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British Journal of Clinical Pharmacology logoLink to British Journal of Clinical Pharmacology
. 2015 Jan 20;79(2):316–329. doi: 10.1111/bcp.12480

Effect of ursodeoxycholic acid treatment on the altered progesterone and bile acid homeostasis in the mother-placenta-foetus trio during cholestasis of pregnancy

Maria C Estiú 1, Maria J Monte 2, Laura Rivas 2, Maria Moirón 1, Laura Gomez-Rodriguez 3, Tomas Rodriguez-Bravo 3, Jose JG Marin 2, Rocio IR Macias 2,✉
PMCID: PMC4309637  PMID: 25099365

Abstract

Aim

Intrahepatic cholestasis of pregnancy (ICP) is characterized by pruritus and elevated bile acid concentrations in maternal serum. This is accompanied by an enhanced risk of intra-uterine and perinatal complications. High concentrations of sulphated progesterone metabolites (PMS) have been suggested to be involved in the multifactorial aetiopathogenesis of ICP. The aim of this study was to investigate further the mechanism accounting for the beneficial effect of oral administration of ursodeoxycholic acid (UDCA), which is the standard treatment, regarding bile acid and PMS homeostasis in the mother-placenta-foetus trio.

Method

Using HPLC-MS/MS bile acids and PMS were determined in maternal and foetal serum and placenta. The expression of ABC proteins in placenta was determined by real time quantitative PCR (RT-QPCR) and immunofluorescence.

Results

In ICP, markedly increased concentrations of bile acids (tauroconjugates > glycoconjugates >> unconjugated), progesterone and PMS in placenta and maternal serum were accompanied by enhanced concentrations in foetal serum of bile acids, but not of PMS. UDCA treatment reduced bile acid accumulation in the mother-placenta-foetus trio, but had no significant effect on progesterone and PMS concentrations. ABCG2 mRNA abundance was increased in placentas from ICP patients vs. controls and remained stable following UDCA treatment, despite an apparent further increase in ABCG2.

Conclusion

UDCA administration partially reduces ICP-induced bile acid accumulation in mothers and foetuses despite the lack of effect on concentrations of progesterone and PMS in maternal serum. Up-regulation of placental ABCG2 may play an important role in protecting the foetus from high concentrations of bile acids and PMS during ICP.

Keywords: bile acids, cholestasis of pregnancy, progesterone, ursodeoxycholic acid

What is Already Known about this Subject

  • Intrahepatic cholestasis of pregnancy (ICP) is characterized by an increase in maternal serum concentrations of bile acids and sulphated progesterone metabolites.

What this Study Adds

  • The accumulation of sulphated progesterone metabolites in the serum of ICP patients is associated with enhanced concentrations of progesterone. Although these compounds may be involved in the aetiopathogenesis of ICP, the magnitude of this accumulation does not correlate with the degree of hypercholanaemia in ICP patients.

  • The beneficial effect of ursodeoxycholic acid (UDCA) treatment includes a reduction of maternal hypercholanaemia, which is not dependent on the correction of progesterone metabolites accumulation.

  • In spite of marked increases in maternal serum concentrations of sulphated progesterone metabolites and bile acids in ICP patients, the concentrations of these compounds in foetal serum remain normal or are only moderately elevated, respectively. The latter can be corrected by treatment with UDCA.

  • The ABCG2 export pump located at the apical membrane of trophoblast cells plays an important role in the placental barrier for sulphated progesterone metabolites and bile acids. ABCG2 up-regulation during ICP, and further by UDCA treatment, is probably involved in the protection of the foetus against the accumulation of these compounds in the maternal compartment.

Introduction

Intrahepatic cholestasis of pregnancy (ICP) is a multifactorial liver disease that usually appears during the third trimester of pregnancy and resolves soon after delivery 1,2. ICP is associated with maternal pruritus, altered liver function and fasting hypercholanaemia (≥10 μmol l−1), while for the foetus the severity of adverse events, such as foetal distress, meconium-stained amniotic fluid, premature delivery and intrauterine foetal death, is higher. The risk of these complications is proportional to the degree of maternal hypercholanaemia 3. In addition to impaired bile acid homeostasis sulphated progesterone metabolites (PMS), whose concentrations are increased even in asymptomatic hypercholanaemia during pregnancy 4, may play a role in ICP owing to their ability to trans-inhibit the bile salt export pump (BSEP) from the canalicular lumen 5. PMS can also interfere with bile acid homeostasis through interactions with the bile acid nuclear receptor FXR 6. Moreover, PMS can affect bile acid-mediated signalling through the plasma membrane-bound bile acid receptor TGR5, which is expressed in several tissues, including liver and placenta 7.

The standard treatment for ICP patients is oral administration of ursodeoxycholic acid (UDCA), which reduces maternal hypercholanaemia and pruritus, normalizes liver function parameters and is associated with a better prognosis of pregnancy outcome 8. Some studies have reported that UDCA administration also reduces maternal plasma concentrations of some PMS 9,10, and this has been related to the improved maternal hepatobiliary function 11. During intra-uterine life, the placenta is essential for this function in the foetus. Bile acids are transferred across the placenta for elimination by the maternal liver. The concerted action of several members of the organic anion transporting polypeptide (OATP) and the ATP-binding cassette (ABC) families located at the foetal and maternal poles of the trophoblast 12,13 accounts for this vectorial transfer (for reviews, see 14,15). Among transporters involved, ABCG2, also known as breast cancer resistance protein (BCRP), plays a key role 16. Previous studies using experimental models of maternal cholestasis in rats 17 have reported an impairment in the placental transfer of bile acids due to trophoblast atrophy and a lack of apical membrane microvilli. This damage was partially prevented by UDCA administration 18.

Although a few studies have already addressed the issue of the effect of maternal cholestasis on foetal bile acid homeostasis in humans 19 or in animal models 17,18, the whole picture is incomplete, which has prompted us to investigate further the impairment in bile acid homeostasis in the mother-placenta-foetus trio during ICP. We have also addressed the question of whether removal of PMS is included among the mechanisms of action accounting for the beneficial effect of UDCA in ICP. Finally, an additional aim of the present study was to determine the effect of ICP and UDCA treatment on ABC proteins accounting for the human placental barrier for bile acids and PMS.

Methods

Chemicals

Free bile acids: cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), α/β muricholic acids (α/β MCA), taurosulfolithocholic acid (TSLCA) and UDCA, as well as tauroconjugate and glycoconjugate forms were from Sigma-Aldrich (Madrid, Spain) and sulphated progesterone metabolites (PMS), allopregnanolone sulphate (PM4-S), epiallopregnanolone sulphate (PM5-S), pregnanolone sulphate (PM6-S) and epipregnanolone sulphate (PM7-S) were from Steraloids (Newport, RI, USA). According to the suppliers, the purity of these compounds used as standards in HPLC-MS/MS analyses was ≥97%. All other chemicals were of analytical grade. The nomenclature of drugs, enzymes and transport proteins conforms to BJP's Guide to Receptors and Channels 20.

Human samples

All samples were collected at the Ramón Sardá Mother' and Children's Hospital, Buenos Aires, Argentina. Maternal and cord blood samples were used for routine biochemical tests and the serum obtained was stored at −20°C until use. Pieces of placentas were collected immediately after delivery, shock frozen in liquid nitrogen and stored at −80°C until used. Women were diagnosed as having ICP when they had pruritus, altered liver function tests and fasting bile acid concentrations higher than 10 μmol l−1 in the absence of any other hepatic dysfunction. Exclusion criteria were (i) patients with any other hepatic disease (confirmed by laboratory tests or ultrasound imaging), (ii) patients with autoimmune or infectious diseases and (iii) the use of any drug/therapy known to induce cholestasis. Controls were selected among the pregnant women without hepatic dysfunction and no previous history of diagnosed ICP or pruritus. The control population and ICP patients were matched for age, race and geographic location.

The research protocol complied with the ethical guidelines of the 1975 Declaration of Helsinki and was reviewed and approved by the Human Subjects Committees of the Ramón Sardá Mother' and Children's Hospital and the Salamanca University Hospital. Samples were collected after obtaining written consent from the patients.

In a first descriptive, cross-sectional study, the effect of UDCA on maternal cholanaemia was determined in women with ICP before and after treatment for 1 week with 900 mg day−1 UDCA. In a second study, bile acid and PMS molecular species were analyzed by HPLC-MS/MS in paired samples collected at term from maternal serum, placenta and foetal (umbilical cord) serum. The cohort of the second study included 24 pregnancies with ICP without (n = 9) or with (n = 15) treatment with 900 mg day−1 UDCA for a dissimilar period (between 9 to 40 days) depending on the individual requirements, and 25 control pregnancies attended in the same period at the hospital. Table 2 shows the demographical and clinical information obtained at delivery.

Table 2.

Clinical data at delivery of pregnancies with ICP without or with UDCA treatment

Control (n = 25) ICP (n = 9) ICP+UDCA (n = 15)
Maternal age (years) 24.1 ± 1.0 30 ± 2.5 26.3 ± 1.8
Gestational age at delivery (weeks) 36.0 ± 0.8 37.6 ± 0.2 35.9 ± 0.4
Number of pregnancies 1.7 ± 0.2 2.6 ± 0.9 2.9 ± 0.3
Male foetuses (%) 54.1 55.6 60
Fetal weight at birth (kg) 3.0 ± 0.2 3.2 ± 0.1 2.8 ± 0.1
UDCA treatment (days) 0 0 range 9–40
Maternal serum markers
ALT (U l−1) 24.5 ± 2.0 86.3 ± 31.7† 80.2 ± 21.1†
AST (U l−1) 15.2 ± 1.2 99.6 ± 29.3† 157 ± 51†
AP (U l−1) 362 ± 34 740 ± 77 509 ± 31.2‡
GGT (U l−1) 24.4 ± 8.6 26.7 ± 6.1 18.0 ± 1.3
Total bilirubin (mg dl−1) 0.7 ± 0.1 1.0 ± 0.1 0.9 ± 0.1
Direct bilirubin (mg dl−1) 0.6 ± 0.1 0.6 ± 0.1 0.5 ± 0.1
Cholesterol (mg dl−1) 249 ± 21 293 ± 25 309 ± 16
Total bile acids (μm) 3.6 ± 0.7 48.3 ± 11.8† 25.6 ± 3.8†‡
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Values are mean ± SEM.

†

P < 0.05 on comparing with control,

‡

P < 0.05 on comparing ICP untreated and UDCA-treated mothers by the Bonferroni test.

Analyses of bile acids and progesterone metabolites

Silica-based bonded phase cartridges (Sep-Pack Plus C18, Waters, Madrid, Spain) were used to extract bile acids and PMS from 1 ml serum or ≈0.5 g of placental tissue 4. Methanolic extracts were analyzed using an adaptation 21 of a previously described method for bile acid measurement by HPLC-MS/MS 22 on a 6410 Triple Quad LC/MS device (Agilent Technologies, Santa Clara, CA). Twenty bile acid species were quantified by this method.

Chromatographic separation of PMS was carried out by gradient elution on a Zorbax C18 column (30 mm × 2.1 mm, 3.5 μm) kept at 35°C. The flow rate was 300 μl min−1. The initial mobile phase was 50:50 methanol/water, both containing 5 mm ammonium acetate and 0.01% formic acid, and it was changed at 4.5 min to 95:5 methanol/water over 2 min and then returned to 50:50 for 1.5 min. Electrospray ionization (ESI) in the negative mode was used, with the following conditions: gas temperature 350°C, gas flow 10 l min−1, nebulizer 20 psi, capillary voltage 2500 V. MS/MS acquisition was performed in multiple reaction monitoring (MRM) mode using the m/z transition 397.1 ([M-H]− molecular ion) to 97.2 (sulphate) for all four PMS analyzed. The method permits the separation of PM-4S and PM-6S, whereas co-elution of PM-5S and its epimer PM-7S does not permit their chromatographic separation. Therefore these were quantified together, keeping in mind that, under normal conditions, maternal serum concentrations of PM5-S are 10-fold higher than those of PM7-S 23. The limit of quantification (LOQ) was 0.05 μm both for bile acid species and progesterone metabolites, for an injection volume of 2 μl (0.1 pmol in column). Coefficients of variation (CVs) were within day CVs ≤4.3% and between day CVs ≤7.5%. Total serum bile acid concentrations were also measured by an enzymatic, colorimetric method (Randox Laboratories, Crumlin, UK) and progesterone and estriol serum concentrations were determined using ELISA kits (Abnova, Heidelberg, Germany).

RT-QPCR

Total RNA extraction was carried out using the illustra RNAspin Mini RNA Isolation Kit (GE Healthcare Life Sciences, Barcelona, Spain) and retrotranscription using a high capacity cDNA reverse transcription kit (Applied Biosystems, Madrid, Spain). Real-time quantitative PCR (QPCR) was performed using AmpliTaq Gold polymerase (Applied Biosystems) in an ABI Prism 7300 Sequence Detection System (Applied Biosystems) with the following thermal conditions: 1 cycle of 95°C for 10 min and 40 cycles of 95°C for 15 s and 60°C for 60 s. The primer oligonucleotide sequences to carry out QPCR are described in Table S1. The results of mRNA abundance for target genes in each sample were normalized on the basis of GAPDH mRNA abundance. Detection of amplification products was carried out using a SYBR Select kit (Applied Biosystems). Total RNA from control placentas was used as a calibrator. Expression levels were calculated as 2−ΔΔCt, where ΔCt was the difference of threshold cycle in each sample between the target gene and the normalizer. This was used to calculate ΔΔCt as the difference of this value between control RNA and ICP groups.

Immunofluorescence assays

Immunostaining was carried out in 5 μm placenta cryosections air-dried before fixation and permeabilization in ice-cold methanol. Mouse monoclonal antibody against ABCG2 (BXP-34, Enzo Life Sciences, Lausen, Switzerland) was diluted in 2% foetal calf serum in PBS. Secondary anti-mouse IgG Alexa-488 antibody (Life Technologies) was diluted 1:1000 and nuclei were counterstained with DAPI (10 μm). Confocal laser-scanning immunofluorescence microscopy was performed using a Zeiss LSM 510 apparatus. For better comparison, samples obtained from the three groups were prepared and analyzed on the same slide. The relative intensity of immunofluorescence was measured and corrected by trophoblast area using ImageJ software.

Statistical analysis

Data are presented as means ± SEM. After performing an anova test, the Bonferroni method of multiple-range testing or the paired t-test were used to calculate the statistical significance of differences among groups.

Results

Groups of patients

The present study was divided into two objectives to be achieved in two completely different cohorts: (i) to analyze the response of ICP patients to UDCA administration (900 mg day−1) daily for 1 week and (ii) to elucidate the effect of UDCA treatment on progesterone and bile acid homeostasis in the mother-placenta-foetus trio, which was investigated at term after different periods of treatment, depending on the individual patient management during the time between diagnosis and delivery.

For the first objective, 47 women were recruited. Twelve were later excluded, nine with cholelithiasis, one with gallbladder polyps and two with very early diagnosis of ICP (18 and 24 weeks of gestation) and a marked increase in cholanaemia during UDCA treatment, probably due to the cholestatic effect of the lorazepam they were taking 24. The remaining 35 ICP patients had a mean gestational age of 29.8 ± 0.8 weeks at the start of treatment. Maternal age at delivery was 26.1 ± 1 years (range 18–40 years) and no stillbirth occurred. The effect of treatment on serum biochemical parameters is shown in Table 1. A significant decrease in AST was only observed after 1 week of UDCA treatment, while other biochemical parameters determined were not changed at this time (Table 1).

Table 1.

Effect of UDCA on serum markers of liver function in ICP patients

Before UDCA treatment After 1 week UDCA treatment
AST (U l−1) 75.5 ± 10.8 47.5 ± 7.3†
ALT (U l−1) 120 ± 21 111 ± 21
AP (U l−1) 499 ± 27 428 ± 24
GGT (U l−1) 25.2 ± 2.3 21.7 ± 2.1
Total bilirubin (mg dl−1) 0.8 ± 0.1 0.7 ± 0.1
Direct bilirubin (mg dl−1) 0.3 ± 0.1 0.2 ± 0.1
Cholesterol (mg dl−1) 292 ± 11 275 ± 10
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ICP patients (n = 35) received UDCA (900 mg day−1) for 1 week. Values are mean ± SEM.

†

P < 0.05 on comparing with values before treatment by the paired t-test.

The clinical and demographic characteristics of the patients recruited for the second objective are shown in Table 2. Maternal age, gestational age at delivery, birth weight and percentage of male foetuses were similar in the control group and in the pregnancies with ICP, both treated and untreated with UDCA. One triplet pregnancy was included in the control group while in the ICP and ICP+UDCA groups all were singleton pregnancies. The inclusion of the triplet pregnancy accounts for the slightly lower gestational age at delivery and mean foetal weight at birth in the control group.

The women suffering from ICP had raised levels of transaminases and alkaline phosphatase, together with a marked hypercholanaemia and no alteration in other parameters, although a tendency for high cholesterol concentrations was observed. UDCA treated patients presented lower serum bile acid and alkaline phosphatase concentrations than untreated patients, while serum transaminase activity was similar (ALT) or higher (AST) in treated patients (Table 2).

Effect of UDCA treatment on bile acid concentrations

In the cross-sectional study, no response to UDCA (<15% reduction in cholanaemia) was observed in some ICP patients (Figure 1A). However, most of them did respond to such treatment (Figure 1B). The responder rate was 77% (Figure 1C). In this group, the UDCA-induced decrease in serum bile acid concentrations was approximately 50% (Figure 1D).

Figure 1.

Figure 1

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Effect in patients with ICP (n = 35) of treatment with UDCA (900 mg day−1) in serum total bile acid concentrations after 1 week of treatment. Patients were classified as non-responders (A) and responders (B), depending on whether their hypercholanaemia was reduced by at least 15%. Proportion of responder and non-responder ICP patients (C). Mean ± SEM change in serum total bile acid concentrations in responder patients (D). *, P < 0.05, compared by the paired t-test

When bile acid species were determined in maternal and foetal serum at term (Table 3), an increase in CA (74-fold), CDCA (17-fold) and DCA (6-fold) families (unconjugated plus conjugated forms) was observed in ICP women. As a consequence, the mean ratios of the serum CA: CDCA: DCA families changed from 1.2:1:0.5 in controls to 5.2:1:0.2 in ICP patients. This was partly corrected (3.3:1:0.4) by UDCA treatment. This was due to a more marked reduction in CA and CDCA than in DCA levels, together with an enrichment (up to 47%) in the UDCA family. Regarding LCA in ICP, only TSLCA, which accounted for only 0.5% of total bile acids, was significantly increased in maternal, but not in foetal, serum. UDCA administration did not affect maternal serum TSLCA concentrations.

Table 3.

Profiles of bile acid species in maternal and foetal sera at term

Bile acids (μm) Mothers Foetuses
Control ICP ICP+UDCA Control ICP ICP+UDCA
 TCA 0.21 ± 0.04 21.3 ± 7.5† 2.88 ± 0.73†,‡ 0.90 ± 0.19 6.10† ± 2.66 0.95 ± 0.41‡
 GCA 0.25 ± 0.06 16.5 ± 3.8† 5.58 ± 1.36†,‡ 0.41 ± 0.06 5.16† ± 2.43 1.59 ± 0.84‡
 CA 0.06 ± 0.03 0.78 ± 0.57† 0.27 ± 0.15† 0.06 ± 0.01 0.13†± 0.02 0.10 ± 0.06
Total CA 0.52 ± 0.10 38.6 ± 10.6† 8.7 ± 1.8†,‡ 1.16 ± 0.20 11.4†± 5.0 2.6 ± 1.3‡
 TCDCA 0.20 ± 0.04 4.30 ± 1.00† 0.87 ± 0.17† 1.49 ± 0.30 1.89 ± 0.62 0.56 ± 0.08‡
 GCDCA 0.21 ± 0.04 3.07 ± 0.47† 1.67 ± 0.37† 0.36 ± 0.03 1.87 ± 0.65 0.67 ± 0.17
 CDCA 0.03 ± 0.01 0.08 ± 0.05 0.05 ± 0.02 0.02 ± 0.00 0.03 ± 0.01 0.04 ± 0.02
Total CDCA 0.43 ± 0.08 7.4 ± 1.2† 2.6 ± 0.5†,‡ 1.73 ± 0.30 3.8 ± 1.2 1.3 ± 0.2‡
 TDCA 0.05 ± 0.01 0.63 ± 0.30† 0.26 ± 0.05† 0.04 ± 0.01 0.02 ± 0.01 0.01 ± 0.00
 GDCA 0.10 ± 0.02 0.68 ± 0.30† 0.67 ± 0.12† 0.03 ± 0.01 0.04 ± 0.02 0.05 ± 0.02
 DCA 0.07 ± 0.03 0.05 ± 0.02 0.15 ± 0.04 0.02 ± 0.00 0.01 ± 0.01 0.02 ± 0.00
Total DCA 0.22 ± 0.05 1.4 ± 0.6† 1.08 ± 0.18† 0.10 ± 0.02 0.08 ± 0.02 0.08 ± 0.02
 TLCA 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00
 GLCA 0.00 ± 0.00 0.00 ± 0.00 0.02 ± 0.01 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00
 LCA 0.00 ± 0.00 0.00 ± 0.00 0.01 ± 0.00 0.01 ± 0.01 0.00 ± 0.00 0.00 ± 0.00
 TSLCA 0.01 ± 0.00 0.11 ± 0.04† 0.09 ± 0.02† 0.01 ± 0.01 0.02 ± 0.01 0.01 ± 0.00
Total LCA 0.01 ± 0.00 0.12 ± 0.04† 0.12 ± 0.02† 0.02 ± 0.01 0.02 ± 0.01 0.02 ± 0.01
 TUDCA 0.01 ± 0.01 0.01 ± 0.00 1.03 ± 0.36†,‡ 0.06 ± 0.01 0.03 ± 0.01 0.09 ± 0.04
 GUDCA 0.06 ± 0.05 0.03 ± 0.01 7.50 ± 1.65†,‡ 0.03 ± 0.01 0.08 ± 0.03 0.87 ± 0.55†,‡
 UDCA 0.06 ± 0.04 0.03 ± 0.01 5.63 ± 1.71†,‡ 0.06 ± 0.02 0.08 ± 0.02 1.34 ± 0.32†,‡
Total UDCA (U) 0.13 ± 0.09 0.07 ± 0.02 14.2 ± 2.6†,‡ 0.13 ± 0.02 0.19 ± 0.05 2.3 ± 0.7†,‡
 TMCA 0.02 ± 0.01 0.00 ± 0.00 0.02 ± 0.01 0.11 ± 0.01 0.09 ± 0.01 0.16 ± 0.03
Total BAs 1.3 ± 0.3 47.5 ± 11.6† 26.7 ± 3.6† 3.6 ± 0.5 15.5 ± 6.2† 6.5 ± 2.1†,‡
Endogenous BAs (Total BAs-U) 1.3 ± 0.3 47.5 ± 11.6 12.5 ± 2.2 3.6 ± 0.5 15.5 ± 6.2 4.2 ± 1.4
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BAs, bile acids; CA, cholic acid; CDCA, chenodeoxycholic acid; DCA, deoxycholic acid; LCA, lithocholic acid; TMCA, tauromuricholic acids; TSLCA, taurosulfolithocholic acid; UDCA (U), ursodeoxycholic acid. G and T prefixes indicate conjugation with glycine and taurine, respectively. Vales are means ± SEM.

†

P < 0.05; on comparing with appropriate controls by the Bonferroni test;

‡

P < 0.05; on comparing ICP untreated or UDCA-treated mothers or their foetuses. Control (n = 25), ICP (n = 9), ICP+UDCA (n = 15). U, unconjugated and conjugated forms of UDCA. HyoDCA, αMCA and βMCA were not detected.

In cord serum from control pregnancies, the mean value of the CA: CDCA ratio was 0.7, similar to that previously reported by our group 25, whereas the concentrations of DCA were very low (0.1 μm) (Table 3). As compared with the control group, in foetuses from mothers with ICP the families of CA and CDCA species were markedly higher (10- and two-fold, respectively), while no change in the DCA family was observed. In the group of ICP patients treated with UDCA, cord serum concentrations of CA and CDCA were close to those found in control pregnancies. In the group of patients receiving UDCA, this bile acid and its derivatives constituted one third of the total bile acids found in foetal serum (Table 3).

Relationship between cholanaemia in mothers and foetuses

As previously described in healthy pregnancies 15,25, the transplacental gradient for total bile acids was in the foetus-to-mother direction (Figure 2A) with a mother : foetus ratio of 0.36. In pregnancies with ICP (Figure 2B, C), there was a reversal in the gradient resulting in a mother : foetus ratio of 3.1. Treatment with UDCA induced a decline in bile acid concentration, which was more marked in foetal than in maternal serum. This resulted in a mother : foetus ratio of 4.1.

Figure 2.

Figure 2

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Individual paired values of maternal-foetal gradients for serum total bile acids at term in control pregnancies (n = 25) (A) or complicated by ICP without (n = 9) (B) or with UDCA treatment (n = 15) (C). Mean values of bile acid concentrations in control pregnancies or complicated by ICP without or with UDCA treatment (ICP+U) as determined in serum samples from mothers and foetuses and expressed as total bile acids (D), or as families of taurine-conjugated (E), glycine-conjugated (F) and unconjugated (G) bile acids. Endogenous bile acid concentrations in the ICP+UDCA group were calculated by subtracting UDCA family. Values are means ± SEM, *, P < 0.05, compared with appropriate controls and †, P < 0.05, comparing ICP groups by the Bonferroni method of multiple range testing. (D–G) □, All species; ■, All species – UDCA

The marked elevation in maternal and foetal serum bile acids observed in ICP (Figure 2D) was mainly due to an increased amount of conjugated bile acids, with either taurine (Figure 2E) or glycine (Figure 2F), and quantitatively less important amounts of unconjugated bile acids (Figure 2G). The impact of ICP on the serum concentrations of bile acids was markedly lower in foetuses than in their mothers (Figure 2). Interestingly, in agreement with a recent study 19, UDCA treatment partly corrected this alteration in the mothers but almost completely so in their foetuses. The correction affected unconjugated and conjugated bile acids. The beneficial effect of UDCA treatment was seen more clearly when changes in endogenous bile acids were explored by calculating total bile acids without taking into account UDCA family (Figure 2D–G).

To complete the picture of the bile acid balance in the maternal-placenta-foetal trio, bile acids were measured in placental tissue. The total bile acid content in placentas was markedly enhanced in ICP as compared with the controls, and it was significantly reduced by UDCA treatment (Figure 3A), mainly due to the normalization of conjugated bile acid concentrations (Figure 3B), while unconjugated bile acids were significantly increased. This can be explained in terms of the accumulation in this organ of unconjugated UDCA (Figure 3C). When bile acid species in ICP placentas were analyzed, an accumulation of CA >> CDCA≈UDCA > LCA (mainly as TSLCA) ≈ DCA forms was observed. The placenta levels of all bile acid species were normalized by UDCA treatment, except UDCA forms, which were increased.

Figure 3.

Figure 3

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Placenta bile acid content at term expressed as total bile acids (A), or as families of taurine-conjugated, glycine-conjugated and unconjugated bile acids (B) and as families of bile acid species (C). Relative concentrations of bile acids in paired samples of maternal serum, placenta and foetal serum collected from control (D, n = 25), ICP (E, n = 9) and ICP+UDCA (F, n = 15) pregnancies. Values are means ± SEM, *, P < 0.05, compared with control; †, P < 0.05, on comparing ICP with and without UDCA treatment. ‡, P < 0.05, compared with placenta by the Bonferroni method of multiple range testing. □, control; ■, ICP; Inline graphic, ICP+UCDA

Assuming a homogeneous distribution of bile acids in the placental tissue and a tissue density value of approximately 1 g ml−1, our results indicated that in the controls the levels of total bile acids in placenta were between values found in maternal and foetal serum but closer to the concentrations found in maternal serum (Figure 3D). In ICP, despite the dramatic increase in bile acid concentrations in the maternal compartment, placental concentrations were maintained at relatively low values (Figure 3E, F). Interestingly, in both the ICP and ICP+UDCA groups the concentrations of bile acids were lower in placenta than in foetuses (Figure 3E, F).

Balance between progesterone and its metabolites in ICP

Since ICP develops during gestation when oestrogens and progesterone concentrations increase rapidly, a role for these hormones and their metabolites in the aetiopathogenesis of ICP has been suggested. In the present study, the concentrations of PMS were found to be markedly elevated in the serum of patients with ICP (Figure 4A). This was mainly due to the increased amount of PM4-S (Figure 4B), together with a minor contribution from the rest of the metabolites assayed (Figure 4C, D). In contrast, the concentrations of PMS in foetal serum were not affected by ICP (Figure 4E–H). In placental tissue, a trend towards enhanced concentrations of PMS in ICP was found (Figure 4I–L). Comparison of the relative values of PMS in maternal-placental-foetal compartments (Figure 4M–O) revealed that the placenta was the element of this trio with the highest concentrations of PMS. ICP was accompanied by an accumulation of PMS in maternal serum up to but not significantly different from placental concentrations, whereas in foetal serum PMS concentrations remained consistently low. Treatment with UDCA had no significant effect on the concentrations or balance of PMS in the mother-placenta-foetus trio found in ICP (Figure 4).

Figure 4.

Figure 4

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Concentrations of total sulphated progesterone metabolites (PMS), allopregnanolone sulphate (PM4-S), epiallopregnanolone sulphate plus epipregnanolone sulphate (PM5-S + PM7-S) and pregnanolone sulphate (PM6-S) in maternal serum (A–D), foetal serum (E–H) and placenta at term (I–L). Relative concentrations of PMS in paired samples of maternal serum, placenta and foetal serum collected from control (M, n = 25), ICP (N, n = 9) and ICP+UDCA (O, n = 15) pregnancies. Values are means ± SEM, *, P < 0.05, compared with control; †, P < 0.05, compared with placenta by the Bonferroni method of multiple range testing. □, control; ■, ICP; Inline graphic, ICP+UCDA

Since UDCA was able to correct the accumulation of bile acids in maternal serum of ICP patients without affecting PMS balance, the relationship between bile acids and progesterone or PMS concentrations in maternal serum was further investigated. As expected, no correlation between bile acids and either progesterone (Figure S1A) or PMS was found (Figure S1B). In contrast, similar and significant correlations between progesterone and PMS in the serum from the women belonging to the control, ICP and ICP+UDCA groups were found (Figure 5A). This was consistent with elevated serum concentrations of progesterone in ICP patients, which was not corrected by UDCA treatment (Figure 5B). To investigate whether, in addition to progesterone production, the biotransformation rate due to sulphotransferase activity was also enhanced, SULT2B1 and SULT1E1 expression was measured. Indeed, the abundance of SULT1E1 mRNA in placenta was higher in ICP pregnancies, while for SULT2B1 only a trend to increase was found. UDCA treatment did not correct these values (Figure 5C). SULT2B1 mRNA levels were much more elevated than those of SULT1E1 in placenta. To elucidate whether UDCA-induced beneficial effect might be mediated by changes in different steroid hormones that are also enhanced in pregnancy and are potentially cholestatic, such as oestrogens, the maternal serum concentrations of estriol were measured. No significant differences in the maternal serum concentrations of estriol between the control, ICP and ICP+UDCA groups were found (Figure 5D).

Figure 5.

Figure 5

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Relationship between the concentrations of progesterone and total sulphated progesterone metabolites (PMS) in maternal serum obtained at term from control (n = 25) or ICP pregnancies without (n = 9) or with UDCA (n = 15) treatment (A). Mean values of progesterone (B) and estriol (D) concentrations in maternal serum. Abundance of SULT2B1 and SULT1E1 mRNA in placenta (C). Values are means ± SEM, *, P < 0.05, compared with controls and †, P < 0.05, on comparing ICP with and without UDCA treatment by the Bonferroni method of multiple range testing. Values of correlation coefficient (A) were as follows: Control: r = 0.632, P < 0.05; ICP: r = 0.891, P < 0.05; ICP+UDCA: r = 0.702, P < 0.05. The value of the threshold cycle (Ct) for SULT1E1 was 32 and for SULT2B1 was 23. □, control; ■, ICP; Inline graphic, ICP+UCDA

Effect of ICP and UDCA treatment on the placental barrier for bile acids and PMS

To determine whether the beneficial effect of UDCA involved an enhancement of the placental barrier for bile acids and PMS, changes in the expression of OATP1B3, main responsible for bile acid transport into the placenta 26, as well as the export pumps which have been suggested to play a role in the hepatobiliary function of the placenta 13,16,27,28 were investigated. Relative SLCO1B3 mRNA levels determined by quantitative PCR in control, ICP and ICP+UDCA placentas were similar, as shown in Table 4. In the placentas of the control group, mRNA abundance of ABCG2 was high and similar to that of ABCB1 and ABCC1, whereas the levels of ABCC3 and ABCC2 mRNA were lower. ABCG2 and MDR1 expression was significantly increased in the ICP and ICP+UDCA groups. Regarding ABCC1-3, although a certain trend was observed, no significant up-regulation by ICP or UDCA treatment was found. As ABCG2, but not MDR1, is believed to play an important role in the placental transport of bile acids 16, we focused our interest on this pump. Thus, protein expression was further analyzed by immunofluorescence-confocal microscopy in human placenta cryosections (Figure 6). Consistent with data from the mRNA determinations, the abundance of ABCG2 at its characteristic location, i.e. the apical membrane of trophoblast 13 (Figure 6A–C), was enhanced in ICP placentas (Figure 6D, E). The immunoreactivity signal for ABCG2 was further increased in the trophoblasts of ICP+UDCA placentas (Figure 6F).

Table 4.

Effect of ICP and UDCA treatment in the expression of ABC export pumps in human term placenta

Gene/Protein Ct Control Control ICP ICP+UDCA
SLCO1B3/OATP1B3 31 100 ± 19 106 ± 26 85 ± 12
ABCG2/BCRP 20 100 ± 14 268 ± 42† 257 ± 44†
ABCC1/MRP1 21 100 ± 14 143 ± 19 146 ± 19
ABCC2/MRP2 26.7 100 ± 27 125 ± 31 84 ± 14
ABCC3/MRP3 24 100 ± 15 170 ± 51 157 ± 45
ABCB1/MDR1 19.5 100 ± 16 301 ± 33† 232 ± 38†
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Values are mean ± SEM (n ≥ 6 placentas analyzed in triplicate) expressed as relative abundance of mRNA vs. control. Human control placenta was used as calibrator. Results from RT-QPCR were normalized with values from GAPDH mRNA.

†

P < 0.05, on comparing with controls by the Bonferroni test. Ct, Threshold cycle.

Figure 6.

Figure 6

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Immunofluorescence localization of ABCG2 in placentas collected at term from control (A,D) or ICP pregnancies without (B,E) or with (C,F) UDCA treatment. Cryosections were stained with anti-ABCG2 antibody (green) and nuclei were stained with DAPI (A–C). Preparations from different placentas showing only ABCG2 immunoreactivity (D–F). (The green fluorescence was transformed into grey scale to facilitate the quantification of signal intensity). Relative integrated fluorescence in trophoblast (G). Values are means ± SEM, *, P < 0.05, compared with controls and †, P < 0.05, on comparing ICP with and without UDCA treatment by the Bonferroni method of multiple range testing

Discussion

The present study confirms previous reports regarding the amelioration of maternal serum bile acid concentrations by treatment with UDCA 19,29,30. These studies have shown that, this is mainly due to a reduction in the accumulation of endogenous bile acids, probably by improving maternal hepatobiliary function owing to the choleretic effect of UDCA together with an up-regulation of export pumps in liver cells 31. Moreover one week of treatment is sufficient to observe the response as regards changes in bile acid concentrations, whereas among the rest of routine biochemical parameters assayed only the maternal serum transaminase AST was corrected in this time.

Since bile acid species are differentially able to cause tissue damage, the profile of bile acid species accumulated in the maternal-placental-foetal trio during ICP provides valuable information. Regarding primary bile acids, an important elevation in the CA and CDCA forms, mainly conjugated with taurine and glycine, was found in maternal serum from women with ICP, whereas the increase of these bile acids in foetal serum was much lower. Regarding more toxic secondary bile acids, DCA was increased in the maternal compartment, but only a very small amount of this bile acid was detected in foetal serum and placenta. No increase in LCA concentrations in the serum of women with ICP, with or without UDCA treatment, was found, except for the LCA derivative TSLCA, whose concentrations were significantly enhanced in maternal serum and placenta in ICP. Our results are in agreement with the recent report by Geenes et al. 19 although different from others who have found enhanced concentrations of LCA in ICP 32.

On considering the beneficial effect of UDCA treatment in reducing the concentrations of toxic bile acids it is important to take into account that part of the maternal bile acid pool is replaced by UDCA during the administration of this bile acid. Accordingly, from the clinical point of view a more interesting analysis than considering total bile acids is to calculate the concentrations of endogenous bile acids without including the UDCA family. With this corrected calculation, a more marked beneficial effect of UDCA was observed in maternal and foetal sera and in the placenta. These results are in agreement with those previously obtained using animal models showing, in pregnant rats with obstructive cholestasis, an impairment in the placental ability to carry out foetal bile acid transfer, which was restored by treatment with UDCA 17.

In normal pregnancies, bile acids are synthesized by the foetal liver since very early in gestation and are mainly transferred across the placenta to the maternal blood, where the concentrations are lower than in the foetal compartment 25. Thus, in the control group there was a moderate gradient in the foetal-to-maternal direction, with the placenta placed in between. The rise in maternal bile acids concentrations in ICP produces a reversal in the direction of the transplacental gradient of these molecules from the maternal blood towards the foetal circulation, which affects the placenta-mediated detoxification pathway 33. The results regarding bile acid contents in placental tissue provide useful information to understanding the handling of bile acids under these circumstances of altered homeostasis. The fact that bile acid concentrations in the placenta were lower than in maternal and foetal sera in ICP, despite the important increase of bile acids in the maternal circulation, and with no changes in SLCO1B3, the main bile acid transporter involved in the uptake by the placenta, further supports the concept of the existence of active transporters, such as ABCG2 16,34, that efficiently exclude these molecules from the placenta, mainly in the foetus-to-mother direction.

ABCG2, located in the apical side of the trophoblast is able to transport bile acids 16 and oestrogen sulphated derivatives, but not free oestrogens 35. The enhanced expression of placental ABCG2 in ICP could be responsible for the maintenance of low bile acid concentrations in the placenta and, subsequently, in the foetal compartment, despite the marked increase in these compounds in the maternal circulation. The abundance of this protein was further increased in the placenta of ICP patients treated with standard doses of UDCA. Enhanced expression of placental ABCG2 has been found previously in ICP patients treated with high doses of UDCA 28. The fact that mRNA was not enhanced in the UDCA treated group suggests that mechanisms other than transcription were involved. In a recent study we have demonstrated that placental ABCG2 protein expression was decreased under acetaminophen treatment, even in absence of changes in ABCG2 mRNA 36, which was related to drug-induced enhanced oxidative stress in trophoblast cells. Thus, the protective effect of UDCA may include intracellular conditions leading to decreased degradation of ABCG2 protein in the placenta.

Previous reports have shown that progesterone and its sulphated derivatives decrease in maternal and foetal sera after UDCA treatment due to stimulation of their biliary secretion by the strong choleretic activity of this bile acid 31. Thus, an interesting question arises as to whether the elimination of these cholestatic compounds could account, in part, for the beneficial effect of UDCA. Our results suggest that this is not the case, because UDCA treatment was successful in reducing hypercholanaemia without affecting the elevated concentrations of PMS. These are probably related to an increased rate of progesterone synthesis and biotransformation. This was consistent with the correlation found between the maternal serum concentrations of progesterone and PMS. Moreover, enhanced sulphotransferase activity could be expected from the up-regulation of SULT2B1 and SULT1E1 found in ICP placentas. SULT2B1, very abundant in the placenta, preferentially sulphonates pregnenolone 37, while SULT1E1 is able to sulphonate steroid hormones, including pregnenolone 38. PMS generation could be secondary to enhanced progesterone synthesis because progesterone has been found to stimulate SULT1E1 expression in human endometrial adenocarcinoma cells 39. Interestingly, progesterone is able to inhibit ABCG2 40 and hence may affect the ability of the placenta to export ABCG2 substrates toward the maternal circulation.

In summary, UDCA reduces the accumulation of bile acids in the maternal-placental-foetal trio during ICP probably by improving maternal hepatobiliary function but not by reducing the production or enhancing the elimination of cholestatic oestrogens and progesterone metabolites. In addition, UDCA treatment further enhances the ICP-induced stimulation of the placental barrier for bile acids and PMS through up-regulation of one of the major actors in this function, i.e. ABCG2.

Contributors

MCE, MJM, LR, MM, LGR, TRB, JJGM and RIRM performed the experiments. MCE, MJM, LR, MM, LGR, TRB, JJGM and RIRM discussed and interpreted the data and drafted the manuscript.

Funding

This study was supported by the Instituto de Salud Carlos III, FIS (Grant PI1100337), the Plan Nacional de Investigacion Cientifica, Desarrollo e Innovacion Tecnologica and the European Regional Development Fund (ERDF) (Grant SAF2010-15517) and Junta de Castilla y Leon (Grants SA015U13, BIO/SA64/13 and BIO/SA65/13), Spain. The group is member of the Network for Cooperative Research on Membrane Transport Proteins (REIT) and belongs to the CIBERehd (Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas) Instituto de Salud Carlos III, Spain.

Competing Interests

All authors have completed the Unified Competing Interest form at http://www.icmje.org/coi_disclosure.pdf (available on request from the corresponding author) and declare no support from any organization for the submitted work, no financial relationships with any organizations that might have an interest in the submitted work in the previous 3 years and no other relationships or activities that could appear to have influenced the submitted work.

The authors thank N. Skinner for a revision of the English spelling, grammar and style of the manuscript.

Supporting Information

Figure S1

Relationship between the concentrations of progesterone and total bile acids (A) and total sulphated progesterone metabolites (PMS) and total bile acids (B) in maternal serum obtained at term from control (n = 25) or ICP pregnancies without (n = 9) or with UDCA (n = 15) treatment

Table S1

Gene-specific oligonucleotide sequences for primers used in real-time RT-QPCR

bcp0079-0316-sd1.zip (202.4KB, zip)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1

Relationship between the concentrations of progesterone and total bile acids (A) and total sulphated progesterone metabolites (PMS) and total bile acids (B) in maternal serum obtained at term from control (n = 25) or ICP pregnancies without (n = 9) or with UDCA (n = 15) treatment

Table S1

Gene-specific oligonucleotide sequences for primers used in real-time RT-QPCR

bcp0079-0316-sd1.zip (202.4KB, zip)

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