3.5 Diagnostic utility of ICP placenta differential lipids
We constructed ROC curves and calculated AUCs to evaluate the
probability of these lipids being diagnosed with ICP. As shown in Fig
5A-C, the AUCs of SM (d42:1), SM (d18:1/24:1) and PC (17:0/18:2) were
0.718, 0.768 and 0.748, respectively. And the AUCs of PE (18:0p/20:1),
dMePE (20:1p/16:0) and PE (16:0p/22:1) were 0.735, 0.723 and 0.764 (Fig
5D-F). We also evaluate the diagnostic utility of 5 lipids identified
from autophagy pathway analysis, results showed the AUCs of PE
(16:0/18:1), PE (16:0/20:2), PE (18:0/22:4), PE (18:1/20:3) and PE
(18:1/20:4) were 0.682, 0.649, 0.665, 0.659 and 0.657, respectively (Fig
S4). Using multiple logistic regression analysis, we found that a good
diagnostic model for ICP could be constructed by combining these lipids.
Analysis results of combined biomarker data yielded an AUC of 0.904 for
PE (16:0/20:2), SM (d42:1) and PC (17:0/18:2) (Fig 5G). The AUCs even
reach 0.933 when combining PE (16:0/20:2), SM (d42:1), PC (17:0/18:2)
and PE (16:0p/22:1) (Fig 5H). Therefore, the combination of these lipids
could be reliable, and a novel biomarker in predicting the risk of ICP.
Discussion
Intrahepatic cholestasis of pregnancy is associated with increased risk
of perinatal mortality and morbidity, however, the pathogenesis of ICP
is still unclear. There is no effective treatment for ICP currently,
once ICP is diagnosed, the only effective way to prevent adverse
pregnancy outcomes is delivery27. As an emerging field
that branches from metabolomics, more and more lipidomics proved that
lipids have multiple functions and are involved in many biological
processes. Previous studies suggested that placental lipid profiles, for
example, LDL cholesterol and sphingolipids differed between normal
pregnant women and ICP patients23, 28. Given that
lipid metabolic dysregulation potentially plays a role in pregnancy, a
better understanding of lipid metabolism could have significant clinical
implications for the diagnosis and treatment of ICP.
In this study, we found ICP placenta has a significantly different lipid
profile from normal pregnancy. A total of 61 lipids were found to be
dysregulated in mild ICP placenta, and the differentially expressed
lipids mainly include PE, PC, PS and SM species. We also identified 88
differential lipids between severe ICP placenta and normal pregnancy,
and these lipids mainly include PE, PC, PS, SM and TG species. Among
these lipids, we identified 44 lipids differentially expressed both in
mild and severe ICP group compared with the control group, mainly of
them were SM, PE, PC and PS species. Moreover, pathway analysis of the
placentas of patients with ICP identified autophagy regulation,
glycerophospholipid metabolism and GPI-anchor biosynthesis, suggesting
these pathways might be related to the pathogenesis of ICP.
Autophagy is an evolutionally conserved process that targets cytoplasmic
components such as organelles, protein aggregates or proteins for
degradation into lysosomes to maintain cellular homeostasis under
environmental stress29, 30. Autophagy plays a pivotal
role in implantation, embryogenesis and maintenance of
pregnancy31. A previous study demonstrated the
expression of LC3-II is elevated in ICP human placenta and placentas of
ICP rats, indicating autophagy is closely related to
ICP25. Zhang et al used TCA-treated term placental
villous for proteomic analysis, their results revealed protein
expression difference in ICP mainly related to autophagy and cell
metabolism32. Consistent with their result, Fang et al
analyzed 10 ICP and 10 normal placental tissue through quantitative
proteomics and their results showed the differentially expressed
proteins mainly participated in autophagy, autophagosome formation and
metabolism33. All of these indicate a close
correlation between ICP and autophagy. In our lipidomic analysis, we
also found the differentially expressed lipids mainly enriched in the
autophagy pathway, and PE (16:0/18:1), PE (16:0/20:2), PE (18:1/20:3),
PE (18:1/20:4) and PE (18:0/22:4) participated in this process.
Phosphatidylethanolamine (PE) is the second most abundant phospholipid
in the membranes of all mammalian cells34, 35. As a
fundamental component of biological membranes, PE is essential for many
cellular functions and the dysregulation of PE metabolism has been
implicated in many diseases, such as nonalcoholic liver disease,
atherosclerosis and obesity36, 37. In addition, PE is
essential for the formation of LC3-II in the autophagolysosomal bilayer
membrane. During autophagy, a cytosolic form of LC3-I is conjugated to
phosphatidylethanolamine to form LC3- phosphatidylethanolamine
conjugate, that is LC3-II, which is recruited to autophagosomal
membranes. The autophagosomal marker LC3-II could reflect autophagic
activity, and detecting LC3-II has become a reliable method for
monitoring autophagy and autophagy-related
processes38. Therefore, excessive accumulation of PE
(16:0/18:1), PE (16:0/20:2), PE (18:1/20:3) and PE (18:1/20:4) in ICP
placenta may lead to the formation of LC3-II and eventually the
activation of autophagy pathway, which has been confirmed to be involved
in the pathogenesis and development of ICP.
Through combined analysis with previous studies, we also identified SM
(d42:1), SM (d18:1/24:1) and PC (17:0/18:2) are differentially expressed
both in ICP placenta and plasma samples. And their good diagnostic
utility was also accessed through the construction of ROC curves. Two of
these three lipids were sphingolipids. The sphingolipid metabolic
pathway produces bioactive metabolites like sphingosine 1-phosphate
(S1P), ceramide and sphingosine, they can participate in various
biological processes such as cell survival, growth, vascular integrity
and inflammation. Sphingolipids have been proven to be related to
pregnancy-associated complications such as preeclampsia, recurrent
pregnancy loss and intrauterine growth
restriction39-41. A previous study reported the median
values of C16-Cer and C18-Cer were significantly increased in the plasma
of ICP patients, and after treatment with UDCA for a week, the
concentration of C16-Cer and C18-Cer decreased
considerably42. Consistent with their finding, Sun et
al also reported ICP is associated with disordered sphingolipid
homeostasis23. Collectively, this evidence proved that
sphingolipids may act as new diagnostic targets for ICP.
There are still several limitations in our study. First, further animal
and cellular experiments are needed to elucidate the specific mechanisms
of lipid changes. Second, many studies on lipidomics and ICP have been
conducted with a small number of patients, which limits the
generalizability of the findings. Larger studies are needed to confirm
the role of lipids in ICP. Finally, the diagnosis of ICP is currently
based on clinical symptoms and biochemical tests. However, there is a
lack of standardized criteria for the diagnosis of ICP, which can lead
to variability in patient selection and make it challenging to compare
results across studies.
In conclusion, we used UHPLC to characterize the placental lipidomics
profiling for women complicated with ICP. Our results confirmed there
were significant changes in lipid profile in the mid and severe ICP
placenta, the metabolic pathway was primarily associated with autophagy
and glycerophospholipid metabolism. And ICP placenta is associated with
dysregulation of sphingolipid homeostasis. Our research highlighted some
important mechanisms involved in the onset of ICP and might
provide new insight into the
treatment and diagnosis of ICP.