Non-visualisation of the fetal gallbladder (NVFGB) in the second trimester is a rare condition affecting 0.1% of pregnancies.1 It might be a transient finding—the gallbladder is visible later in pregnancy or after birth in 70% of cases.2 Persistent NVFGB may be associated with benign conditions (eg, gallbladder agenesis); it may also be a manifestation of serious disorders, such as biliary atresia (BA), cystic fibrosis, or chromosomal abnormalities.3 Although chromosomal abnormalities and cystic fibrosis can be identified prenatally via chromosomal microarray (CMA) and sequencing of the cystic fibrosis transmembrane conductance regulator gene, respectively, it remains challenging to diagnose BA before birth because no diagnostic prenatal test is currently available. Biliary atresia is a devastating condition and is the leading indication for liver transplantation in childhood.4 Prenatal suspicion of BA allows prompt postnatal assessment and early diagnosis, permitting timely intervention via Kasai hepatoportoenterostomy and resulting in improved outcomes.4

The measurement of gamma-glutamyl transferase (GGT) in amniotic fluid (AF) has been suggested as a method for prenatal detection of BA. This transferase is secreted by the fetal biliary tract, passes into the intestines, and is ultimately excreted into the amniotic cavity. It becomes detectable in AF at around 14 weeks of gestation upon maturation of the intestinal villi and opening of the cloacal membrane.5 6 7 Biliary tract obstructions, such as BA, hinder the passage of GGT into the intestines and AF, leading to reduced levels of amniotic fluid gamma-glutamyl transferase (AFGGT). Muller et al5 first reported extremely low AFGGT levels in three fetuses with extrahepatic bile duct obstruction at 18 to 19 weeks of gestation; they concluded that a low AFGGT level could be a useful indicator of BA. Subsequently, Burc et al,6 Dreux et al,8 and Bardin et al9 reported similar findings. Nevertheless, existing platforms for analysis of GGT in serum or plasma samples have not been thoroughly validated for use with AF samples. Furthermore, an appropriate reference range, essential for the interpretation of AFGGT results, is difficult to establish due to the limited availability of AF samples from normal pregnancies. Thus, publications regarding gestational age–specific reference ranges for AFGGT have been scarce.

Burc et al6 established reference values for five AF enzymes (AFEs), including GGT, using the Hitachi 911 analyser (Roche Diagnostics). Despite a large sample size of 508, no separate reference range was constructed for each gestational age from 20 to 24 weeks, limiting clinical use of the findings.6 Bardin et al7 established another reference range for AFGGT, from 16 to 22 weeks, using the Integra 800 analyser (Roche). However, the numbers of samples at weeks 16, 21, and 22 were small and had relatively large standard deviations, precluding clinical application.7 Both reference ranges were derived from Caucasian populations. Because the GGT level in adult blood varies according to ethnicity,10 it is likely that AFGGT levels also vary according to ethnicity; thus, there is a need to establish a local AFGGT reference range. Furthermore, both previous reference ranges covered the 5th to 95th percentiles. A wider reference range, from the 2.5th to 97.5th percentiles, would allow greater flexibility in selecting cut-off values for clinical application.

In this study, we aimed to validate a serum/plasma matrix–based GGT assay for AF samples, establish a local gestational age–specific reference range for AFGGT (from the 2.5th to 97.5th percentiles), and evaluate the efficacy of AFGGT for predicting fetal BA in pregnancies with NVFGB using the constructed reference range.

This retrospective study used archived AF supernatant obtained from amniocentesis procedures that had been conducted to exclude fetal chromosomal abnormalities between January 2012 and December 2021. All amniocenteses were performed under ultrasound guidance with aseptic technique by fetal-maternal medicine specialists who also performed detailed ultrasound examinations to document the presence or absence of fetal structural abnormalities. All AF samples were centrifuged at 100 g for 10 minutes; 1 mL of the resulting supernatant was stored at -80℃ in plain aliquots without additives (Axygen; BioGene, Union City [CA], United States), whereas the pellet was used for either CMA and/or karyotyping by G-banding analysis. The cytogenetic laboratory information system recorded the ultrasound findings, indication for amniocentesis, gestational age at amniocentesis, and CMA and/or karyotype results. Pregnancy and fetal outcomes, including the presence of any abnormalities at birth or autopsy findings, were recorded in each patient’s electronic records.

Archived AF samples with known fetal and pregnancy outcomes, taken at 16⁺⁰ to 22⁺⁶ weeks of gestation from singleton pregnancies with a euploid fetus (confirmed by CMA or karyotype) during the period from January 2018 to December 2020, were retrieved to establish a gestational age–specific reference range for GGT. Pregnancies with fetal chromosomal, gastrointestinal, or hepatobiliary anomalies (particularly BA) polyhydramnios, or oligohydramnios were excluded to eliminate potential confounding effects of these conditions.

Non-visualisation of the fetal gallbladder was incidentally detected during fetal morphology scans in pregnancies with risk factors for fetal abnormalities because the fetal gallbladder was not assessed in low-risk routine anomaly scans, in accordance with the International Society of Ultrasound in Obstetrics and Gynecology guideline.11 It was defined as failure to visualise the fetal gallbladder on two targeted ultrasound examinations performed 1 week apart. Isolated NVFGB was defined as NVFGB in the absence of other abnormal ultrasound findings. Pregnant women were counselled regarding possible differential diagnoses and offered amniocentesis for chromosomal analysis, as well as repeated ultrasound scans until the fetal gallbladder was visible. After birth, babies with persistent NVFGB were referred to paediatricians for hepatobiliary ultrasound and liver function tests. If the gallbladder was visible during prenatal scans, paediatricians did not order further tests in the absence of clinical suspicion. We followed the progress of all babies until the time of writing, using electronic hospital records for those delivered in public hospitals and phone calls for those delivered in private hospitals. With parental consent, post-mortem examinations were arranged in pregnancies terminated for serious associated fetal abnormalities.

Amniotic fluid samples were removed from -80℃ storage in batches, thawed, and equilibrated to room temperature immediately prior to analysis. Gamma-glutamyl transferase levels were determined using an International Federation of Clinical Chemistry and Laboratory Medicine–standardised L-gamma-glutamyl-3-carboxy-4-nitroanilide (GGCNA) enzymatic colorimetric assay on a Cobas c502 analyser (Roche, Basel, Switzerland). Internal quality controls were performed before and after each batch.

Details of the AFGGT assay validation and analytical performance, including matrix effects; linearity; intra- and inter-run precision at various GGT levels; interference due to haemolysis, icterus, and lipaemia; and sample stability, are summarised in online supplementary Appendix 1.

An AFGGT reference range was developed using the Generalised Additive Models for Location (μ), Scale (υ) and Shape (σ) [GAMLSS] package in R statistical software (version 3.3.2; R Foundation for Statistical Computing, Vienna, Austria). All GGT values were transformed to their natural log equivalent before model construction; the final model balanced between percentile smoothness, goodness-of-fit, and simplicity. Model fit was assessed using the generalised Akaike information criterion and by inspection of residuals with quantile-quantile plots for all measurements, detrending of quantile-quantile plots, and comparison of empirical percentiles to fitted percentiles. Empirical percentiles were determined for comparative purposes by grouping GGT levels according to gestational age (in weeks).

The final model was used to determine reference values for the 2.5th, 5th, 50th, 95th (z = ± 1.645), and 97.5th percentiles (z = ± 1.964). Percentiles were determined using the expression μ × (1 + z_pυσ)1/υ, where z_p is the percentile of interest and μ, υ, and σ are dependent on the time covariate, gestational age. The GGT reference range was constructed using R statistical software and Microsoft Excel (Microsoft Corporation, Redmond [WA], United States).

We estimated a priori that 293 AFGGT measurements were needed to achieve a standard error of 10% of the gestational age–specific standard deviation for the 2.5th and 97.5th (z=1.96) reference percentiles, assuming that the standard error of the percentile of interest is expressed as a multiple of standard deviation using the formula , where each gestational age between 16⁺⁰ and 22⁺⁶ weeks has a minimum of 42 measurements.

Measured levels of AFGGT in pregnancies with NVFGB were transformed to their gestational age–specific percentile values using the final model. We then determined the positive predictive value (PPV) and negative predictive value (NPV) of AFGGT level <2.5th percentile for identifying BA in euploid pregnancies with NVFGB.

The performance of the GGT assay using AF is summarised in online supplementary Appendix 2. Validation studies indicated that the GGCNA enzymatic colorimetric assay for determination of GGT activity in AF had linearity, precision, recovery, interference profiles, and stability comparable to the values reported for measurement of GGT in plasma and serum samples. The verified analytical measurement range was 10 to 1200 U/L. No significant interference was observed in the presence of 0.25 g/dL haemoglobin, 103 μmol/L bilirubin, or 16.8 mmol/L triglyceride. The limits of haemolysis/icterus/lipaemia indices above which interference occurred were significantly higher than the degrees of those indices in all analysed samples.

A database search identified 518 stored amniotic samples (502 [97%] Chinese and 16 [3%] other Asian ethnicities) suitable for use in establishing a local gestational age–specific AFGGT reference range. The median number of samples per week was 65; there were 2 weeks with <42 samples (27 and 28 samples in the 16th and 19th week of gestation, respectively). The online supplementary Table lists the regressed values of AFGGT levels according to percentile for each week of gestation.

The simplest best-fit model indicated a linear relationship between natural log-transformed GGT and gestational age. Table 1 and the online supplementary Figure show the final smoothing equations for median, coefficient of variation, and skewness, along with gestational age–specific smoothed percentile curves and values of AFGGT for our local population. Residuals of the final model had a mean skewness of 0 and a variance of 1; they were almost mesokurtic, with kurtosis values close to 3.

The database search identified 32 pregnancies with NVFGB from 2012 to 2021, of which 18 had an available AF sample (four isolated NVFGB and 14 non-isolated NVFGB). There were no cases of cystic fibrosis. Nine cases were excluded from analysis, including five with chromosomal abnormalities, one with renal hypoplasia and oligohydramnios, one with hydrops and polyhydramnios, and two in which the biliary tract anatomy could not be identified. The nine remaining cases for analysis included four with isolated NVFGB and five with non-isolated NVFGB (Table 2). The median gestational age at amniocentesis was 21.0 weeks (interquartile range=20.7-22.1).

The Figure depicts the AFGGT levels in the nine pregnancies for analysis compared with our local gestational age–specific AFGGT reference range. Four cases had AFGGT level <2.5th percentile, including one with BA (AFGGT level of 27 U/L at 20⁺³ weeks) and three with transient non-visualisation (AFGGT levels of 32, 68, and 37 U/L at 20⁺⁰, 20⁺⁶, and 22⁺² weeks, respectively). Five had AFGGT level ≥2.5th percentile, including one with gallbladder agenesis and four with transient non-visualisation (Table 2). Using AFGGT level <2.5th percentile as the cut-off, the NPV and PPV for identifying fetal BA in pregnancies with NVFGB were 100% and 25% (95% confidence interval=0, 53), respectively (Table 3). Repeated analysis using AFGGT level <5th percentile or the reference ranges from Burc et al6 or Bardin et al7 yielded identical results.

Biliary atresia is a rare congenital anomaly, with a prevalence of 1 in 15 000 to 20 000 live births among Caucasian populations.12 However, BA is more common in East Asians; the prevalence is 1 in 5000 to 7000 among Chinese populations.13 14 Untreated BA is a progressive and devastating disease that can cause cirrhosis and death by 2 years of age.14 This outcome can be prevented by early intervention via palliative Kasai hepatoportoenterostomy, which is essential for re-establishing biliary drainage. If biliary drainage cannot be re-established, liver transplantation is necessary. Indeed, BA is the most common indication for liver transplantation in children, contributing to 75% of transplantations in children before 2 years of age.12 It is therefore imperative to diagnose BA early, preferably during the prenatal period. Nevertheless, prenatal diagnosis of BA is challenging because ultrasound cannot directly examine the patency of fetal bile ducts. When NVFGB is associated with a hepatic hilar cyst or heterotaxy, it is highly suggestive of BA.15 Non-visualisation of the fetal gallbladder with a hepatic hilar cyst is an indicator of cystic BA, a rare subtype representing 5% to 10% of BA cases16; therefore, this prenatal combination is uncommon. Heterotaxy is another rare condition with a prevalence of 1 in 10 000 live births,17 and concurrent BA is present in only 10.4% of left atrial isomerism cases18; therefore, this prenatal combination is even less common. Consequently, NVFGB may be the only prenatal sign indicating the possibility of BA. However, in cases of isolated NVFGB, it is difficult to differentiate between BA and gallbladder agenesis. The discovery of the association between low AFGGT levels and fetal BA has led to interest regarding the role of AFGGT in the management of NVFGB.

To our knowledge, this study is the first to validate the International Federation of Clinical Chemistry and Laboratory Medicine–standardised GGCNA enzymatic colorimetric assay on the Cobas c502 analyser, a common locally available plasma and serum analyser, for use with AF. We have demonstrated that accurate and precise measurements of GGT can be achieved with AF samples, enabling adoption in clinical settings. We have also established that the analytical measurement range is 10 to 1200 U/L. This is particularly important for an AFGGT assay because AFGGT levels in euploid pregnancies can vary across multiple orders of magnitude, whereas plasma GGT levels in healthy individuals usually remain below 100 U/L. This verification of the lower limit of quantification and linearity range improves confidence in our measurements. Furthermore, we have excluded potential interference, particularly from haemoglobin, because AF samples may sometimes contain maternal blood. We were initially concerned about GGT stability because some samples had been stored for several years; however, consistent with the World Health Organization report that GGT is stable for years in frozen serum and plasma samples,19 we found that AF stored frozen in plain bottles without additives at -20℃ or -80℃ remained stable for at least 6 months (online supplementary Appendix 1). This finding indicates that supernatants can be stored and subsequently retrieved for GGT assays, an important consideration if amniocentesis is performed in the early second trimester but an indication for AFGGT testing is identified during a mid-trimester morphology scan.

We have established a reference range for AFGGT levels at 16⁺⁰ to 22⁺⁶ weeks of gestation using a large local reference population of 518 samples (all Asian, 97% Chinese); this reference population is the largest compared with similar previous publications. The presence of an adequate sample size for each week of gestation allowed us to establish a reference range for each gestational age, thus overcoming the aforementioned limitations regarding clinical use of the two previous reference ranges.6 7 With respect to the two previous reference ranges, the 5th, 50th, and 95th percentiles in our study are similar to those reported by Bardin et al7 but higher at most gestational ages than those reported by Burc et al.6 The larger sample size in our study permitted the calculation of the 2.5th and 97.5th percentiles, such that a 95% central reference range could be established; this allows greater flexibility in selecting cut-off values for clinical application.

It has been reported that AFGGT levels are increased in oesophageal atresia and duodenal atresia, whereas they are decreased in anal atresia without fistula.20 21 Although duodenal atresia can easily be diagnosed prenatally by detection of the double bubble sign, this sign usually appears after 24 weeks of gestation.22 Prenatal diagnosis of oesophageal atresia relies on the indirect sign of non-visualisation of the stomach bubble and polyhydramnios; the sensitivities of these ultrasound findings range from 8.9% to 42%.20 Additionally, prenatal detection of anal atresia without fistula depends on the absence of the perianal muscular complex, but the anal sphincter does not fully mature until after 28 weeks of gestation.23 Further research regarding the use of AFGGT for early detection of these congenital gastrointestinal tract obstructions is valuable, and the availability of a local gestational age–specific reference range is crucial for supporting such research.

In the present study, the NPV and PPV of AFGGT level <2.5th percentile for identifying fetal BA in NVFGB were 100% and 25%, respectively (Table 3). Bardin et al9 assessed the efficacy of low AFGGT levels in predicting BA among cases of NVFGB between 17 and 22 weeks of gestation. Of the 26 cases with AFGGT level ≥5th percentile, none had BA; of the four cases with AFGGT level <5th percentile, three had BA. The corresponding NPV and PPV were 100% and 75%, respectively. The PPV in their study may have been higher because amniocentesis was performed before 22 weeks of gestation in all cases.9 In our cohort, after exclusion of the two cases in which amniocentesis was performed after 22 weeks of gestation, the PPV only marginally improved to 33.3%. Our figures are more consistent with those of Dreux et al,8 who analysed the efficacy of AFEs for predicting BA in NVFGB before and after 22 weeks of gestation. In that study, abnormal AFE was defined as GGT and/or intestinal alkaline phosphatase level <0.5 multiples of the median.8 Before 22 weeks of gestation, there were three cases of BA among seven cases with abnormal AFE and no cases of BA among 16 cases with normal AFE. The corresponding NPV and PPV were 100% and 43%, respectively. However, after 22 weeks of gestation, there was only one case of BA among six cases with abnormal AFE and four cases of BA among 56 cases with normal AFE. The corresponding NPV and PPV were 93% and 17%, respectively (Table 3). These findings confirmed the expected decrease in AFE efficacy for predicting BA after 22 weeks of gestation. By that gestational age, the passage of GGT from the intestine into the AF is impeded by mature anal sphincter muscles in normal fetuses; thus, the AFGGT level is very low after 22 weeks of gestation, and it is difficult to distinguish between a low level due to BA and a low level related to normal development.5 6 7

In our cohort, there were three fetuses without BA who had AFGGT level ≤2.5th percentile; one of these fetuses had an extremely low AFGGT level (Case 4) [Table 2]. In addition to its association with fetal BA, low AFGGT is linked to chromosomal abnormalities, cystic fibrosis, anal atresia without fistula, and polyhydramnios. However, none of the three fetuses had any of these conditions. Although one fetus (Case 2) [Table 2] had a small choledochal cyst which might have impeded biliary drainage and caused a mild decrease in AFGGT, we could not find a potential explanation for the low AFGGT levels in the other two fetuses.

Limitations of our study include its retrospective nature and small cohort size. The incidence of NVFGB is low (0.1%).1 In the largest systematic review concerning the outcomes of NVFGB, encompassing seven studies, the total number of cases was 280; among 170 cases of isolated NVFGB, only six (3.5%) had BA.2 In Hong Kong, the fetal gallbladder is not routinely assessed in low-risk routine anomaly scans, in accordance with the International Society of Ultrasound in Obstetrics and Gynecology guideline.11 24 Among the 32 cases of NVFGB in our cohort, one (3.1%) had BA; this incidence is comparable to the findings in the aforementioned review. However, only 18 cases had an AF sample available for AFGGT testing; after the exclusion of cases with abnormalities that might affect the AFGGT level, we included nine cases in the analysis. Because there was only one case of BA in our cohort and 11 more were described in the literature (Table 3), clinical application of these research findings requires caution, as well as careful pre- and post-test counselling.

In addition to the one case of BA in this cohort, two additional cases of BA were not included in this study because prenatal ultrasound scans did not indicate NVFGB. The AFGGT levels in all three cases were <30 U/L and <2.5th percentile (27, 28, and 29 U/L at 20⁺³, 21⁺⁵, and 21⁺⁶ weeks, respectively), whereas the AFGGT levels in all samples used to establish our reference range were >30 U/L. Thus, an AFGGT level <30 U/L may be a useful absolute cut-off for the prediction of BA. Notably, all three cases of extrahepatic bile duct obstruction reported by Muller et al5 also had an AFGGT level <30 U/L (20 U/L for all three cases [<1st percentile]). Further research with a larger cohort is required to confirm the efficacy of using an AFGGT level <30 U/L as the absolute cut-off for predicting BA in NVFGB.

Based on the present findings and published literature, we conclude that AFGGT testing is useful for the exclusion of fetal BA in pregnancies with NVFGB. With a consistent NPV of 100% across all published series, AFGGT level ≥2.5th percentile can provide reassurance for parents that the fetus is unlikely to have BA. However, considering its PPV of 33.3% to 75% before 22 weeks of gestation and 17% to 43% after 22 weeks of gestation, AFGGT level <2.5th percentile cannot be considered diagnostic for BA. Instead, it serves as a warning sign, indicating the need for prompt postnatal investigation of possible BA. Because NVFGB is also associated with chromosomal abnormalities, amniocentesis is recommended; the advantages of detecting underlying chromosomal abnormalities by CMA and excluding BA through AFGGT testing likely outweigh the 0.3% risk of procedure-related miscarriage.25 Follow-up prenatal ultrasound scans to visualise the fetal gallbladder should be arranged. Paediatricians should also be alerted for prompt postnatal assessment to facilitate early detection of BA. Timely performance of the Kasai operation can reduce the need for liver transplantation in childhood and improve the rate of overall survival into adulthood to 90%.26 27

Concept or design: YH Ting, DS Sahota, FCK Wong, TYT Cheung.
Acquisition of data: TYT Cheung, SR Subramaniam, FCK Wong, MHM Chan, YH Ting, NKL Wong, SL Lau, X Zhu, WT Lui, EKW Chan, YKY Kwok, KW Choy, TY Leung.
Analysis or interpretation of data: TYT Cheung, DS Sahota, FCK Wong, YH Ting, NKL Wong.
Drafting of the manuscript: YH Ting, TYT Cheung, DS Sahota, NKL Wong, FCK Wong, SR Subramaniam.
Critical revision of the manuscript for important intellectual content: DS Sahota, YH Ting, TYT Cheung, FCK Wong, NKL Wong.

All authors had full access to the data, contributed to the study, approved the final version for publication, and take responsibility for its accuracy and integrity.

All authors have disclosed no conflicts of interest.

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

This research was approved by the Joint Chinese University of Hong Kong—New Territories East Cluster Clinical Research Ethics Committee, Hong Kong (Ref No.: CRE 2020.060). Informed consent for amniocentesis in the current study and storage and use of excess amniotic fluid in future research was obtained from the patients at the time of amniocentesis.

The supplementary material was provided by the authors and some information may not have been peer reviewed. Accepted supplementary material will be published as submitted by the authors, without any editing or formatting. Any opinions or recommendations discussed are solely those of the authors and are not endorsed by the Hong Kong Academy of Medicine and the Hong Kong Medical Association. The Hong Kong Academy of Medicine and the Hong Kong Medical Association disclaim all liability and responsibility arising from any reliance placed on the content.

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