Roth-Schulze et al. Microbiome (2021) 9:167 https://doi.org/10.1186/s40168-021-01104-y RESEARCH Open Access Type 1 diabetes in pregnancy is associated with distinct changes in the composition and function of the gut microbiome Alexandra J. Roth-Schulze1,2, Megan A. S. Penno3, Katrina M. Ngui1,2, Helena Oakey3, Esther Bandala-Sanchez1,2, Alannah D. Smith1,2, Theo R. Allnutt1,2, Rebecca L. Thomson3, Peter J. Vuillermin4, Maria E. Craig5,6, William D. Rawlinson7,8, Elizabeth A. Davis9, Mark Harris10,11, Georgia Soldatos12, Peter G. Colman13, John M. Wentworth1,2,13, Aveni Haynes9, Simon C. Barry3, Richard O. Sinnott14, Grant Morahan15, Naiara G. Bediaga1,2, Gordon K. Smyth1,16, Anthony T. Papenfuss1,16,17,18, Jennifer J. Couper3,19, Leonard C. Harrison1,2* and on behalf of the ENDIA Study Group Abstract Background: The gut microbiome changes in response to a range of environmental conditions, life events and disease states. Pregnancy is a natural life event that involves major physiological adaptation yet studies of the microbiome in pregnancy are limited and their findings inconsistent. Pregnancy with type 1 diabetes (T1D) is associated with increased maternal and fetal risks but the gut microbiome in this context has not been characterized. By whole metagenome sequencing (WMS), we defined the taxonomic composition and function of the gut bacterial microbiome across 70 pregnancies, 36 in women with T1D. Results: Women with and without T1D exhibited compositional and functional changes in the gut microbiome acrosspregnancy.ProfilesinwomenwithT1Dweredistinct,withanincreaseinbacteriathatproducelipopolysaccharides and a decrease in those that produce short-chain fatty acids, especially in the third trimester. In addition, women with T1D had elevated concentrations of fecal calprotectin, a marker of intestinal inflammation, and serum intestinal fatty acid- binding protein (I-FABP), a marker of intestinal epithelial damage. Conclusions: Women with T1D exhibit a shift towards a more pro-inflammatory gut microbiome during pregnancy, associated with evidence of intestinal inflammation. These changes could contribute to the increased risk of pregnancy complications in women with T1D and are potentially modifiable by dietary means. Keywords: Microbiome,Gut,Type1diabetes,Pregnancy, Metagenomics, Quantitative PCR, Inflammation markers * Correspondence: [email protected] 1Walter and Eliza Hall Institute of Medical Research, Melbourne, VIC 3052, Australia 2Department of Medical Biology, University of Melbourne, Melbourne, VIC 3010, Australia Full list of author information is available at the end of the article © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Roth-Schulze et al. Microbiome (2021) 9:167 Page 2 of 21 Background T1D, dietary butyrate supplementation promoted an in- The gut microbiome provides essential metabolites, vita- crease in regulatory T cells and a decrease in the inci- mins, co-factors and hormones, protects against patho- dence of spontaneous diabetes [20]. Increased gut genic microorganisms and has a key role in the leakiness has been described in established T1D [21] and development of the immune and other systems [1, 2]. recently by ourselves in association with gut microbiome Changes in the composition of the gut microbiome are changes in children with islet autoimmunity who pro- associated with ageing, environmental conditions, life gressed to T1D [22]. Gut leakiness with translocation of events and disease states [2–4]. In pregnancy, women toxins and dietary antigens into the blood may result in undergo significant physiological changes, but only re- systemic inflammation, reported with T1D in pregnancy cently has the gut microbiome been studied in this con- complicated by pre-eclampsia [10]. Because a consensus text [5, 6]. Koren et al. [5] sampled the gut microbiome about the gut microbiome in pregnancy is lacking, even in the first and third trimesters and found that the taxo- in the absence of T1D, we applied shotgun whole meta- nomic composition in the first trimester was similar to genomic sequencing (WMS) to analyse the gut micro- that of non-pregnant women but in the third trimester biome across pregnancy in women with and without the abundance of Actinobacteria and Proteobacteria T1D participating in the Australia-wide Environmental phyla increased along with an overall decrease in bacter- Determinants of Islet Autoimmunity (ENDIA) study. ial richness (alpha diversity). In studies in germ-free mice, they observed that inoculation with third com- Results pared to first trimester feces led to greater weight gain, Study population insulin resistance and gut inflammation and suggested Fecal samples were collected between February 2013 and this was an adaptive proinflammatory response to de- October 2017 from women enrolled in the ENDIA fend the fetus from pathogens and provide it with nutri- study, a prospective, pregnancy-birth cohort study that ents. In contrast, after analysing fecal samples weekly follows 1500 Australian children who have a first-degree across pregnancy, DiGiulio et al. [6] found no significant relative with T1D [23]. Thirty-five women (36 pregnan- temporal differences in diversity or composition of the cies) with T1D and 31 women (34 pregnancies) without gut microbiome. These contrary findings and the dearth T1D had each provided from one to three fecal samples of studies warrant further investigation of the gut micro- across pregnancy (total 134 samples) for analysis by shot- biome in pregnancy. gun WMS (Fig. 1). Table 1 summarizes and compares Type 1 diabetes (T1D) is an autoimmune disease in characteristics of the T1D and non-T1D pregnancies. which insulin-producing β cells in the islets of the pan- creas are destroyed by T lymphocytes leading to insulin Whole metagenomic sequencing deficiency [7]. In pregnancy, T1D is associated with sys- The WMS dataset, 47,766,763 ± 10,956,057 (mean ± temic and intra-uterine markers of sub-clinical inflam- SD) paired-end reads per sample, was obtained using an mation and higher risks of complications for mother and Illumina NovaSeq 6000. Raw reads (SRA accession: fetus [8–10]. Alterations in the bacterial gut microbiome PRJNA604850) were pre-processed using KneadData have been reported in T1D, mainly in children at high bioBakery tool [24] to eliminate human DNA sequences risk and at diagnosis (reviewed in [11], [12–17]). They and filter sequences with poor quality which on average include a decrease in alpha diversity (richness) [12–14] removed 6% of the reads. After quality control and read and in the abundance of lactate- and butyrate-producing filter steps, 44,940,628 ± 10,572,188 (mean ± SD) paired- and mucin-degrading bacteria [13–17], and an increase end reads per sample were obtained (Excel file E0). in the abundance of the Bacteroides genus [13, 14]. Functionally, these compositional changes are reflected Taxonomic diversity and composition of the gut by a decreased abundance of genes encoding related microbiome in women with and without T1D during metabolic pathways and enzymes, e.g. butyryl-coenzyme pregnancy A (CoA)-CoA transferase [15] and butyryl-CoA de- Sequences were analysed with MetaPhLan2 imple- hydrogenase for butyrate synthesis [16]. These changes mented within the HUMAnN2 pipeline. Overall, 340 are not necessarily specific for T1D but nevertheless, bacterial species were identified, with an average of 93 ± they may have clinical consequences, including in preg- 13 (mean ± SD) species per sample. The top 25 most nancy. Gut butyrate is a key determinant of gut health abundant species accounted for more than 50% of the and regulator of gene expression and homeostatic im- gut microbiome composition of each subject in any munity [18–20]. It is the major energy source for the co- given trimester (Figure S1). lonic mucosa, induces the synthesis of mucin and it Alpha diversity (observed richness or number of spe- promotes gut epithelial integrity, preventing ‘gut leaki- cies) per sample was calculated and generalized estimat- ness’. In the non-obese diabetic (NOD) mouse model of ing equations (GEE) were applied to test for differences Roth-Schulze et al. Microbiome (2021) 9:167 Page 3 of 21 Fig. 1 Fecal samples obtained in pregnancy. n: number of samples; T1: trimester 1; T2: trimester 2; T3: trimester 3; T1D: women with type 1 diabetes; Non-T1D: women without T1D between women without and with T1D, and between tri- Only taxa for which the prevalence (i.e. proportion of mesters, and to determine if there was an interaction be- samples with those taxa) was above 50% in at least one tween T1D status and trimester. No differences were group and with a log2 fold-change (logFC) greater than found in richness due to T1D status or time, or interac- 0.5 or less than − 0.5 were considered. Across all trimes- tions (Figure S2, Excel file E1). ters, the species Bacteroides caccae (FDR 0.03) and its For analysis of beta diversity, Bray-Curtis coefficients unique strain (unclassified) in the dataset (FDR 0.03), as were calculated between sample pairs, ordinated and well as the order Enterobacteriales (FDR 0.07) were in- plotted by principal coordinate analysis (PCoA) for each creased in women with T1D (Fig.