Jenny Chambers

Contribution of Variant Alleles of ABCB11 to Susceptibility to Intrahepatic Cholestasis of Pregnancy.

Background: Intrahepatic cholestasis of pregnancy (ICP) has a complex aetiology with a significant genetic component. ABCB11 encodes the bile salt export pump (BSEP); mutations cause a spectrum of cholestatic disease, and are implicated in the aetiology of ICP. Methods: ABCB11 variation in ICP was investigated by screening for five mutant alleles (E297G, D482G, N591S, D676Y and G855R) and the V444A polymorphism (c.1331T>C, rs2287622) in two ICP cohorts (n = 333 UK, n = 158 continental Europe), and controls (n = 261) for V444A. PCR primers were used to amplify and sequence patient and control DNA. The molecular basis for the observed phenotypes was investigated in silico by analysing the equivalent residues in the structure of the homologous bacterial transporter Sav1866. Results: E297G was observed four times and D482G once. N591S was present in two patients; D676Y and G855R were not observed. The V444A polymorphism was associated with ICP (allelic analysis for C vs T: OR 1.7 (95% CI 1.4 to 2.1, p<0.001)). In addition, CC homozygotes were more likely to have ICP than TT homozygotes: OR 2.8 (95% CI 1.7 to 4.4 p<0.0001). Structural analyses suggest that E297G and D482G destabilise the protein fold of BSEP. The molecular basis of V444A and N591S was not apparent from the Sav1866 structure. Conclusions: Heterozygosity for the common ABCB11 mutations accounts for 1% of European ICP cases; these two mutants probably reduce the folding efficiency of BSEP. N591S is a recurrent mutation; however, the mechanism may be independent of protein stability or function. The V444A polymorphism is a significant risk factor for ICP in this population.

Ursodeoxycholic acid versus placebo, and early term delivery versus expectant management, in women with intrahepatic cholestasis of pregnancy: semifactorial randomised clinical trial

Objectives To test whether ursodeoxycholic acid reduces pruritus in women with intrahepatic cholestasis of pregnancy, whether early term delivery does not increase the incidence of caesarean section, and the feasibility of recruiting women with intrahepatic cholestasis of pregnancy to trials of these interventions. Design First phase of a semifactorial randomised controlled trial. Setting Nine consultant led maternity units, United Kingdom. Participants 125 women with intrahepatic cholestasis of pregnancy (pruritus and raised levels of serum bile acids) or pruritus and raised alanine transaminase levels (>100 IU/L) recruited after 24 weeks’ gestation and followed until delivery. 56 women were randomised to ursodeoxycholic acid, 55 to placebo, 30 to early term delivery, and 32 to expectant management. Interventions Ursodeoxycholic acid 500 mg twice daily or placebo increased as necessary for symptomatic or biochemical improvement until delivery; early term delivery (induction or delivery started between 37+0 and 37+6) or expectant management (spontaneous labour awaited until 40 weeks’ gestation or caesarean section undertaken by normal obstetric guidelines, usually after 39 weeks’ gestation). Main outcome measures The primary outcome for ursodeoxycholic acid was maternal itch (arithmetic mean of measures (100 mm visual analogue scale) of worst itch in past 24 hours) and for the timing of delivery was caesarean section. Secondary outcomes were other maternal and perinatal outcomes and recruitment rates. Results Ursodeoxycholic acid reduced itching by −16 mm (95% confidence interval −27 mm to −6 mm), less than the 30 mm difference prespecified by clinicians and women as clinically meaningful. 32% (14/44) of women randomised to ursodeoxycholic acid experienced a reduction in worst itching by at least 30 mm compared with 16% (6/37) randomised to placebo. The difference of 16% (95% confidence interval −3 to 34); this would represent a number needed to treat of 6, but it failed to reach significance. Early term delivery did not increase caesarean sections (7/30 (23%) in the early term delivery group versus 11/32 (33%) in the expectant management group (relative risk 0.70, 95% confidence interval 0.31 to 1.57). No serious harms were noted in either trial. 22% (73/325) of eligible women participated in the drug trial and 19% (39/209) in the timing of delivery trial; both groups had a similar spectrum of disease severity to non-participants. Conclusions Ursodeoxycholic acid significantly reduces pruritus, but the size of the benefit may be too small for most doctors to recommend it, or for most women to want to take it. Women are, however, likely to differ in whether they consider the benefit to be worthwhile. Planned early term delivery seems not to increase incidence of caesarean section, although a small increase cannot be excluded. A trial to test whether ursodeoxycholic acid reduces adverse perinatal outcomes would have to be large, but is feasible. A trial to test the effect of early term delivery on adverse fetal outcomes would have to be significantly larger and may not be feasible. Trial registration Current Controlled Trials ISRCTN37730443.

ABCB4 gene sequence variation in women with intrahepatic cholestasis of pregnancy

Intrahepatic cholestasis of pregnancy (ICP), also known as obstetric cholestasis, is a liver disease of pregnancy that complicates 0.7% of pregnancies in the UK.1,2 ICP causes maternal pruritus and hepatic impairment and can cause fetal death, spontaneous prematurity, and sudden intrauterine death.3–6 A diagnosis of ICP is made by the demonstration of abnormal liver function test results, and in particular the serum bile acids are raised.7–10 This is thought to be a consequence of abnormal bile transport across the hepatocyte canalicular membrane. Clinical features are heterogeneous and the aetiology is likely to be complex. Insights to the genetic aetiology of ICP have come from studies of the childhood liver disease progressive familial intrahepatic cholestasis (PFIC), a condition which is divided into three subtypes. Children with PFIC1 and 2 have low concentrations of biliary bile acids and low to normal gamma-glutamyl transpeptidase (GGT) in the serum. PFIC3 patients have high serum levels of GGT and bile which lacks phospholipid but has a normal biliary bile acid concentration.11 Homozygous mutations of the ABCB4 (also called MDR3 or mdr2 in the mouse) gene have been described in pedigrees with PFIC3.11–13 The ABCB4 protein is a member of the ATP binding cassette (ABC) family of membrane transporters.14–16 One of the normal functions of ABCB4 is to transport phosphatidylcholine across the hepatocyte canalicular membrane. The fact that expression is not only found in hepatocytes but also in B lymphocytes, heart, and muscle suggests that it may also transport other substrates. However, homozygous knockouts of the homologous (>90% identity at the amino acid level) murine mdr2 only had hepatic effects.17 Several heterozygous mothers of children with PFIC3 have symptoms consistent with ICP.11–13 In a large consanguineous pedigree with coexisting PFIC3 and ICP, …

Intrahepatic cholestasis of pregnancy levels of sulfated progesterone metabolites inhibit farnesoid X receptor resulting in a cholestatic phenotype

Intrahepatic cholestasis of pregnancy (ICP) is the most prevalent pregnancy‐specific liver disease and is associated with an increased risk of adverse fetal outcomes, including preterm labor and intrauterine death. The endocrine signals that cause cholestasis are not known but 3α‐sulfated progesterone metabolites have been shown to be elevated in ICP, leading us to study the impact of sulfated progesterone metabolites on farnesoid X receptor (FXR)‐mediated bile acid homeostasis pathways. Here we report that the 3β‐sulfated progesterone metabolite epiallopregnanolone sulfate is supraphysiologically raised in the serum of ICP patients. Mice challenged with cholic acid developed hypercholanemia and a hepatic gene expression profile indicative of FXR activation. However, coadministration of epiallopregnanolone sulfate with cholic acid exacerbated the hypercholanemia and resulted in aberrant gene expression profiles for hepatic bile acid‐responsive genes consistent with cholestasis. We demonstrate that levels of epiallopregnanolone sulfate found in ICP can function as a partial agonist for FXR, resulting in the aberrant expression of bile acid homeostasis genes in hepatoma cell lines and primary human hepatocytes. Furthermore, epiallopregnanolone sulfate inhibition of FXR results in reduced FXR‐mediated bile acid efflux and secreted FGF19. Using cofactor recruitment assays, we show that epiallopregnanolone sulfate competitively inhibits bile acid‐mediated recruitment of cofactor motifs to the FXR‐ligand binding domain. Conclusion: Our results reveal a novel molecular interaction between ICP‐associated levels of the 3β‐sulfated progesterone metabolite epiallopregnanolone sulfate and FXR that couples the endocrine component of pregnancy in ICP to abnormal bile acid homeostasis. (HEPATOLOGY 2013;)

A placental phenotype for intrahepatic cholestasis of pregnancy

Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy specific liver disease associated with significant risk of fetal complications. It is hypothesised that the risk of adverse fetal outcomes relates to the toxic effects of bile acids, the levels of which are increased in both maternal and fetal serum. Human and rodent studies have shown that transplacental transfer of bile acids is impaired in ICP. Furthermore, the morphology of placentas from the rodent model of ICP is markedly abnormal, and is associated with increased expression of apoptotic markers and oxidative stress. Using placental tissue from ICP cases and normal pregnancies and cultured placental explant fragments we investigated the histological and molecular effects of cholestasis. We also examined the influence of ursodeoxycholic acid (UDCA) administration on these parameters. Here we report that ICP is associated with several morphological abnormalities of the placenta, including an increase in the number of syncytial knots, and that these can be reproduced in an in vitro (explant) model exposed to the bile acids taurocholic acid and taurochenodoexycholic acid. Furthermore, we demonstrate that ursodeoxycholic acid, a drug commonly used in the management of ICP, has a protective effect on placental tissue both in vivo and in vitro.

Obstetric cholestasis with elevated gamma glutamyl transpeptidase: Incidence, presentation and treatment

Background:  Obstetric cholestasis (OC) may cause severe pruritus in the mother and lead to fetal distress and stillbirth. The etiology of OC is multifactorial, but includes inherited dysfunction of bile canalicular transporters. One of these, multidrug resistant protein 3 (MDR3), a phospholipid transporter, when dysfunctional is associated with elevated levels of gamma glutamyl transpeptidase (GGT). The aim of the present study was to assess the incidence of OC associated with elevated GGT. We compared the natural history of a cholestatic pregnancy and the efficacy of ursodeoxycholic acid (URSO) in OC patients grouped according to a normal or raised GGT level.

Prognostic and mechanistic potential of progesterone sulfates in intrahepatic cholestasis of pregnancy and pruritus gravidarum

A challenge in obstetrics is to distinguish pathological symptoms from those associated with normal changes of pregnancy, typified by the need to differentiate whether gestational pruritus of the skin is an early symptom of intrahepatic cholestasis of pregnancy (ICP) or due to benign pruritus gravidarum. ICP is characterized by raised serum bile acids and complicated by spontaneous preterm labor and stillbirth. A biomarker for ICP would be invaluable for early diagnosis and treatment and to enable its differentiation from other maternal diseases. Three progesterone sulfate compounds, whose concentrations have not previously been studied, were newly synthesized and assayed in the serum of three groups of ICP patients and found to be significantly higher in ICP at 9‐15 weeks of gestation and prior to symptom onset (group 1 cases/samples: ICP n = 35/80, uncomplicated pregnancy = 29/100), demonstrating that all three progesterone sulfates are prognostic for ICP. Concentrations of progesterone sulfates were associated with itch severity and, in combination with autotaxin, distinguished pregnant women with itch that would subsequently develop ICP from pruritus gravidarum (group 2: ICP n = 41, pruritus gravidarum n = 14). In a third group of first‐trimester samples all progesterone sulfates were significantly elevated in serum from low‐risk asymptomatic women who subsequently developed ICP (ICP/uncomplicated pregnancy n = 54/51). Finally, we show mechanistically that progesterone sulfates mediate itch by evoking a Tgr5‐dependent scratch response in mice. Conclusion: Our discovery that sulfated progesterone metabolites are a prognostic indicator for ICP will help predict onset of ICP and distinguish it from benign pruritus gravidarum, enabling targeted obstetric care to a high‐risk population. Delineation of a progesterone sulfate‐TGR5 pruritus axis identifies a therapeutic target for itch management in ICP. (Hepatology 2016;63:1287–1298)

Autotaxin activity has a high accuracy to diagnose intrahepatic cholestasis of pregnancy

BACKGROUND & AIMS Intrahepatic cholestasis of pregnancy (ICP) is defined by pruritus, elevated total fasting serum bile salts (TBS) and transaminases, and an increased risk of adverse fetal outcome. An accurate diagnostic marker is needed. Increased serum autotaxin correlates with cholestasis-associated pruritus. We aimed at unraveling the diagnostic accuracy of autotaxin in ICP. METHODS Serum samples and placental tissue were collected from 44 women with uncomplicated pregnancies and 105 with pruritus and/or elevated serum transaminases. Autotaxin serum levels were quantified enzymatically and by Western blotting, autotaxin gene expression by quantitative PCR. RESULTS Serum autotaxin was increased in ICP (mean ± SD: 43.5 ± 18.2 nmol ml(-1)min(-1), n=55, p<0.0001) compared to other pruritic disorders of pregnancy (16.8 ± 6.7 nmol ml(-1)min(-1), n=33), pre-eclampsia complicated by HELLP-syndrome (16.8 ± 8.9 nmol ml(-1)min(-1), n=17), and pregnant controls (19.6 ± 5.7 nmol ml(-1)min(-1), n=44). Longitudinal analysis during pregnancy revealed a marked rise in serum autotaxin with onset of ICP-related pruritus. Serum autotaxin was increased in women taking oral contraceptives. Increased serum autotaxin during ICP was not associated with increased autotaxin mRNA in placenta. With a cut-off value of 27.0 nmol ml(-1)min(-1), autotaxin had an excellent sensitivity and specificity in distinguishing ICP from other pruritic disorders or pre-eclampsia/HELLP-syndrome. Serum autotaxin displayed no circadian rhythm and was not influenced by food intake. CONCLUSIONS Increased serum autotaxin activity represents a highly sensitive, specific and robust diagnostic marker of ICP, distinguishing ICP from other pruritic disorders of pregnancy and pregnancy-related liver diseases. Pregnancy and oral contraception increase serum autotaxin to a much lesser extent than ICP.

The Reversed Feto-Maternal Bile Acid Gradient in Intrahepatic Cholestasis of Pregnancy Is Corrected by Ursodeoxycholic Acid

Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy-specific liver disorder associated with an increased risk of adverse fetal outcomes. It is characterised by raised maternal serum bile acids, which are believed to cause the adverse outcomes. ICP is commonly treated with ursodeoxycholic acid (UDCA). This study aimed to determine the fetal and maternal bile acid profiles in normal and ICP pregnancies, and to examine the effect of UDCA treatment. Matched maternal and umbilical cord serum samples were collected from untreated ICP (n = 18), UDCA-treated ICP (n = 46) and uncomplicated pregnancy (n = 15) cases at the time of delivery. Nineteen individual bile acids were measured using HPLC-MS/MS. Maternal and fetal serum bile acids are significantly raised in ICP compared with normal pregnancy (p = <0.0001 and <0.05, respectively), predominantly due to increased levels of conjugated cholic and chenodeoxycholic acid. There are no differences between the umbilical cord artery and cord vein levels of the major bile acid species. The feto-maternal gradient of bile acids is reversed in ICP. Treatment with UDCA significantly reduces serum bile acids in the maternal compartment (p = <0.0001), thereby reducing the feto-maternal transplacental gradient. UDCA-treatment does not cause a clinically important increase in lithocholic acid (LCA) concentrations. ICP is associated with significant quantitative and qualitative changes in the maternal and fetal bile acid pools. Treatment with UDCA reduces the level of bile acids in both compartments and reverses the qualitative changes. We have not found evidence to support the suggestion that UDCA treatment increases fetal LCA concentrations to deleterious levels.

Rifampicin in the treatment of severe intrahepatic cholestasis of pregnancy.

OBJECTIVE To describe the use of combined ursodeoxycholic acid (UDCA) and rifampicin treatment in intrahepatic cholestasis of pregnancy (ICP). STUDY DESIGN A questionnaire survey of 27 women with 28 affected pregnancies identified via the UK and International Obstetric Medicine forum. The clinical case notes of women with ICP treated with combined UDCA and rifampicin therapy were reviewed, and data regarding maternal and perinatal outcomes extracted. RESULTS Serum bile acids remained high whilst taking UDCA as monotherapy. In 14 pregnancies (54%) serum bile acids decreased following the introduction of rifampicin. In 10 pregnancies (38%), there was a 50% reduction in serum bile acids. There were no adverse effects reported with either drug. CONCLUSIONS This is the first report of the use of rifampicin in ICP. The data suggest that combined treatment with UDCA and rifampicin is an effective way of treating women with severe ICP who do not respond to treatment with UDCA alone.