Herein we analysed the influence of early life factors, including breast milk composition, on the development of the intestinal microbiota of infants born to mothers with and without IBD. The MECONIUM [Exploring MEChanisms Of disease traNsmission In Utero through the Microbiome] study is a prospective cohort study consisting of pregnant women with or without IBD and their infants. Longitudinal stool samples were collected from babies and analysed using 16s rRNA sequencing and faecal calprotectin. Breast milk proteomics was profiled using Olink inflammation panel. We analysed gut microbiota of 1034 faecal samples from 294 infants [80 born to mothers with and 214 to mothers without IBD]. Alpha diversity was driven by maternal IBD status and time point. The major influencers of the overall composition of the microbiota were mode of delivery, feeding, and maternal IBD status. Specific taxa were associated with these exposures, and maternal IBD was associated with a reduction in Bifidobacterium. In 312 breast milk samples [91 from mothers with IBD], mothers with IBD displayed lower abundance of proteins involved in immune regulation, such as thymic stromal lymphopoietin, interleukin-12 subunit beta, tumour necrosis factor-beta, and C-C motif chemokine 20, as compared with control mothers [adjusted p = 0.0016, 0.049, 0.049, and 0.049, respectively], with negative correlations with baby´s calprotectin, and microbiome at different time points. Maternal IBD diagnosis influences microbiota in their offspring during early life. The proteomic profile of breast milk of women with IBD differs from that of women without IBD, with distinct time-dependent associations with baby's gut microbiome and feacal calprotectin.

Abstract Background Monitoring faecal calprotectin (fCal) levels is important in treating patients with inflammatory bowel disease (IBD). Analysis of faecal samples can be divided into sample collection and sample extraction, of which the former is very time-consuming and tedious work for the lab personnel. This step can easily be improved by allowing the patient to perform the sample collection at home with required amount of sample directly into the extraction buffer. Any home faecal collection device needs to show conformity to the golden standard of weighing method. Home sampling presents a solution but requires clear instructions to the user. The collected sample is also required to maintain stability during transportation. Methods The present study aimed to determine the usability of EasyExtractTM and a quick guide for home sampling performed by lay users. The usability study was conducted according to FDA guidelines on usability engineering. Twenty-four participants without prior knowledge of the product or procedure received an EasyExtractTM, a simulated human faecal sample, and a quick-guide to perform the home sampling. Matched participant-observer questionnaires were assessed to determine the usability. Performance studies of EasyExtractTM included consistent sample uptake, agreement with the weighing method, and stability (at room temperature, after transportation, and after freeze-thaw cycles). All samples were analysed with CalproLab ELISA (CALP0170) to determine fCal concentration. Appropriate statistical methods were used to quantify and visualize the results. Results 100% of the participants in the usability study completed sampling successfully. EasyExtractTM and the quick guide got an average rating of 4.8 on a scale of 1-5 (1=difficult, 5= easy). In-house performance studies show that the average sample weight collected 29.2 mg with a CV of 2.7%. It also showed that the extracts remained stable during transport at RT for 5 days, and no changes after repeated freeze-thaw cycles. The collection device also shows strong agreement with the established weighing method. Conclusion EasyExtractTM and the quick guide enabled participants to sample accurately without prior experience with the device. Additionally, EasyExtractTM allows for consistent weight and agrees with the golden standard of weighing method. Furthermore, fCal stability is maintained at RT for 5 days and repeated freeze-thawing cycles. EasyExtractTM is a useful tool for home-sampling of fCal.

Abstract Background There are increasing data on changes in intestinal inflammation and microbiome diversity during pregnancy in women with inflammatory bowel disease (IBD) with implications towards individual and offspring immune function. However, differences in intestinal inflammation, as measured by fecal calprotectin (FC) and microbial a-diversity, in consecutive pregnancies are not known. Methods We prospectively enrolled a cohort of women, 37 with IBD and 39 without IBD, during two consecutive pregnancies, and their offspring. We collected serial stool samples and clinical data, and measured FC and bacterial abundance during each trimester of each pregnancy. We further performed correlation analysis between FC in consecutive pregnancies and between microbial a-diversity in consecutive pregnancies among women with and without IBD. Results Compared to healthy controls, IBD pregnancies had significantly lower gestational age at birth and higher frequency of Cesarean section. Mode of delivery, the status of Group B Streptococcus (GBS) infection and GBS infection prophylaxis were significantly associated with the pregnancy order in both IBD and controls (Table). Furthermore, we observed strong correlations of FC (r=0.56, p-value=0.093) and microbial alpha-diversity assessed as operational taxonomic unit (OTU) richness (r=0.88, p=0.0087) between paired consecutive pregnancies in women with IBD, but not in those without IBD (Figure). There were no differences in microbial alpha-diversity using the Shannon and Simpson indices when comparing consecutive pregnancies of women with and without IBD. Conclusion In this study, we demonstrate that intestinal inflammation and microbiome diversity are more conserved in consecutive pregnancies of women with IBD compared to healthy controls. These findings may have implications towards understanding the impact of pregnancy on host-microbiome interactions in IBD as well as the potential impact on offspring health.

The effect of pregnancy on inflammatory bowel disease (IBD) remains poorly understood. We aimed to monitor intestinal inflammation using fecal calprotectin (FC) in pregnant women and their babies during early life. Pregnant women with or without IBD and their infants were prospectively enrolled. FC levels were measured at each trimester of pregnancy and in babies throughout the first 3 years of life. Repeated-measures analysis was applied to investigate changes in FC levels while adjusting for confounders. The FC levels were correlated with the bacterial abundance in both mothers and babies. Six hundred and fourteen fecal samples from 358 mothers (98 with IBD) and 1005 fecal samples from 289 infants (76 born to IBD mothers) were analyzed. Pregnant Patients with IBD maintained higher FC levels through pregnancy compared with controls (P= 7.5× 10-54). FC gradually increased in controls and declined in Patients with IBD throughout pregnancy (P for interaction= 5.8× 10-7). Babies born to mothers with IBD presented with significantly higher FC levels than those born to controls up to 3 years of age, after adjusting for sex, delivery mode, feeding behavior, and antibiotics exposure (2 weeks to 3 months of age, P= .015; 12-36 months of age, P= .00003). Subdoligranulum, Roseburia, Fusicatenibacter, and Alistipes negatively correlated, and Streptococcus, Prevotella, Escherichia-Shigella, and Bifidobacterium positively correlated with maternal FC levels at T3. Faecalibacterium, Bifidobacterium, and Alistipes showed negative correlations, and Streptococcus were positively correlated with FC levels within 3 months of birth. Pregnancy is associated with decreased inflammatory activity in mothers with IBD. Higher FC levels in babies born to mothers with IBD suggest subclinical inflammation in early life, the long-term consequences of which are uncertain.

Abstract Background IBD often affects women during their reproductive years; however, the effect of pregnancy on disease course remains poorly understood. We aimed to assess intestinal inflammation in IBD patients compared with controls as measured by faecal calprotectin (FC). We also investigated whether maternal IBD diagnosis was associated with altered FC in the offspring and if there were particular bacterial taxa that correlated with FC levels. Methods Pregnant women with or without IBD and their infants were prospectively enrolled in the MECONIUM study during 2015–2018 years. FC levels at each trimester of pregnancy and in babies throughout the first 3 years of life were measured using a quantitative enzyme immunoassay (CALPRO AS, Norway). Multivariate regression analysis was applied to investigate FC levels. Stool microbiota composition in the maternal and baby stool was assessed using 16s rRNA sequencing. Results 617 faecal samples from 342 mothers (91 IBD, 251 control) and 1005 faecal samples from 288 infants (born to 76 mothers with and 212 without IBD) were analysed. FC levels in pregnant women with IBD were significantly higher than in control pregnant women regardless of IBD type (Crohn’s disease vs. ulcerative colitis). In IBD mothers FC levels showed a decline during pregnancy (µg/g, median (interquartile range); first trimester 130.8 (102.3–147.3) vs. third trimester 85.9 (44.5–125.4), p = 0.02) (Figure 1). Patients with flare at stool collection had the highest, while those treated with anti-TNF plus thiopurine showed the lowest FC at each trimester. Babies born to mothers with IBD presented higher FC levels than those born to control mothers at multiple time points between 2 and 36 months of age (Figure 2A); the median FC levels were the highest after 1 year up to 3 years in those babies whose mothers presented active disease during pregnancy (Figure 2B). Microbial α-diversity showed an inverse relationship with FC in both mothers (r = −0.25, p = 0.032) and babies (r = −0.31, p = 0.0035). Blautia and Streptococcus abundance were positively correlated with FC in babies. Conclusion FC levels in IBD patients decreased throughout pregnancy. Maternal IBD, lower microbiome diversity and the abundance of certain microbial genera were associated with higher FC in the offspring up to 3 years of life. These findings suggest a potential favourable impact of pregnancy on IBD activity and highlight a possible effect of IBD during pregnancy on the intestinal inflammation in offspring, which could be mediated through altered microbiome.

Monitoring IBD activity during pregnancy is challenging because clinical and laboratory markers may be altered due to the physiological adaptation that occurs, and endoscopy use is limited. Fecal Lactoferrin (FL) is a non-invasive biomarker of gut inflammation used for the diagnostics and management of IBD; however, little is known about its use in pregnant women. Herein, we investigated FL concentrations in IBD and control women, participating in the MECONIUM (Exploring MEChanisms Of disease traNsmission In Utero through the Microbiome) study, prior to (T0) and during each trimester of pregnancy. 405 faecal samples [32 at T0; 50 at first trimester (T1); 134 at second trimester (T2) and 189 at third trimester (T3)] from 76 IBD women and 175 controls were analysed using a quantitative enzyme immunoassay (LACTOFERRIN SCANTM, TECHLAB®). Correlation analyses with clinical scores collected prospectively [physician global assessment (PGA), modified Harvey-Bradshaw index (HBI) for CD and partial Mayo score for UC] and with fecal calprotectin analysed using a quantitative enzyme immunoassay (CalproLabTM Calprotectin ELISA, Norway) were performed. Statistical analyses were conducting using R software. The median FL (µg/ml) for pregnant was not different to that of non-pregnant women in controls (1.52 at T1 vs. 1.08 at T0; p = 0.08) and in IBD group (3.58 at T1 vs. 2.64 at T0; p = 0.53). FL was significantly higher in IBD women compared with controls at each trimester of pregnancy (Figure 1) and differed by disease activity (active vs. in remission) but showed significance at only T3 (p = 0.002). At T3, FL significantly correlated with PGA (spearman r = 0.42; p = 0.001), partial Mayo score in UC patients (r = 0.41; p = 0.04) and with HBI in CD patients (r = 0.36; p = 0.05). Finally, FL correlated closest with fecal calprotectin (r = 0.63; p < 0.0001), especially in IBD women (r = 0.78; p < 0.0001, Figure 2) at T3. Fecal Lactoferrin concentrations in pregnant women with and without IBD (T0: prior to pregnancy, T1: first trimester, T2: second trimester, T3: third trimester Correlation between fecal Lactoferrin and fecal Calprotection concentrations at third trimester (T3) in pregnant women with or without IBD FL is not affected by pregnancy regardless of maternal IBD status. IBD women had higher FL levels than controls at each trimester of pregnancy. Suggesting that FL could be a reliable, non-invasive biomarker of gut inflammation to monitor IBD activity during this period.

Prognostic biomarkers are lacking in primary sclerosing cholangitis, hampering patient care and the development of therapy. We aimed to identify novel protein biomarkers of disease severity and prognosis in primary sclerosing cholangitis (PSC). Using a bead-based array targeting 63 proteins, we profiled a derivation panel of Norwegian endoscopic retrograde cholangiography bile samples (55 PSC, 20 disease controls) and a Finnish validation panel (34 PSC, 10 disease controls). Selected identified proteins were measured in serum from two Norwegian PSC cohorts (n=167 [1992-2006] and n=138 [2008-2012]), inflammatory bowel disease (n=96) and healthy controls (n=100). In the bile derivation panel, the levels of 14 proteins were different between PSC patients and controls (p<0.05); all were confirmed in the validation panel. Twenty-four proteins in the bile derivation panel were significantly (p<0.05) different between PSC patients with mild compared to severe cholangiographic changes (modified Amsterdam criteria); this was replicated for 18 proteins in the validation panel. Interleukin (IL)-8, matrix metallopeptidase (MMP)9/lipocalin (LCN)2-complex, S100A8/9, S100A12 and tryptophan hydroxylase (TPH)2 in the bile were associated with both a PSC diagnosis and grade of cholangiographic changes. Stratifying PSC patients according to tertiles of serum IL-8, but not MMP9/LCN2 and S100A12, provided excellent discrimination for transplant-free survival both in the serum derivation and validation cohort. Furthermore, IL-8 was associated with transplant-free survival in multivariable analyses in both serum panels independently of age and disease duration, indicating an independent influence on PSC progression. However, the Enhanced Liver Fibrosis (ELF®) test and Mayo risk score proved to be stronger predictors of transplant-free survival. Based on assaying of biliary proteins, we have identified novel biliary and serum biomarkers as indicators of severity and prognosis in PSC. Prognostic biomarkers are lacking in primary sclerosing cholangitis, hampering patient care and the development of therapy. We have identified inflammatory proteins including calprotectin and IL-8 as important indicators of disease severity and prognosis in bile and serum from patients with primary sclerosing cholangitis.