The fetal gallbladder can be observed by antenatal ultrasound scan at 14 weeks of gestation.1 During the morphology scan at approximately 20 weeks of gestation, >99% of fetal gallbladders can be observed; in 75% of cases of non-visualisation of fetal gallbladder (NVFGB), a gallbladder is clearly present during subsequent scans.2 However, the visualisation rate drops to 75% to 85% after 32 weeks of gestation when the gallbladder becomes contractile.3 4

Although sonographic examination of the fetal gallbladder is not technically difficult, the International Society of Ultrasound in Obstetrics and Gynecology and other professional bodies have not yet included the gallbladder as a routine component of the mid-trimester anatomical survey.5 6 7 8 The problem with routine examination of the fetal gallbladder is that non-visualisation of the gallbladder can lead to challenging counselling and antenatal diagnosis because NVFGB is related to a wide spectrum of fetal conditions. While NVFGB may be a transient phenomenon in a normal fetus or the result of gallbladder agenesis (a benign congenital anomaly), it can also be associated with more serious underlying conditions such as biliary atresia, cystic fibrosis, or chromosomal abnormalities. While it is generally simple to identify chromosomal abnormalities and cystic fibrosis by amniocentesis, the antenatal diagnosis of biliary atresia is challenging because no diagnostic antenatal test is currently available. Because biliary atresia can be fatal without early postnatal intervention and may eventually require liver transplantation, uncertainty regarding the antenatal diagnosis of such a condition may cause significant parental anxiety; some parents may even consider termination of pregnancy to avoid the risk of a severe abnormality in their child.9 10

A systematic review of isolated NVFGB in Western populations revealed that the incidences of transient non-visualisation, gallbladder agenesis, biliary atresia, cystic fibrosis, and chromosomal abnormalities were 69.4%, 24.7%, 3.5%, 2.4%, and 1.4%, respectively. The incidences of biliary atresia, cystic fibrosis, and chromosomal abnormalities were higher in cases of non-isolated NVFGB with additional sonographic abnormalities: 18.2%, 23.1%, and 20.4%, respectively.11 However, the incidences may differ considerably among Chinese women with NVFGB, as cystic fibrosis is uncommon in Asian populations, while biliary atresia is more prevalent in Chinese individuals.12 13 14 15 16 The aim of this study was to investigate the outcomes of fetuses with NVFGB in a cohort of Chinese women; the findings may provide guidance for the management of NVFGB.

This retrospective review included cases of NVFGB among Chinese pregnant women at five public fetal medicine clinics in Hong Kong from 2012 to 2019. In these clinics, fetal morphology scans were limited to high-risk cases and fetal gallbladder assessment was not routinely performed, in accordance with guidelines from the International Society of Ultrasound in Obstetrics and Gynecology.5 17 When cases of NVFGB were detected incidentally or referred from private clinics, the pregnant women were provided counselling regarding possible differential diagnoses and offered amniocentesis for chromosomal analysis. In cases of serious fetal abnormalities where parents decided for legal termination of pregnancy before 24 weeks of gestation, post-mortem examinations were arranged with parental consent. After birth, babies with NVFGB were referred to paediatricians for further evaluation.

The following data were reviewed: demographic information, gestational age at detection of NVFGB, findings during the morphology scan and subsequent scans, results of all amniotic fluid investigations if amniocentesis had been performed, pregnancy outcome, all neonatal imaging reports, operations performed on the baby and intra-operative findings, and autopsy findings in case of termination of pregnancy. The cases were segregated into isolated and non-isolated groups according to the absence or presence of additional sonographic findings. The incidences of subsequent visualisation of gallbladder, gallbladder agenesis, biliary atresia, cystic fibrosis, and chromosomal abnormalities were compared between the two groups.

The study protocol was approved by the Joint Chinese University of Hong Kong–New Territories East Cluster Clinical Research Ethics Committee on 3 March 2020 (CREC Ref. No.: 2020.060). This manuscript was written in accordance with STROBE reporting guidelines.

Fisher’s exact test was used for comparisons between the isolated and non-isolated groups. All statistical analyses were performed using SPSS software (Windows version 22.0; IBM Corp., Armonk [NY], United States). P values <0.05 were considered statistically significant.

Among 19 cases of NVFGB detected at a median gestational age of 21.3 weeks (interquartile range, 20.0-22.3 weeks), 10 (52.6%) were isolated and nine (47.4%) were non-isolated. Eleven (58.0%) cases had transient non-visualisation, four (21.0%) had gallbladder agenesis, three (15.8%) had chromosomal abnormalities (trisomy 18, trisomy 21, and 22q11.2 de novo microduplication), and one (5.2%) had biliary atresia. There were no cases with features suggestive of cystic fibrosis (Tables 1 and 2).

Amniocentesis was performed in eight of the nine non-isolated cases; three chromosomal abnormalities (33.3%) were found, including trisomy 18 (case 1), trisomy 21 (case 2), and 22q11.2 de novo microduplication (case 3). Case 1 ended in neonatal death and the parents declined post-mortem investigation. Case 2 was a dichorionic twin pregnancy; selective feticide was performed on the fetus with trisomy 21, but post-mortem investigation could not be performed because of the long interval between selective feticide and delivery of the normal co-twin. Termination of pregnancy was performed in case 3 and gallbladder agenesis was confirmed at autopsy. Termination of pregnancy was also performed in case 4 because of multiple structural abnormalities; chromosomal microarray (CMA) findings were normal and post-mortem investigation revealed normal gallbladder. Live birth occurred in the remaining five cases; in one case, the gallbladder was observed during a subsequent antenatal scan and the postnatal outcome was normal, while NVFGB persisted in the other four cases. Among the four cases with persistent NVFGB, one (11.1%) had biliary atresia that required liver transplantation (case 6); antenatal scans had shown a hepatic hilar cyst (Fig 1), which was highly suggestive of cystic biliary atresia.18 Postnatal examination showed a normal gallbladder in the other three cases. None of the five live births had features suggestive of cystic fibrosis (Table 1 and Fig 2).

Among the 10 cases of isolated NVFGB, amniocentesis was performed in one, while chorionic villi sampling was performed in the first trimester because of positive Down syndrome screening result in another; CMA findings were normal in both cases. In four cases, gallbladders were observed during subsequent antenatal scans at a mean follow-up interval of 1.0 week (range, 0.3-2.0); NVFGB persisted in the other six cases. Among the six cases with persistent NVFGB, gallbladder agenesis was confirmed in four (66.7%) and gallbladders were observed after birth in two (33.3%). None of the 10 cases had features suggestive of cystic fibrosis after birth (Table 1 and Fig 2).

The characteristics of isolated and non-isolated groups are compared in Table 2. The incidence of serious abnormalities (chromosomal abnormalities, biliary atresia) was significantly higher in the non-isolated group than in the isolated group (44.4% vs 0%; P=0.029). Notably, all serious conditions in the cohort (all three cases of chromosomal abnormalities and the only case of biliary atresia) were observed in the non-isolated group, while all benign conditions (all four cases of isolated gallbladder agenesis) were observed in the isolated group. The incidences of transient non-visualisation did not significantly differ between the isolated and non-isolated groups (60.0% vs 55.6%; P=1.000).

In our cohort, cases of isolated NVFGB had a good overall prognosis, with a 60% probability that the gallbladder would be observed in a subsequent scan and a 40% probability of gallbladder agenesis. This incidence of transient non-visualisation (60%) is consistent with findings by Yayla and Bayik2 (75%) and Di Pasquo et al11 (69.4%). In our cases of transient NVFGB, the gallbladder was observed during a subsequent antenatal scan in 40%, and during the postnatal period in the remaining 20%. The mean interval between NVFGB and subsequent antenatal detection of the gallbladder was 1 week. Therefore, a second sonographic examination within 1 week after NVFGB would help to alleviate parental anxiety and avoid the need for further investigations in nearly half of such cases. Even in cases with persistent isolated NVFGB, the prognosis remains good, because the gallbladder is likely to be observed after birth in one-third of cases; while gallbladder agenesis is likely in the remaining cases. Our findings are similar to the results of a recent systematic review of seven studies in Western populations, including 217 cases of isolated NVFGB; most cases had transient non-visualisation (69.4%) and gallbladder agenesis (24.7%), but some cases had serious conditions (biliary atresia [3.5%], cystic fibrosis [2.4%], and chromosomal abnormalities [1.4%]).11 Therefore, further investigations to rule out such serious abnormalities remain important in cases of isolated NVFGB.

In the aforementioned review, the incidences of biliary atresia, cystic fibrosis, and chromosomal abnormalities were much higher when NVFGB occurred in combination with other ultrasound abnormalities (18.2%, 23.1%, and 20.4%, respectively).11 Copy number variants were observed in one of three cases with chromosomal abnormalities in our study and two of 11 such cases in the study by Di Pasquo et al11; these findings support the recommendation for the use of CMA, rather than karyotyping.19 20 21 However, our results differ from the findings reported by Di Pasquo et al11 in that none of our cases had cystic fibrosis, which is unsurprising because cystic fibrosis is rare in Chinese individuals; moreover, our incidence of biliary atresia (11.1%) was much lower than expected, considering that biliary atresia is reportedly threefold more common in Chinese individuals than in Caucasian individuals.12 13 14 15 16

Based on the data described above, in cases of non-isolated NVFGB or persistent isolated NVFGB, amniocentesis may help to rule out chromosomal abnormalities; this approach is generally simple with current CMA technology.19 20 However, the antenatal diagnosis of biliary atresia is challenging because fetal bile duct patency cannot be determined by sonographic examination; NVFGB may be the only suggestive sign of biliary atresia. When NVFGB is associated with a hepatic hilar cyst or heterotaxy, a diagnosis of biliary atresia is likely.18 However, it is difficult to differentiate biliary atresia from gallbladder agenesis in cases of isolated NVFGB. Thus, a low amniotic fluid gamma-glutamyl transpeptidase (AFGGT) level has been proposed as an indicator of biliary atresia.22 23 24 Gamma-glutamyl transpeptidase (GGT) is initially derived from the fetal biliary tract, passed into the gastrointestinal tract, and finally excreted into the amniotic fluid. The AFGGT level decreases with increasing gestational age because progressive maturation of the anal sphincter impairs the passage of GGT from the gastrointestinal tract into the amniotic fluid.22 25 26 The anal sphincter muscles become fully mature by 20 weeks of gestation, and the AFGGT level becomes very low after 22 weeks of gestation. Therefore, it may be difficult to distinguish between a low level related to biliary atresia and a low level related to normal development after 22 weeks of gestation.25 26 Using an AFGGT level below the 5th centile, Bardin et al23 reported 100% sensitivity in the detection of biliary atresia, with a false positive rate of 4%, between 17 and 22 weeks of gestation (Table 3). Using AFGGT level and/or intestinal alkaline phosphatase <0.5 multiples of the median, Dreux et al24 also reported 100% sensitivity before 22 weeks of gestation; however, their false positive rate was 20%. Notably, when the test was performed after 22 weeks of gestation, the sensitivity decreased to 20%. Therefore, gestational age at amniocentesis is a critical consideration during the assessment of biliary atresia; if NVFGB is first detected near 22 weeks of gestation, amniocentesis should be performed immediately, rather than waiting for sonographic examination to be repeated. Another limitation of using the AFGGT level to identify biliary atresia is that it has a moderately low positive predictive value: 43% to 75% before 22 weeks of gestation, and 17% thereafter.23 24 Accordingly, a positive AFGGT test result is not diagnostic of biliary atresia, particularly in cases of isolated NVFGB where the incidence of biliary atresia is presumably low. Conversely, the negative predictive value of AFGGT is near 100%; a negative test result is very reassuring, which can help to alleviate parental anxiety and avoid unwarranted termination of pregnancy.23 24 When NVFGB is detected after 22 weeks of gestation, the fetal blood GGT level may be useful for identification of biliary atresia.27 28 However, cordocentesis may be unwarranted, as the procedure-related risk outweighs the possible diagnostic benefit, particularly in cases of isolated NVFGB where the risk of biliary atresia is presumably low.

In Caucasian populations, the incidence of cystic fibrosis is 1:2500-3500 live births and the carrier rate is 1:50; in contrast, this hereditary disease is extremely rare among East Asian individuals (1:350000 people in Japan and 1:300000 live births in Hong Kong).15 16 29 Unsurprisingly, we did not observe cystic fibrosis in either group of NVFGB cases. Therefore, in the absence of significant family history, consanguinity, or concurrent ultrasound features suggestive of cystic fibrosis (eg, echogenic or dilated bowel), amniocentesis for genetic testing for cystic fibrosis is not recommended in cases of NVFGB in Hong Kong. Assessment of the parental CFTR gene mutation status may be a useful alternative.

Based on our findings and the results of previous studies, we propose the following approach for the management of NVFGB. When NVFGB is detected, a detailed morphology scan should be performed to identify associated abnormalities, such as hepatic hilar cyst and heterotaxy (indicative of biliary atresia) or echogenic and dilated bowel (suggestive of cystic fibrosis). A sonographic examination of the gallbladder should be repeated within 1 week. Considering the potential for chromosomal abnormalities (even in cases of isolated NVFGB), amniocentesis is recommended for CMA analysis in cases of persistent NVFGB. The AFGGT assay can also be performed before 22 weeks of gestation; counselling prior to the test should involve an explanation of the moderately low positive predictive value for identification of biliary atresia. Beyond 22 weeks of gestation, the AFGGT level is not useful for identifying biliary atresia, but cordocentesis for GGT level may be useful. However, cordocentesis is generally not recommended because the procedure-related risk outweighs the possible diagnostic benefit, particularly in cases of isolated NVFGB where the risk of biliary atresia is presumably low. Further testing for cystic fibrosis may be unnecessary in Chinese fetuses unless the NVFGB is associated with other ultrasound features suggestive of cystic fibrosis, significant family history, or consanguinity. Further research is needed concerning AFGGT reference values and the ability of the AFGGT level to identify biliary atresia in Chinese fetuses with NVFGB.

Similar to other reports regarding NVFGB, our study was limited by its retrospective design and small cohort size. Because fetal gallbladder examination has not been a routine practice in Hong Kong, we cannot calculate the prevalence of NVFGB. To our knowledge, this is the first report of NVFGB in a Chinese cohort. Moreover, our results differed from findings in Caucasian populations in that we did not observe cystic fibrosis in our cohort; such information may be useful during antenatal counselling in cases of NVFGB.

The prognosis of isolated NVFGB is generally good because the non-visualisation is either transient or related to gallbladder agenesis. While investigations of chromosomal abnormalities and biliary atresia are reasonable in cases of NVFGB, testing for cystic fibrosis may be unnecessary in Chinese fetuses unless the NVFGB is associated with consistent ultrasound features, significant family history, or consanguinity.

Concept or design: YH Ting, TY Leung.
Acquisition of data: YH Ting, PL So, KW Cheung, TK Lo, TWL Ma.
Analysis or interpretation of data: YH Ting.
Drafting of the manuscript: YH Ting, TY Leung.
Critical revision of the manuscript for important intellectual content: All authors.

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.

The research was presented as poster in the 30th World Congress on Ultrasound in Obstetrics and Gynecology, 16-18 October 2020, and was published as an abstract in Ultrasound in Obstetrics & Gynecology (Ting Y, Leung T, Law K, Lo T, Cheung K, So P. VP09.12: Non-visualisation of fetal gall bladder in a Chinese cohort. Ultrasound Obstet Gynecol 2020;56(S1):84).

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

The study protocol was approved by the Joint Chinese University of Hong Kong–New Territories East Cluster Clinical Research Ethics Committee on 3 March 2020 (CREC Ref. No.: 2020.060).

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