Strengths and limitations
The main strength is the large cohort of pregnancies analysed (612 patients), which to our knowledge is the largest sample of healthy pregnant women that has been examined for TSBA so far.
We believe the strengths also include the prospective design, the accurate selection of eligible patients, the generalizability, and the clinical relevance of the results. Since no correlation has been reported between TSBA levels and gestational age within the third trimester26, our results could be applicable to the third trimester of pregnancy. The duration of the study over one year allowed us to evaluate the seasonality of TSBA levels.
Finally, all blood samples were analysed in the same laboratory and were taken ad hoc for the study purposes.
The low variability of ethnicity was due both to the majority of Caucasian patients in our hospital and to the difficulty of collecting informed consent from non-Italian speakers. This limited our ability to evaluate variations in normal TSBA levels between different ethnic groups.
Even though widely used in clinical laboratories, the most important limitation of the enzymatic method is low sensitivity, as the lowest concentration of TSBA measurable is around 1.5 µmol/L18, 29. Nevertheless, 100% of the measured values ​​resulted above the evaluation limit.
Another limitation is that common hospital food was provided, not standardized for macronutrients and potentially different from each other.

Interpretation

Evidence that normal pregnancy may be associated with a mild sub-cholestatic state has been described in early studies. Although based on a limited number of patients, an increase has been demonstrated in the mean of single and total BA concentration in uncomplicated pregnancies with no other evidence of ICP13-16.
Our results are consistent with early observations that pregnancy is a sub-cholestatic state13-16, and this can be explained by the cholestatic effect of reproductive hormones. Normal pregnancy is an hyperestrogenic state and therefore is associated with a physiological elevation of TSBA. Thus, obstetricians need to be aware that healthy pregnant women have increased levels of TSBA when assessing women who may have ICP.
Early studies described that ICP have a seasonal pattern with increased incidence in some countries during winter months, suggesting a possible association with an environmental trigger. This pattern, however, was not yet demonstrated in clinical studies. Two possible explanations are low levels of natural selenium and of vitamin D during the winter, as both deficiencies have been reported in women with ICP1,5,19,20.
It has recently been suggested that higher thresholds should be used for the diagnosis of ICP, also given the low risk of stillbirth demonstrated recently for TSBA levels < 100 µmol/L26, 29.
The assessment of diagnostic criteria for ICP is complex, as the classic methodologies used to establish diagnostic thresholds for other pathologies are difficult to apply here. Currently, in clinical practice, ICP is diagnosed by elevated TSBA levels that are measured after the insurgence of pruritus, a non-specific symptom that is not necessarily associated with ICP. Thus, the diagnosis is based on TSBA alterations (as pruritus without TSBA elevation will not be diagnosed as ICP) by using non-pregnant reference ranges. In non-pregnant patients TSBA levels are used as a biomarker for hepatic injury, while in pregnant patients, the outcome of interest is not liver dysfunction but rather the absolute elevation in circulating TSBA levels4. Beside pruritus, TSBA elevation is not associated with adverse outcome unless reaching a severe ICP (> 100 µmol/L for stillbirth and > 40 µmol/L for other adverse outcomes), thereby the diagnostic threshold cannot be tested by maternal or neonatal outcomes either.
For these reasons, an important limitation when studying the normal distribution of TSBA levels in pregnancy is that the accuracy (sensibility and specificity) of the upper limit value of normality among the ICP population cannot be calculated, as ICP was originally diagnosed by the same criteria that needs to be tested.
Our results demonstrate that the non-pregnant reference range cannot be used in the pregnant population. In accordance with the RCOG clinical guidelines for ICP25, we suggest using in clinical practice the upper limit value of our normal pregnancy-specific refence range. Specifically, 14 µmol/L for fasting TSBA values and 20 µmol/L for postprandial TSBA values. A similar threshold (19 µmol/L) was suggested by a recent study that re-evaluated the diagnostic thresholds for ICP. Although using different methodologies, our postprandial threshold was similar to the random threshold described by Mitchell et al26. In accordance, we also suggest that patients with otherwise unexplained pruritus with TSBA levels below the proposed thresholds should repeat the TSBA measurements since pruritus often precedes an elevation in TSBA levels2,34.
There is ongoing debate regarding which value should be used in clinical practice to diagnose ICP. Postprandial TSBA assessment may be a more sensitive test, whereas elevated fasting TSBA levels are a more specific indicator of severe liver disease11,25. Some authors suggest measuring TSBA levels in the fasting state due to the large overlap between normal non-fasting values and commonly used thresholds for diagnosis29. On the other hand, considering that the adverse perinatal outcomes of ICP are associated with peak TSBA concentration, a non-fasting measurement has greater clinical relevance26.
We suggest using both values ​​of TSBA. We recommend distinguishing the fasting and postprandial status and avoiding random samplings due to different reference ranges and upper limit values that should be applied. Fasting TSBA levels may be the most specific for the diagnosis of ICP and therefore better for confirming ICP. Fasting measurements are also more predictable, have less variability, and correlate better with certain risk factors. The postprandial measurement, on the other hand, is essential for risk stratification. We suggest using postprandial TSBA levels to assess the severity of the disease and to follow-up patients with ICP for subsequent management of the pregnancy and eventual active management.