Obeticholic acid improves fetal bile acid profile in a mouse model of gestational hypercholanemia

27 Intrahepatic cholestasis of pregnancy (ICP) is characterized by elevated maternal circulating bile acid 28 levels and associated dyslipidemia. ICP leads to accumulation of bile acids in the fetal compartment 29 and the elevated bile acid concentrations are associated with an increased risk of adverse fetal 30 outcomes. The farnesoid X receptor agonist, obeticholic acid (OCA) is efficient in the treatment of 31 cholestatic conditions such as primary biliary cholangitis. We hypothesized that OCA administration 32 during hypercholanemic pregnancy will improve maternal and fetal bile acid and lipid profiles. 33 Female C57BL/6J mice were fed either: a normal chow diet, a 0.5% cholic acid (CA)-supplemented 34 diet, a 0.03% OCA-supplemented diet, or a 0.5% CA + 0.03% OCA-supplemented diet for 1 week prior 35 to mating and throughout pregnancy until euthanization on day 18. The effects of CA and OCA 36 feeding on maternal and fetal morphometry, bile acid and lipid levels, and cecal microbiota were 37 investigated. OCA administration during gestation did not alter the maternal or fetal body weight or 38 organ morphometry. OCA treatment during hypercholanemic pregnancy reduced bile acid levels in 39 the fetal compartment. However, fetal dyslipidemia was not reversed, and OCA did not impact 40 maternal bile acid levels or dyslipidemia. In conclusion, OCA administration during gestation had no 41 apparent detrimental impact on maternal or fetal morphometry and improved fetal 42 hypercholanemia. As high serum bile acid concentrations in ICP are associated with increased rates 43 of adverse fetal outcomes, further investigations into the potential use of OCA during cholestatic We used a mouse model of gestational hypercholanemia to investigate the use of obeticholic acid 48 (OCA), a potent FXR agonist, as a treatment for the hypercholanemia of intrahepatic cholestasis of 49 pregnancy (ICP). The results demonstrate that OCA can improve the fetal bile acid profile. This is 50 relevant not only to women with ICP, but also for women who become pregnant while receiving 51 OCA treatment for other conditions such as primary biliary cholangitis and non-alcoholic 52 steatohepatitis.

Intrahepatic cholestasis of pregnancy (ICP) is characterized by elevated maternal circulating bile acid 28 levels and associated dyslipidemia. ICP leads to accumulation of bile acids in the fetal compartment 29 and the elevated bile acid concentrations are associated with an increased risk of adverse fetal 30 outcomes. The farnesoid X receptor agonist, obeticholic acid (OCA) is efficient in the treatment of 31 cholestatic conditions such as primary biliary cholangitis. We hypothesized that OCA administration 32 during hypercholanemic pregnancy will improve maternal and fetal bile acid and lipid profiles. 33 Female C57BL/6J mice were fed either: a normal chow diet, a 0.5% cholic acid (CA)-supplemented 34 diet, a 0.03% OCA-supplemented diet, or a 0.5% CA + 0.03% OCA-supplemented diet for 1 week prior 35 to mating and throughout pregnancy until euthanization on day 18. The effects of CA and OCA 36 feeding on maternal and fetal morphometry, bile acid and lipid levels, and cecal microbiota were 37 investigated. OCA administration during gestation did not alter the maternal or fetal body weight or 38 organ morphometry. OCA treatment during hypercholanemic pregnancy reduced bile acid levels in 39 the fetal compartment. However, fetal dyslipidemia was not reversed, and OCA did not impact 40 maternal bile acid levels or dyslipidemia. In conclusion, OCA administration during gestation had no 41 apparent detrimental impact on maternal or fetal morphometry and improved fetal 42 hypercholanemia. As high serum bile acid concentrations in ICP are associated with increased rates 43 of adverse fetal outcomes, further investigations into the potential use of OCA during cholestatic 44 gestation are warranted. 45

Introduction 55
Intrahepatic cholestasis of pregnancy (ICP) is a cholestatic condition that affects 0.4-2.2% of 56 pregnancies in North America and Western Europe, but is more common in Chile and Bolivia where 57 it can affect 1.5-4% of pregnancies (11,13,44). ICP typically presents from 30 weeks of gestation and 58 the main symptom is persistent generalized itch. Diagnosis is made in women with an elevation of 59 serum bile acids. ICP is associated with maternal dyslipidemia (12,27) and increased risk of 60 gestational diabetes mellitus (26,27,49). The most common treatment for ICP is ursodeoxycholic 61 acid (UDCA) administration, but not all patients respond (8,9,18) and a recent trial revealed no 62 benefit for adverse perinatal outcomes (8). 63 The adverse fetal outcomes that occur in ICP include preterm birth, fetal hypoxia, meconium-stained 64 amniotic fluid, stillbirth and prolonged admission to the neonatal unit (19). Maternal bile acid levels 65 have been reported to be positively correlated to fetal bile acid levels, and incremental rises in 66 maternal serum bile acids above 40 µmol/l are associated with higher risk of adverse fetal outcomes 67 (7,19,21). The fetal lipid profile has also been shown to be affected by maternal cholestasis, with 68 increased cholesterol accumulation in the fetal liver and placenta in a mouse model of gestational 69 cholestasis and in the umbilical cord of neonates exposed to maternal ICP (41). 70 It has previously been described that during normal pregnancy, the activity of farnesoid X receptor 71 (FXR), the master nuclear receptor regulating bile acid homeostasis, is decreased allowing for a 72 maternal pro-cholestatic profile even during normal gestation (31,33,39). However, it is thought 73 that in ICP, the combination of genetic susceptibility, elevated reproductive hormones and 74 environmental factors may lead to an exacerbation of the pro-cholestatic profile found in pregnancy 75 and result in a pathological rise of bile acid levels (17). 76 In recent years, synthetic FXR agonists have been developed. In particular, the semi-synthetic bile 77 acid, obeticholic acid (OCA) has over 100x higher affinity for FXR than its most potent natural ligand, 78 chenodeoxycholic acid (CDCA), and has been shown to promote bile acid efflux and reduce bile acid 79 synthesis (51). Clinical trials of OCA have shown promising results for the treatment of primary 80 biliary cholangitis (PBC) and non-alcoholic steatohepatitis (NASH) (2). 81 In this study, we used a previously established model of 0.5% cholic acid (CA) feeding in pregnancy 82 to mimic the hypercholanemia of ICP (32, 41). Due to the key role of FXR in bile acid synthesis, 83 transport and excretion, as well as regulation of lipid metabolism, we hypothesized that activation of 84 FXR by OCA could improve maternal and fetal hypercholanemia and dyslipidemia. 85

Animal experiments 87
Six to eight-week-old C57BL/6J mice were purchased from Envigo, UK and allowed to acclimatize for 88 one week before any experimental procedures were carried out. All mice were kept on a 12h/12h 89 light/dark cycle with access to food and water ad libitum. All procedures were approved by the 90 All values are shown as mean ± standard error of the mean (SEM). Statistical analysis was performed 165 using GraphPad Prism 7 software. One-way ANOVA followed by a Newman-Keuls post-hoc test was 166 used, with a significance cut-off of P ≤ 0.05. Statistical analysis of 16S rRNA gene sequencing data is 167 detailed in the relevant section above. 168

OCA administration during pregnancy does not negatively impact maternal or fetal morphometry 170
We first aimed to establish the effect of hypercholanemia and OCA supplementation during 171 pregnancy on body weight and organ morphometry. During pregnancy, no body weight differences 172 were seen between groups, except on D7 when CA and CA+OCA-fed females were significantly 173 lighter than OCA-fed females ( Figure 1A). Although no body weight differences were registered on 174 D18 gestation, pregnant females fed a CA diet had increased liver weight and decreased gWAT 175 weight, regardless of OCA co-feeding ( Figure 1B). A trend for decreased sWAT weight was also seen 176 in pregnant CA and CA+OCA groups ( Figure 1B). OCA supplementation alone did not affect body 177 weight or organ morphometry ( Figure 1B). 178 Despite the changes in maternal liver and gWAT morphometry in the CA and CA+OCA-fed groups, no 179 changes in pup number, pup weight or placental weight were registered ( Figure 1C). 180 Outside of pregnancy, both CA and CA+OCA non-pregnant females were lighter than NC-and OCA-181 These results demonstrate that OCA administration either alone or to hypercholanemic pregnant 185 females did not negatively impact maternal or fetal body or organ morphometry. 186

OCA administration during hypercholanemic pregnancy reduces fetal hypercholanemia 188
We next investigated whether OCA administration ameliorated the maternal and fetal bile acid 189 profiles during hypercholanemic gestation. In pregnant females, CA feeding led to a significant 190 increase in total serum bile acid levels, CA, deoxycholic acid (DCA), taurocholic acid (TCA) and 191 taurodeoxycholic acid (TDCA) compared to NC controls, confirming that CA-feeding induces 192 maternal hypercholanemia, as has previously been described (32, 41). CA+OCA co-supplementation 193 did not ameliorate total serum bile acid levels, although total unconjugated bile acids were 194 significantly reduced compared to CA alone, due to changes in CA (P > 0.05) and DCA (P ≤ 0.05) 195 ( Figure 2A) . 196 In non-pregnant females, total bile acids, DCA, TCA and TDCA levels were significantly elevated by CA 197 feeding and were not reduced by CA+OCA co-feeding ( Supplementary Fig. S2). 198 In the fetal compartment, maternal hypercholanemia led to a significant rise in fetal serum total bile 199 acids ( Figure 2B). However, total serum bile acid levels were 29.9% lower in fetuses from mothers 200 fed a CA+OCA diet compared to CA alone, although still higher than NC controls ( Figure 2B). This was 201 due to decreased concentrations of DCA, TCA, TDCA and in particular, CA ( Figure 2B). Maternal OCA 202 feeding alone did not change fetal bile acid concentrations although the presence of OCA and T-OCA 203 in the fetal circulation suggests that OCA is able to cross the placenta ( Figure 2B). 204 Overall, OCA administration to hypercholanemic females did not significantly ameliorate maternal 205 hypercholanemia, but improved the fetal bile acid profile. 206

OCA administration alone reduces cecal bile acid levels 208
Cecal bile acid concentrations were also measured. As expected in the cecum, bile acids were 209 largely unconjugated ( Figure 3). Total cecal bile acid levels were significantly increased in mice fed 210 CA+OCA compared to CA alone, however this was largely due to enrichment with OCA, and also with 211 DCA that also increased in the CA-fed group ( Figure 3A,B). Muricholic acids levels were markedly 212 reduced in both CA and CA+OCA groups ( Figure 3B,C). OCA administration alone significantly 213 reduced total cecal bile acid levels compared to all other groups, which was due to an overall 214 reduction in bile acids ( Figure 3A). Interestingly, as seen in the serum, T-OCA levels were significantly 215 lower in CA+OCA co-fed mice compared to females supplemented with OCA only, while OCA levels 216 were increased ( Figure 3B,C). 217 218

Bile acid supplementation impacts the cecal microbiome's microbiota composition 219
Conversion of primary to secondary bile acids, as well as bile acid deconjugation, are performed by 220 intestinal bacteria. Since changes in bile metabolizing bacteria will affect the host bile acid pool, the 221 cecal bacterial community was investigated by 16S rRNA gene sequencing. Non-metric 222 multidimensional scaling (NMDS) analysis of weights UniFrac distances, which shows how the 223 microbial communities vary between the groups, demonstrates significant differences between all 224 the dietary groups in pregnant mice ( Figure 4A, Supplementary Table S2). OCA supplementation 225 alone was the least different to NC, with CA and then CA+OCA being more dissimilar. Differences in 226 the relative proportion of phyla were observed between pregnant groups ( Figure 4B); specifically, 227 both CA feeding and CA+OCA co-feeding significantly increased the relative abundance of 228 Proteobacteria in the cecum of pregnant mice, compared to NC groups ( Figure 4C). OCA feeding 229 alone did not significantly impact Proteobacteria, but the relative abundance of Bacteroidetes was 230 significantly decreased in pregnant females ( Figure 4C). Significant changes were also observed at 231 genus level, with an increase in the relative proportion of Bilophila and Bacteroides in CA+OCA-fed 232 mice compared to all other groups ( Figure 4D). This was reinforced by correlation analysis between 233 microbiota and bile acid concentrations in the cecum, which showed that Proteobacteria and 234 Bacteroidetes positively correlated with OCA, and negatively with T-OCA, concentrations 235 ( Supplementary Fig. S3A). Alpha diversity (Shannon diversity index) plots showed that CA 236 supplementation alone or co-fed with OCA resulted in decreased bacterial diversity (Supplementary 237 Fig. S3B). Pregnancy caused a significant increase in an unclassified class of Bacteroidetes in NC 238 controls ( Supplementary Fig. S3C). In non-pregnant mice, NMDS analysis and alpha diversity plots 239 were similar to pregnant mice ( Supplementary Fig. S4A,B). However, changes between the dietary 240 groups differed at phylum level; in particular, significant differences were observed in Bacteroidetes, 241 Firmicutes and Proteobacteria ( Supplementary Fig. S4C). 242

OCA administration represses maternal hepatic Cyp7a1 expression via intestinal FXR 244
To further assess the effects of hypercholanemia and OCA administration on bile acid homeostasis 245 during pregnancy, the expression of key genes for bile acid homeostasis in the liver and terminal 246 ileum was investigated. 247 The hepatic FXR target Shp was significantly upregulated in pregnant females fed a CA or a CA+OCA 248 diet and this change was concomitant with the repression of hepatic Cyp7a1 ( Figure 5A). Both CA 249 and CA+OCA diet increased the hepatic expression of the bile acid transporters Bsep, Mrp3 and 250 Mrp4 in pregnant females ( Figure 5A). Whilst OCA supplementation alone did not induce significant 251 hepatic Shp upregulation, Cyp7a1 expression was significantly decreased in D18 pregnant females 252 ( Figure 5A). In parallel, intestinal Shp expression was upregulated in OCA-fed females and intestinal 253 Fgf15 expression was significantly increased by maternal CA, OCA and CA+OCA supplementation 254 ( Figure 5B). 255 In non-pregnant females, relative mRNA expression followed a very similar pattern to pregnant mice 256 ( Supplementary Fig. S5A,B). Of note, lower hepatic gene expression of several FXR targets was 257 observed in pregnant mice compared to non-pregnant, regardless of diet (Table 1). Expression of 258 FXR targets in the terminal ileum was similarly affected by pregnancy. In pregnant CA-fed females, 259 Shp and Fgf15 expression was lower than outside pregnancy (Table 2). Shp expression levels were 260 also lower in CA+OCA-fed pregnant females compared to non-pregnant (Table 2). 261 Overall, we conclude that despite decreased expression of FXR target genes during pregnancy, 262 activation of intestinal rather than hepatic FXR can mediate OCA-induced suppression of hepatic 263 Cyp7a1 expression. 264

Maternal OCA administration represses fetal hepatic Cyp7a1 expression 266
Given the decrease in fetal serum bile acid concentrations in maternal CA+OCA feeding groups, the 267 expression of key bile acid homeostasis genes in the fetal liver and placenta were assessed. Maternal 268 CA feeding alone or co-supplemented with OCA induced an upregulation of Shp expression, and a 269 concomitant reduction in Cyp7a1 and Ntcp, in the fetal liver ( Figure 6A). Of note, while maternal 270 OCA diet alone did not have an impact on fetal hepatic Shp expression, a significant downregulation 271 of hepatic Cyp7a1 expression was observed, although to a lesser extent than in groups with 272 maternal CA supplementation ( Figure 6A). Maternal bile acid feeding did not have an impact on 273 hepatic fetal Mrp3, Mrp4 or Oatp1b2 expression ( Figure 6A). 274 As the placenta plays a crucial role in bile acid transport between maternal and fetal circulations, we 275 further sought to determine whether maternal OCA administration had an impact on placental bile 276 acid transporter gene expression. Interestingly, all maternal bile acid feeding groups showed a 277 significant upregulation of Abcg2 expression in the placenta ( Figure 6B). Moreover, maternal 278 CA+OCA feeding increased placental Mrp2 expression when compared against all other feeding 279 groups, and Oatp1b2 expression was increased compared to NC and CA groups ( Figure 6B). Overall, 280 we conclude that OCA modulates the expression of Cyp7a1 in the fetal liver and bile acid 281 transporters in the placenta. 282

OCA administration during hypercholanemic pregnancy does not reverse maternal dyslipidemia 284
Cholestasis is commonly accompanied by dyslipidemia. Hence, we next studied the effect of OCA 285 administration during hypercholanemic pregnancy on maternal and fetal serum and hepatic lipid 286 levels. No changes in total serum cholesterol levels were seen in pregnant CA and CA+OCA-287 supplemented groups ( Figure 7A). However, females exposed to a CA or CA+OCA diet had raised 288 serum LDL-cholesterol and decreased HDL-cholesterol levels compared to NC females ( Figure 7A), 289 also outside of pregnancy ( Supplementary Fig. S6A). Conversely, OCA feeding resulted in decreased 290 total serum cholesterol levels compared to NC controls which was associated with a reduction in 291 serum HDL-cholesterol concentrations ( Figure 7A). Serum HDL-cholesterol was also reduced in non-292 pregnant OCA-fed mice (Supplementary Fig. S6A). CA feeding did not alter serum triglyceride levels 293 in pregnant females, but OCA diet reduced serum triglyceride levels and a further decrease was 294 observed in CA+OCA fed females ( Figure 7A). In contrast, no significant changes were observed in 295 serum triglyceride levels in non-pregnant females (Supplementary Figure S6A). 296 In the liver, CA, OCA and CA+OCA supplementation of pregnant females led to hepatic cholesterol 297 accumulation compared to NC control group ( Figure 7B). In non-pregnant females, hepatic 298 cholesterol levels were significantly lower with OCA supplementation alone compared to CA and 299 CA+OCA-fed mice ( Supplementary Fig. S6B). 300 Taken together, these data lead us to conclude that OCA administration does not ameliorate 301 maternal dyslipidemia during hypercholanemic gestation. 302

OCA administration during hypercholanemic pregnancy does not reverse fetal dyslipidemia 304
As maternal dyslipidemia is commonly associated with fetal dyslipidemia, we next investigated the 305 fetal lipid profile. Maternal CA feeding significantly increased fetal serum cholesterol levels, 306 including LDL-cholesterol, and this was not altered by maternal CA+OCA supplementation ( Figure  307 8A). In parallel, fetal serum HDL-cholesterol concentrations were reduced in maternal CA and 308 CA+OCA supplementation groups. Fetal circulating triglycerides were increased in fetuses from CA-309 fed mothers and were not improved by maternal CA+OCA feeding ( Figure 8A). Of note, maternal 310 OCA-feeding alone had no effect on fetal total and LDL-or HDL-cholesterol levels or triglyceride and 311 FFA concentrations ( Figure 8A). 312 Fetal hepatic cholesterol and FFA content were increased in fetuses from CA+OCA-fed mothers 313 compared to NC mothers ( Figure 8B). However, maternal OCA diet alone did not affect fetal 314 cholesterol and FFA accumulation in the liver ( Figure 8B). A trend for increased hepatic cholesterol 315 and FFAs was also observed in fetuses from CA-fed mothers compared to NC controls, albeit not 316 reaching statistical significance ( Figure 8B). 317 To assess a potential relationship between fetal and placental lipid levels, the placental lipid content 318 on D18 of gestation was also evaluated. However, no significant changes in placental cholesterol, 319 triglycerides or FFAs content were registered between different groups ( Figure 8C). 320 We subsequently aimed to establish whether the changes in the fetal lipid profile on D18 of 321 gestation were due to shifts in lipid de novo biosynthesis and transport in the fetal liver or placenta. 322 Maternal bile acid feeding did not impact fetal hepatic Hmgcr, Fas or Fatp4 expression ( Figure 9A). 323 However, maternal CA+OCA feeding led to a significant increase in placental expression of the 324 cholesterol transporter Abca1 compared to NC placentas ( Figure 9B). Interestingly, maternal CA and 325 CA+OCA supplementation, but not maternal OCA alone, resulted in a significant increase in Fatp4 326 placental expression compared to NC controls ( Figure 9B). Taken together, these data lead us to 327 conclude that OCA administration does not ameliorate fetal dyslipidemia during hypercholanemic 328

gestation. 329
Discussion 330 ICP is the commonest gestational liver disease and can lead to adverse fetal outcomes (19,21,40). 331 Increased rates of stillbirth, spontaneous preterm birth, and meconium-stained amniotic fluid have 332 been reported in pregnancies with high maternal serum concentrations of bile acids (19,21,40), 333 likely related to fetal exposure to high bile acid concentrations (7). While UDCA treatment of ICP has 334 been shown to reduce maternal bile acid levels in some studies (23), it is not effective in all patients 335 (8), and it does not return fetal bile acid levels to normal concentrations (20). The present study 336 shows that OCA administration in a mouse model of hypercholanemia, as seen in ICP, is not 337 detrimental to the mother or fetus and improves fetal hypercholanemia. 338 In our model, CA-feeding led to significantly raised total bile acids in fetal serum. This was largely 339 due to an increase in taurine-conjugated CA and DCA. While the fetus synthesizes bile acids from 340 early pregnancy onwards, maternal bile acids can also cross the placenta and contribute to the fetal 341 bile acid pool (29). Unconjugated and, at much lower levels, taurine-conjugated CA and DCA were 342 also raised in the serum of CA-fed mothers. In the fetal compartment, DCA must be maternally 343 derived since the fetus cannot synthesize secondary bile acids due to the absence of gut flora, and it 344 is possible that CA is also being transferred from the mother. However, it is not known whether 345 there is preferential transport of more hydrophilic taurine conjugates across the placenta, or 346 increased taurine conjugation occurring in the fetal liver. We have previously observed in humans 347 that the ratio of conjugated to unconjugated bile acids is higher in umbilical cord blood than in 348 maternal serum (20). 349 OCA treatment during hypercholanemic gestation significantly reduced fetal total serum bile acid 350 levels, due to a reduction in DCA, TDCA and TCA, compared to fetuses of untreated hypercholanemic 351 mothers. Furthermore, analysis of fetal serum showed that OCA crosses the placenta and is present 352 in the fetal compartment, predominantly as T-OCA. In line with this, hepatic Cyp7a1 expression was 353 reduced in fetuses from OCA-fed mice, and further reduced in both CA and CA+OCA-fed groups. 354 Interestingly, OCA treatment of hypercholanemic mothers was associated with an upregulation of 355 placental transporters Mrp2 (at the maternal-facing apical membrane) and Oatp1b2 (basolateral 356 membrane), which suggests enhanced elimination of fetal bile acids via the placenta. Increased 357 placental expression of MRP2 has previously been associated with reduced bile acids in the fetal 358 compartment in ICP pregnancies following UDCA treatment (3). Protein expression and bile acid 359 transport studies would be required to confirm whether enhanced placental bile acid detoxification 360 is responsible for this reduction in serum bile acids. The impact of OCA on fetal bile acid levels is of 361 clinical interest due to the recent approval of OCA as a treatment for patients with PBC, as women 362 with PBC may already be receiving OCA treatment when they become pregnant. In our study, we did 363 not observe any detrimental effect of OCA on the fetus, in agreement with a previous study that 364 found no impact on resorptions, number of fetuses, or fetal growth (10). However, detailed 365 pathological investigations are required to assess the safety of fetal exposure to OCA. 366 In contrast to the fetus, maternal total serum bile acid levels were not reduced by OCA treatment. 367 Furthermore, OCA treatment did not induce significant shifts in hepatic mRNA expression of bile acid 368 homeostasis genes. These findings differ from a previous study of an estrogen-induced cholestasis 369 rodent model reporting that OCA treatment induced bile flow and hepatocyte expression of Shp, 370 Bsep and Mrp-2, while repressing Ntcp and Cyp7a1 expression (15). A more recent study of 371 estrogen-induced cholestasis in mice showed that OCA treatment did not upregulate mRNA 372 expression of FXR targets in the liver or placenta but did increase hepatic FXR protein levels. Total 373 serum bile acid levels were reduced in mothers, however serum bile acids were only mildly elevated 374 in this model (10). In contrast, a study investigating the effect of OCA administration to Mdr2 -/mice 375 found that dietary 0.03% OCA supplementation failed to exert any effect on bile flow and 376 composition. This study further reported that both OCA and a dual FXR and TGR5 agonist,377 were effective in reducing Cyp7a1 and Cyp8b1 gene expression, but only INT-767 administration 378 resulted in increased hepatic Shp gene expression and BSEP protein expression (4). A possible 379 explanation is that despite a far higher affinity of FXR for OCA, due to the activation of FXR by CA-380 feeding, this limited the impact of OCA in our study. This is perhaps surprising given that CA is a 381 weak agonist of FXR (EC 50 = 586 µM (25)) in comparison to OCA (EC 50 = 99nM (42)). In line, CA has 382 previously been shown to only partially induce BSEP in vitro, in comparison to the natural FXR ligand, 383 CDCA (25). A possible explanation is the 10-times higher abundance of CA as compared to OCA, at 384 least as measured in serum, which limited the impact of OCA. Regardless, OCA administration alone 385 did not cause the expected robust upregulation of hepatic FXR targets. Of note, OCA alone 386 downregulated hepatic Cyp7a1 expression and this change was associated with an upregulation of 387 Shp and Fgf15 in the terminal ileum rather than hepatic Shp induction. Indeed, previous studies have 388 demonstrated that OCA administration in rats leads to upregulation of Shp in the terminal ileum (46)  389 and that in mice lacking intestinal Fxr, OCA supplementation does not result in repression of hepatic 390 Cyp7a1 expression (50). Taken together with these studies, our findings suggest OCA acts primarily 391 through ileal FXR to stimulate FGF15 secretion into the portal circulation and repress hepatic Cyp7a1 392 expression in the maternal liver, rather than via hepatic FXR to modulate the expression of other 393 hepatic genes involved in bile acid homeostasis. Our study did not assess the effect of OCA on 394 markers of liver damage. However, we are aware that CA feeding in twice the dose in male Swiss 395 Albino mice has previously been shown to increase serum AST, ALT and AP levels, as well as 396 hepatocyte size, mitosis and necrosis (14). 397 Of note, the expression of FXR target genes was decreased overall by pregnancy, both in the liver 398 and terminal ileum, which likely reflects the previously documented decreased gestational FXR 399 activity (31,33,39). Nonetheless, in the liver of pregnant NC-fed females, OCA administration did 400 not appear to efficiently overcome the reduction of FXR activity, and gene expression levels of FXR 401 targets were similar. Conversely, in the maternal terminal ileum, the upregulation of Shp and Fgf15 402 expression suggests an increase in FXR activity induced by OCA administration to NC-fed mice, but 403 levels remained below those observed outside of pregnancy and so similarly indicate that OCA is 404 unable to fully activate FXR in the terminal ileum. In support of this data, we also observed in a 405 mouse model of gestational diabetes mellitus a diminished effect of OCA in pregnant mice compared 406 to non-pregnant controls (30). This highlights the issue that limited efficacy of FXR agonists should 407 be taken into account in treatment of pregnant women. 408 OCA was predominately unconjugated in the serum and the cecum, in contrast to mice fed OCA 409 alone where T-OCA predominated. This indicated a different pattern or activity of bile acid 410 deconjugating microbiota. Indeed, 16S rRNA gene sequencing showed that there was an increase in 411 relative abundance of Bacteroidetes and Proteobacteria (and also Bacteroides and Bilophila, when 412 analysed at genus level) in the cecum of CA+OCA-fed females. We recently reported in pregnant 413 mice that bile salt hydrolase, involved in deconjugation of bile acids, was exclusively detected in 414 Bacteroidetes, with Proteobacteria also enriched in pregnancy, likely secondary to increased taurine 415 made available after bile acid deconjugation (39). Bilophila Wadsworthia is known to be taurine-416 metabolizing (24). These findings suggest that the predominance of unconjugated OCA in the serum 417 of CA+OCA-fed mice could be due to an increase of Bacteroidetes and Proteobacteria in the gut. 418 OCA administration during hypercholanemic gestation did not reverse maternal dyslipidemia. Of 419 note, maternal OCA supplementation alone resulted in a decrease in serum total cholesterol, due to 420 a reduction in HDL-cholesterol. A similar decrease in serum HDL-cholesterol was seen in non-421 pregnant females. This decrease is not unexpected as OCA has previously been shown to reduce 422 HDL-cholesterol in healthy humans, PBC and NASH patients (22,37,43)