Intrahepatic cholestasis of pregnancy (ICP) is a rare but severe complication for both the mother and the unborn child. The diagnosis is primarily based on elevated serum levels of bile acids. In a large ICP cohort, we here study in detail liver stiffness (LS) using transient elastography (TE), now widely used to non-invasively screen for liver cirrhosis within minutes.

To specifically explore LS in a large cohort of women with ICP compared to a control group with uncomplicated pregnancy.

LS and hepatic steatosis marker controlled attenuation parameter (CAP) were measured in 100 pregnant women with ICP using TE (Fibroscan, Echosens, Paris, France) between 2010 and 2020. In 17 cases, LS could be measured postpartum. 450 women before and 38 women after delivery with uncomplicated pregnancy served as control group. Routine laboratory, levels of bile acids and apoptosis marker caspase-cleaved cytokeratin 18 fragment (M30) were also measured.

Women with ICP had significantly elevated transaminases but normal gamma-glutamyl transferase (GGT). Mean LS was significantly increased at 7.3 ± 3.0 kPa compared to the control group at 6.2 ± 2.3 kPa (P < 0.0001). Postpartum LS decreased significantly in both groups but was still higher in ICP (5.8 ± 1.7 kPa vs 4.2 ± 0.9 kPa, P < 0.0001), respectively. In ICP, LS was highly significantly correlated with levels of bile acids and M30 but not transaminases. No correlation was seen with GGT that even increased significantly after delivery in the ICP group. Bile acids were mostly correlated with the liver apoptosis marker M30, LS and levels of alanine aminotransferase, aspartate aminotransferase, and bilirubin. In multivariate analysis, LS remained the sole parameter that was independently associated with elevated bile acids.

In conclusion, LS is significantly elevated in ICP which is most likely due to toxic bile acid accumulation and hepatocyte apoptosis. In association with conventional laboratory markers, LS provides additional non-invasive information to rapidly identify women at risk for ICP.

Approximately 3% of pregnant women have liver disorders that can cause severe problems for the mother and unborn child, e.g., liver failure, preterm labor, and stillbirth[1-3]. Despite intensive research on pregnancy-related liver complications in recent decades, treatment options are still insufficient, and no effective screening tests for early assessment have been established[4-6]. Intrahepatic cholestasis of pregnancy (ICP) with elevated serum bile acid levels higher than 20 μmol/L is the most common pregnancy-specific liver disease. Its etiology is complex and consists of genetic, endocrine (circulating estrogen and progesterone), and environmental factors (reduced vitamin D and selenium levels in winter). Severe forms with bile acids levels higher than > 40 μmol/L are associated with abnormal fetal echocardiography, meconium-stained amniotic fluid, spontaneous preterm labor and fetal asphyxia. Moreover, women with a total serum bile acid level of 100 μmol/L have an increased risk of stillbirth. The incidence varied between 0.05% and 27.6% for all pregnancies[7-12].

ICP typically presents in the third trimester as nocturnal pruritus of the soles and palms[13]. ICP also increases the risk of gestational diabetes and pre-eclampsia[14]. Long-term consequences of ICP include a higher risk of cancer of the liver and biliary tree, diabetes mellitus, thyroid disease, autoimmune diseases (psoriasis, inflammatory polyarthropathies, and Crohn’s disease), and cardiovascular disease[15]. Treatment with ursodeoxycholic acid has been proven to significantly improve itching, blood levels, and fetal outcomes in numerous studies; however, pregnancy termination remains the only causal therapy[2,7]. A rapid diagnosis of ICP is essential to protect mothers and children from (long-term) complications[16]. The diagnosis is normally based on elevated serum levels of bile acids. Unfortunately, these blood tests usually take several hours, even at maximum care facilities and are not available at any time. To date, no screening tests are available for liver disease during pregnancy, except serological testing for viral hepatitis in the third trimester.

Despite the scarcity of ICP data, enormous progress has been made in the molecular understanding of cholestatic liver disease in recent decades[10]. Hepatocytes and cholangiocytes cooperatively produce bile, which is a mixture of organic and inorganic compounds[17]. Cholestasis usually describes the impairment of bile flow caused by defects in hepatocytes, which form and secrete bile, and/or defects in the secretory machinery of cholangiocytes[17]. The detergent properties of bile render it highly toxic to cells and tissues[17]. In addition to drugs, inflammation, liver disease, and hormones, several gene mutations have been discovered that can cause cholestasis and ICP[8-10,18-21].

Measurement of liver stiffness (LS) using elastographic techniques has become the gold standard for the noninvasive diagnosis of liver fibrosis and cirrhosis and it often avoids invasive liver biopsies[22]. Transient elastography (TE) (FibroScan, Echosens, Paris, France), the first elastographic technique, is an ultrasound-based technique that uses a transducer probe to create an elastic shear wave[23]. Pulse-echo ultrasound is used to measure shear wave velocity, which is directly associated with LS expressed in kilopascals (kPa). TE requires only a few minutes, is highly accurate, and has a lower sampling error than biopsy, thus allowing for repetitive measurements[24]. LS values below 6 kPa are considered normal, while the generally accepted cutoff values for liver fibrosis (F3) and cirrhosis (F4) are 8 and 12.5 kPa[25]. However, LS is not only elevated by the fibrosis stage but also by other important confounding factors, including physiological conditions such as food and alcohol intake, or pathological confounders such as inflammation or pressure elevation[22]. Of note, all these confounders and artifacts will always increase LS but never decrease it, which is the most important reason, while normal LS has a very high negative predictive value in excluding liver pathologies[24].

Therefore, liver elastography is an ideal diagnostic tool to address hepatic complications during pregnancy. In the first elastography study of > 500 pregnant women without liver disease, we recently demonstrated that LS increased significantly in the third trimester and was an independent predictive factor for pre-eclampsia[26]. These findings were independently confirmed in a smaller study in Denmark[27]. Moreover, in pregnant women with cirrhosis, LS predicts hepatic decompensation after delivery[28]. The aim of the present study was to specifically explore LS in a large cohort of women with ICP compared with a control group with uncomplicated pregnancies.

In this study, we noninvasively measured LS through TE and steatosis using CAP in a large cohort of pregnant women with diagnosed ICP, primarily through elevated bile acids. Our data clearly show that LS is higher in women with ICP than those in controls. Although LS decreased rapidly after delivery, as described recently[26,27], it remained significantly higher in women with ICP despite identical follow-up observation times. Women with ICP predominantly had elevated ALT levels, followed by those with elevated AST and bilirubin levels. In addition to AP levels, no differences were observed in GGT levels. Although all parameters, including LS, improved after delivery, GGT levels increased significantly in the ICP cohort. In addition, hepatic fat content, as measured using CAP, although within the normal range, was lower in patients with ICP than in controls. Finally, the liver apoptosis marker M30 showed the highest association with bile acid levels in univariate regression analysis, whereas LS remained the strongest independent predictor of bile acid levels > 20 mol/L in multivariate regression analysis.

In confirmation of earlier reports[26-28], the present study demonstrates that noninvasive assessment of LS through elastography is feasible and well accepted in pregnant women. In contrast to reports from internal medicine departments[34], elastography can be performed in all women. Second, we showed that LS is significantly elevated in women with ICP and higher than that in controls, which is remarkable, as we and others showed that LS is generally and reversibly elevated in the third trimester[26,27]. Consequently, and comparable to the previously reported LS elevation in women with preeclampsia, LS can be considered a feasible and noninvasive tool for screening, identifying, and following women with pregnancy-related liver complications.

What are the confounding factors for LS elevation in women with ICP? In contrast to initial beliefs, LS can be elevated because of many confounding factors, including inflammation, arterial and venous pressure elevation, and physiological conditions such as meal intake[22,24]. Mechanic cholestasis has been demonstrated to reversibly increase LS[35]. Of note, however, continued elevation of LS owing to these confounders has a negative impact on the liver, and the first long-term mortality data demonstrated that LS is one of the best long-term predictors of liver-related and all-cause mortality[22]. LS measurement can also be used to identify and monitor pregnant women with preexisting liver cirrhosis and predict hepatic decompensation[28]. The first study in women with uncomplicated pregnancies showed that elevated LS was significantly correlated with AP, leukocytes, gestational age, and an increase in body weight[26]. In the present study, in women with ICP, the confounders were completely different, and LS was tightly associated with elevated serum levels of bile acids and serum markers of liver apoptosis (M30). This is particularly interesting with regard to the fact that in liver diseases, such as alcoholic liver disease or viral hepatitis, LS elevation is typically associated with transaminase levels, namely, AST but not ALT[33]. Although we show here that LS is an independent predictor of elevated bile acids, the performance of LS in predicting ICP was only moderate and lower than that in the previous smaller ICP cohort[26].

To the best of our knowledge, this is the first study to show an exceptionally strong association between the established serum liver apoptosis marker (M30) with levels and bile acid levels in women with ICP and its association with elevated LS. In the multivariate analysis, LS remained the only parameter independently associated with elevated bile acid levels. In patients with liver disease, the association between liver apoptosis and LS elevation has already been shown both at the histological level[36] and using serum markers, such as M30[29]. The tight association of bile acids with LS and M30 levels in pregnancy is new. Bile acids are synthesized in hepatocytes as essential components for bile formation[9,17]. Owing to their detergent nature, however, they are highly cytotoxic and can disrupt cellular membranes if not protected, e.g., by phospholipids[9,17,19]. Specifically, serum cholic acid becomes the primary bile acid in women with ICP, in contrast to normal pregnant and non-pregnant women, whose proportion is similar to that of chenodeoxycholic acid[37]. Typical examples are cholestatic liver diseases, such as primary biliary cirrhosis, which ultimately cause chronic bile duct inflammation, and later cirrhosis and cancer. Even simple mechanical cholestasis through obstruction of the major bile ducts by biliary stones can cause severe tissue damage.

In the last three decades, many gene mutations have been discovered that can cause cholestasis through the impairment of hepatocyte or cholangiocyte transport proteins relevant to bile formation[8-10,18-20]. These findings have resulted in a group of diseases known as progressive familial intrahepatic cholestasis. The normal GGT levels in our ICP cohort are a strong argument for a genetic cause. Hormonal changes/normalization after delivery with subsequent normalization of bile acid export through the hepatocellular apical membrane are considered important for the role of sex hormones in ICP[9]. In line with this, we observed a postpartum increase in GGT in our ICP cohort, suggesting a reinduction of GGT with the onset of bile flow.

Surprisingly, hepatic steatosis, as measured by CAP, which is now widely explored in patients with fatty liver[38], did not show any conclusive data in women with normal pregnancy or with ICP. The reason for this remains unclear because we expected that at least some women would present with steatosis, which can be a severe complication of pregnancy. We also briefly discuss some of the limitations of our study, which are mostly due to the challenging setting of performing clinical studies during late pregnancy, particularly in women with suspected complications. Serum was not available for all women to allow for the subsequent measurement of markers such as M30 and M65. In addition, we managed to measure LS sequentially before and after delivery in only a few cases in the same person. Another limitation with regard to postpartum follow-up measurements was that we only included women 24 h after delivery. This short time may explain why some parameters did not reach statistical significance.

In conclusion, we showed in a large cohort that women with ICP show significantly elevated LS compared with women with uncomplicated pregnancies. In contrast to the large body of evidence in the liver literature, elevated LS in ICP is primarily correlated with the accumulation of bile acids known to be highly toxic to hepatocytes, and liver apoptosis, as measured through M30 levels, but not with transaminases, bilirubin, or GGT. We also showed for the first time that typically low GGT levels in ICP increase after delivery. Consequently, we believe that screening for LS in pregnancy is not “another diagnostic tool” to further complicate the already intensive surveillance during pregnancy, but could provide a novel non-invasive strategy to early identify women at risk for complications.