, Head of the Laboratory of Hereditary Metabolic Diseases and Laboratory of Selective Screening, Research Centre for Medical Genetics named after Academician N.P. Bochkov.

11. Viktor V. Zubkov – MD, PhD, Director of the Institute of Neonatology and Pediatrics, Head of the Department of Neonatology, National Medical Research Center for Obstetrics, Gynecology and Perinatology named after Academician V.I. Kulakov of the Ministry of Health of Russia; Professor, Department of Neonatology, N.F. Filatov Clinical Institute of Children's Health, I.M. Sechenov First Moscow State Medical University of the Ministry of Health of Russia (Sechenov University); Member of the Council of the Russian Society of Neonatologists.

12. Anna A. Puchkova – PhD, Head of Clinical Work, Scientific and Consultative Pediatric Department, Institute of Neonatology and Pediatrics, National Medical Research Center for Obstetrics, Gynecology and Perinatology named after Academician V.I. Kulakov of the Ministry of Health of Russia.

13. Tatyana V. Strokova – MD, PhD, Professor of the Russian Academy of Sciences, Head of the Department of Pediatric Gastroenterology, Hepatology and Dietotherapy, Clinic of the Federal Research Centre of Nutrition and Biotechnology; Head of the Department of Gastroenterology and Dietotherapy, Faculty of Continuous Professional Education, Pirogov Russian National Research Medical University of the Ministry of Health of Russia.

14. Natalia N. Taran – Senior Researcher, Department of Pediatric Gastroenterology, Hepatology and Dietotherapy, Federal Research Centre of Nutrition and Biotechnology; Assistant Professor, Department of Gastroenterology and Dietotherapy, Pirogov Russian National Research Medical University of the Ministry of Health of Russia.

15. Elena L. Tumanova – MD, PhD, Professor, Honored Doctor of the Russian Federation, Chief freelance specialist in pathological anatomy (pediatric network) of Moscow, Head of the Department of Pathology and Clinical Pathology, Pediatric Faculty, Pirogov Russian National Research Medical University of the Ministry of Health of Russia; Head of the Department of Pathology, N.F. Filatov Children's City Clinical Hospital of the Moscow Healthcare Department.

16. Elena A. Filippova – PhD, Head of the Department of Ultrasound Diagnostics in Neonatology and Pediatrics, National Medical Research Center for Obstetrics, Gynecology and Perinatology named after Academician V.I. Kulakov of the Ministry of Health of Russia; Associate Professor, Department of Pediatric Diagnostic Radiology, Russian Medical Academy of Continuous Professional Education of the Ministry of Health of Russia.

There is no conflict of interest.

Appendix A2. Methodology for Developing Clinical Guidelines

Methods used for collecting/selecting evidence: electronic database search.

Description of methods used for evidence collection/selection: the evidence base for the guidelines consisted of publications included in the Cochrane Library, PubMed, EMBASE, MEDLINE, Scopus, Web of Science, e-Library, clinicaltrials.gov databases, electronic libraries, and clinical guidelines published by The National Institute for Health and Care Excellence, The European Association of Perinatal Medicine, The European Society for Pediatric Research, The European Foundation for the Care of Newborn Infants, and The European Society for Neonatology. The search depth was 43 years.

Methods used for assessing the quality and strength of evidence:

• expert consensus;

• assessment of significance in accordance with the rating scheme.

Scales for assessing levels of evidence (LE) (Tables 1, 2) for diagnostic, preventive, therapeutic, and rehabilitation methods (diagnostic, preventive, therapeutic, rehabilitation interventions) with definitions, and the scale for assessing grades of recommendation (GR) (Table 3) for preventive, diagnostic, therapeutic, and rehabilitation methods (preventive, diagnostic, therapeutic, rehabilitation interventions).

Methods used for analyzing evidence:

• reviews of published meta-analyses;

• systematic reviews with evidence tables.

Description of methods used for analysis of evidence.

When selecting publications as potential sources of evidence, the methodology used in each study was reviewed to ensure its validity. Methodological evaluation was based on questions focused on study design features that significantly affect the validity of the results and conclusions.

To minimize potential subjective errors, each study was evaluated independently by at least two members of the working group. Any discrepancies in assessments were discussed by the full working group. If consensus could not be reached, an independent expert was involved.

Economic analysis:

Cost analysis was not performed, and pharmacoeconomic publications were not evaluated.

Method of guidelines validation:

• external expert review;

• internal expert review.

Description of the guidelines validation method:

The preliminary version of these guidelines was reviewed by independent experts who were asked to comment primarily on whether the interpretation of the evidence underlying the recommendations was accessible and understandable.

Comments were received from primary care physicians and district pediatricians regarding the clarity of the presentation of the guidelines and their assessment of the importance of the guidelines as a working tool in daily practice.

The draft version was also sent to a non-medically trained reviewer to obtain comments from a patient perspective.

Comments received from experts were carefully systematized and discussed by the chair and members of the working group. Each point was discussed, and resulting modifications to the guidelines were recorded. If no changes were made, the reasons for declining the changes were recorded.

Consultation and peer review:

Recent changes to these guidelines are presented as a draft version for discussion. The updated version for broad discussion was published on the websites of the Russian Society of Neonatologists (RSN) www.neonatology.pro, www.raspm.ru, and https://portalcr.minzdrav.gov.ru/ so that all stakeholders could participate in discussing and improving the guidelines.

The draft guidelines were reviewed by independent experts who were asked to comment primarily on the clarity and accuracy of the interpretation of the evidence base underlying the recommendations.

For the final revision and quality control, the recommendations were re-evaluated by the members of the working group, who concluded that all comments and feedback from the experts had been taken into account and the risk of bias in the development of the recommendations was minimized.

Target audience for these clinical guidelines:

1. neonatologists,

2. anesthesiologists and intensivists,

3. pediatricians.

Table 1.Scale for assessing levels of evidence (LE) for diagnostic methods (diagnostic interventions)

Table 2. Levels of Evidence (LoE) grading scale for prevention, treatment, and medical rehabilitation methods, including those based on natural therapeutic factors (preventive, therapeutic, and rehabilitation interventions)

Table 3. Scale for assessing grades of recommendation (GR) for prevention, diagnostic, treatment, and medical rehabilitation methods, including those based on natural therapeutic factors (preventive, diagnostic, therapeutic, and rehabilitative interventions)

Procedure for updating clinical guidelines.

The mechanism for updating clinical guidelines provides for their systematic revision at least once every three years, as well as upon the emergence of new evidence-based medicine data on the diagnosis, treatment, prevention, and rehabilitation of specific diseases, or in the presence of justified additions/comments to previously approved clinical guidelines, but no more frequently than once every 6 months.

Appendix A3. Reference Materials

Appendix A3.1 Related Documents

These clinical guidelines have been developed taking into account the following regulatory and legal documents:

1. Federal Law No. 323-FZ dated November 21, 2011, "On the Basics of Protecting the Health of Citizens in the Russian Federation".

2. Order of the Ministry of Health of Russia No. 203n dated May 10, 2017, "On Approval of Criteria for Assessing the Quality of Medical Care".

3. Order of the Ministry of Health of Russia No. 274n dated April 21, 2022, "On Approval of the Procedure for Providing Medical Care to Patients with Congenital and (or) Hereditary Diseases."

4. International Statistical Classification of Diseases and Related Health Problems, 10th Revision (ICD-10) (World Health Organization), 2019 version.

5. Order of the Russian Ministry of Health No. 804n dated October 13, 2017, "On Approval of the Nomenclature of Medical Services."

6. Decree of the Government of the Russian Federation No. 2406-r dated October 12, 2019, "On Approval of the List of Vital and Essential Medicines, as well as Lists of Medicines for Medical Use and the Minimum Assortment of Medicines Required for Medical Care".

7. State Register of Medicines: https://grls.rosminzdrav.ru

8. Decree of the Government of the Russian Federation No. 3551-r dated December 11, 2023, "On Approval of the List of Specialized Medical Nutrition Products for Disabled Children".

9. Order of the Russian Ministry of Health dated April 17, 2025 No. 222n "On Approval of the Procedure for Providing Medical Care in the Profile 'Neonatology'" (Registered with the Russian Ministry of Justice on June 2, 2025, No. 82516).

Appendix B. Physician Action Algorithms

Figure 1 Algorithm for differential diagnosis of liver diseases presenting with neonatal cholestasis.

Appendix C. Patient Information

What is neonatal cholestasis?

Neonatal cholestasis is a condition in infants where bile flow from the liver is impaired. Bile is a dark green or yellowish-brown fluid produced by liver cells. Bile flows from the liver into the bile ducts, gallbladder, and then into the intestine, aiding digestion. Cholestasis occurs either due to liver disease or when there is an obstruction to the flow of bile from the liver into the intestine. When cholestasis is detected at birth or shortly thereafter, it is called neonatal.

What causes cholestasis?

Cholestasis can be caused by many conditions, including infections, anatomical, genetic, metabolic, and immune disorders. Cholestasis can also be caused by medications and toxins. However, in some cases, the cause of cholestasis remains unknown. One of the most common causes of cholestasis is biliary atresia, in which the bile ducts are malformed or damaged. Other conditions that can cause neonatal cholestasis include infections (e.g., cytomegalovirus) or hereditary diseases (e.g., progressive familial intrahepatic cholestasis, Alagille syndrome, cystic fibrosis, alpha-1 antitrypsin deficiency, and others). Your child's doctor will perform an evaluation to determine the cause of neonatal cholestasis.

What are some of the consequences of cholestasis in newborns?

The liver is important for removing toxic substances from the body and also produces proteins such as albumin and clotting factors, which are essential for life. Bile is also important for nutrition by helping to absorb fats and fat-soluble vitamins. Infants with cholestasis may have difficulty digesting food, leading to poor weight and height gain. In severe cases, cholestasis can lead to liver cirrhosis and require a liver transplant.

What are the symptoms of cholestasis in newborns?

Yellow discoloration of the skin and the whites of the eyes, called jaundice, is often one of the first signs of cholestasis. Although most cases of neonatal jaundice are not caused by liver disease, they should be monitored, especially if an infant with jaundice has dark-colored urine and light or pale stools. Later symptoms of cholestasis include fatigue, poor feeding, difficulty gaining weight, and skin itching (pruritus). With progressive liver disease, the child may develop signs of ascites (fluid accumulation in the abdomen) and may experience bleeding from blood collection sites, the gastrointestinal tract, and other organs due to decreased blood clotting.

What should be noted at the birth of a newborn?

Jaundice combined with dark urine or light/pale stools. Jaundice associated with other abnormalities in the body, including the heart, spleen, skin, eyes, and skeleton.

How do doctors evaluate cholestasis in newborns?

If your newborn has these symptoms, they will be examined by a neonatologist and a surgeon. The doctor may prescribe additional tests: blood and urine tests, ultrasound of the liver, gallbladder, and other organs. In some patients, a liver biopsy is required for diagnosis. A liver biopsy involves taking a small piece of liver tissue using a small needle while your child is under anesthesia.

What is the treatment for neonatal cholestasis?

Your child may be prescribed special formulas, which are necessary to improve digestion and weight gain, and can be combined with breast milk. Breastfeeding is contraindicated only in rare cases. In addition, infants with cholestasis syndrome develop a deficiency of fat-soluble vitamins (A, D, E, K) due to impaired fat absorption, and therefore patients receive fat-soluble vitamin supplementation. Medications to improve bile flow will also be prescribed. In some infants, the disease progresses and causes cirrhosis, which may require a liver transplant.

What is the prognosis for neonatal cholestasis?

The outcome of neonatal cholestasis varies depending on the underlying cause. Some patients improve over time, while others may worsen. For certain conditions, medications or surgical interventions are available that lead to recovery or clinical and laboratory remission, where disease manifestations normalize or decrease during treatment. After your child's physician completes the necessary studies, the treatment plan and prognosis will be discussed in more detail.

Appendix G. Assessment Scales, Questionnaires, and Other Evaluation Tools

Not applicable.