Gut microbiota ORIGINAL RESEARCH Gut: first published as 10.1136/gutjnl-2019-319101 on 3 January 2020. Downloaded from Gut dysbiosis induces the development of pre- eclampsia through bacterial translocation Xia Chen ,1 Pan Li,2 Mian Liu,1 Huimin Zheng,2,3 Yan He,2 Mu- Xuan Chen,2 Wenli Tang,2 Xiaojing Yue,1 Yongxin Huang,1 Lingling Zhuang,4 Zhijian Wang,1 Mei Zhong,1 Guibao Ke,5 Haoyue Hu,1 Yinglin Feng,1 Yun Chen,1 Yanhong Yu,1 Hongwei Zhou,2 Liping Huang1 ► Additional material is ABSTRact published online only. To view, Objective Pre-eclampsia (PE) is one of the malignant Significance of this study please visit the journal online metabolic diseases that complicate pregnancy. Gut (http:// dx. doi. org/ 10. 1136/ What is already known on this subject? gutjnl- 2019- 319101). dysbiosis has been identified for causing metabolic diseases, but the role of gut microbiome in the ► Pre- eclampsia (PE) is one of the leading causes For numbered affiliations see of maternal and perinatal morbidity and end of article. pathogenesis of PE remains unknown. Design We performed a case–control study to compare mortality and lacks reliable prediction methods and detailed pathogenesis knowledge. Correspondence to the faecal microbiome of PE and normotensive pregnant Professor Liping Huang, women by 16S ribosomal RNA (rRNA) sequencing. To ► PE can be considered as a type of metabolic Department of Obstetrics and address the causative relationship between gut dysbiosis disease that complicates pregnancy. Gynecology, Southern Medical and PE, we used faecal microbiota transplantation ► Gut microbiota dysbiosis plays a causative role University Nanfang Hospital, (FMT) in an antibiotic-treated mouse model. Finally, we in the development of metabolic syndrome and Guangzhou, Guangdong, China; complicated pregnancies. lphuang2006@ 126. com, determined the microbiome translocation and immune Professor Hongwei Zhou, responses in human and mouse placental samples by What are the new findings? Microbiome Medicine Center, 16S rRNA sequencing, quantitative PCR and in situ ► Inoculation of the gut microbiome from Division of Laboratory Medicine, hybridisation. Zhujiang Hospital, Southern patients with PE triggered a significantly Medical University, Guangzhou, Results Patients with PE showed reduced bacterial higher blood pressure before pregnancy and Guangdong, China; diversity with obvious dysbiosis. Opportunistic pathogens, PE-lik e phenotypes during pregnancy, including hzhou@ smu. edu. cn and particularly Fusobacterium and Veillonella, were enriched, worsened hypertension, exacerbation of Professor Yanhong Yu, whereas beneficial bacteria, including Faecalibacterium http://gut.bmj.com/ Department of Obstetrics and proteinuria and intrauterine growth restriction. and Akkermansia, were markedly depleted in the PE Gynecology, Southern Medical ► Higher levels of total bacteria and University Nanfang Hospital, group. The abundances of these discriminative bacteria Fusobacterium and an altered microbiota Guangzhou, Guangdong, China; were correlated with blood pressure (BP), proteinuria, profile were identified in PE placenta samples yuyh1010@ hotmail. com aminotransferase and creatinine levels. On successful compared with in normotensive samples. colonisation, the gut microbiome from patients with XC, PL and ML contributed ► The dysbiotic gut microbiome induced immune PE triggered a dramatic, increased pregestational BP of equally. imbalances related to T-lymphocytes and on September 28, 2021 by guest. Protected copyright. recipient mice, which further increased after gestation. intestinal barrier dysfunction, facilitating Received 15 May 2019 In addition, the PE-tr ansplanted group showed increased translocation of bacteria to the intrauterine Revised 11 December 2019 proteinuria, embryonic resorption and lower fetal and Accepted 14 December 2019 cavity, ultimately eliciting inflammation in the Published Online First placental weights. Their T regulatory/helper-17 balance placenta and contributing to poor placentation. 3 January 2020 in the small intestine and spleen was disturbed with more severe intestinal leakage. In the placenta of both How might it impact on clinical practice in the patients with PE and PE-FMT mice, the total bacteria, foreseeable future? Fusobacterium, and inflammatory cytokine levels were ► Gut microbiota screening shows potential significantly increased. for predicting PE onset and provides a new Conclusions This study suggests that the gut approach for the prevention and treatment of microbiome of patients with PE is dysbiotic and this disease. contributes to disease pathogenesis. To date, no first-trimester or second- trimester tests can reliably predict the development of all cases of 3 © Author(s) (or their INTRODUCTION PE, and thus, it is of particular interest to find effec- employer(s)) 2020. Re- use Pre- eclampsia (PE) is a disorder of pregnancy asso- tive and accessible clinical biomarkers in the field permitted under CC BY-­NC. No ciated with new- onset hypertension, which occurs of obstetrics. The pathogenesis of this morbidity is commercial re- use. See rights most often after 20 weeks of gestation combined still vague to date.2 Emerging studies have indicated and permissions. Published with proteinuria.1 PE affects 2%–8% of pregnancies that PE should be considered as a type of metabolic by BMJ. and is associated with an increased risk of adverse disease closely associated with metabolic disorders To cite: Chen X, Li P, Liu M, pregnancy outcomes, including placental abruption, such as obesity, diabetes mellitus, insulin resistance, et al. Gut 2020;69:513–522. prematurity and intrauterine growth restriction.2 3 atherogenic dyslipidaemia and hyperglycaemia.4–6 Chen X, et al. Gut 2020;69:513–522. doi:10.1136/gutjnl-2019-319101 513 Gut microbiota The presence of glucose intolerance and dyslipidaemia, along prepared faecal contents. Another group of female mice was with hypertension, can contribute to endothelial dysfunction, gavaged as described previously, but the donor supernatant was Gut: first published as 10.1136/gutjnl-2019-319101 on 3 January 2020. Downloaded from poor placentation and abnormal placental development, which replaced with phosphate- buffered saline (PBS). Six weeks after are common pathological abnormalities in PE.7 the first microbial administration, all female mice were housed Numerous studies have suggested that gut dysbiosis plays with male mice at a 2–3:1 ratio overnight, and pregnancy a causative role in the development of metabolic syndrome, was confirmed by the presence of vaginal spermatozoa. Mice chronic inflammation diseases and complicated pregnancies.8 9 harbouring microbiota from patients with PE and NP, as well as Gut microbiota can facilitate insulin resistance by inducing the PBS, were referred to as PE- FMT, NP- FMT and control, respec- chronic inflammation in the host and causing fat accumulation tively. The group and cage number of the mice involved in the by regulating genes related to energy metabolism, which plays study are shown in online supplementary table S4. an important role in the development of core symptoms in meta- 10 bolic disorders. Gut microbiome dysbiosis is a key factor in Protein expression and biochemical analysis 11 12 blood pressure (BP) regulation and can contribute to the Protein was extracted from tissues with commercial lysis buffer. 17 development of proteinuria and subsequently lead to kidney Western blotting was performed as described previously. 13 disease. Primary antibodies against zonula occluden (ZO)-1 (Invit- The composition of the gut microbiota in patients with PE rogen, Carlsbad, California, USA), ZO-2 (Abcam, Cambridge, and the relationship between the gut microbiota and PE have UK), occludin (Invitrogen), claudin-4 (Abcam) and glyceralde- not been clarified so far. Recent studies suggested a disrupted gut hyde 3-phosphate dehydrogenase (GAPDH, Invitrogen) were 14 15 microbiota composition in patients with PE in late pregnancy, used. Faecal albumin and urinary protein concentrations were but there is lack of causative analysis. In this study, we performed determined with a mouse albumin ELISA kit (Bethyl Labs, 16S ribosomal RNA (rRNA) gene sequencing on faecal samples Montgomery, Alabama, USA) according to the manufacturer’s to investigate the detailed profile of the gut microbiome and to instructions. The endotoxin level was measured with a commer- determine the crucial role of the disordered gut microbiome in cial kit (Bio-Swamp, Beijing, China). triggering PE- like phenotypes by faecal microbiota transplanta- tion (FMT). Gene expression analysis Total RNA was extracted with Trizol reagent (Invitrogen) MatERIALS AND METHODS according to the manufacturer’s instructions. The reverse tran- Study subjects scription reaction was carried out with reverse transcription Women with PE and normotensive pregnant (NP) women without enzyme (Toyobo, Shanghai, China). Quantitative real- time previous treatments were recruited in the third trimester from PCR was carried out on an ABI Q5 real- time PCR system and March 2017 to March 2018 in the Department of Obstetrics the specific primers for quantitative PCR are shown in online of the Nanfang Hospital, Southern Medical University, China. supplementary table S5. According to the current American College of Obstetricians 2 and Gynaecologists criteria, PE was defined as hypertension Flow cytometry http://gut.bmj.com/ (systolic blood pressure (SBP) or diastolic blood pressure (DBP) Isolated immune cells were