Endometrial in women with adenomysosis,

endometriosis, and healthy controls: A pilot study

Cocoro Mori1, Jocelyn M. Wessels, Ph.D.2, Maria Haikalis1, Michael Tsoulis1, Sanjay K. Agarwal3, and Warren G. Foster1,3,†, 1Department of Obstetrics and Gynaecology, 2Department of Pathology and Molecular Medicine, McMaster University, 3Center for Endometriosis Research and Treatment, University of California San Diego, La Jolla, California, USA

Top 20 genera in the Results Adenomyosis Controls endometrial microbiome Introduction 100

Differences in the eutopic of women with endometriosis compared to controls Lactobacillus have been suggested to be important in the pathogenesis of endometriosis (1). Consequently, Paenibacillus we postulate that environmental influences and changes in endometrial function may be 75 important in the pathobiology of endometriosis. To this end, we further reasoned that Dialister interaction of the endometrium with exogenous factors such as the local microbiome could be Methylophilaceae**

Campylobacter important and lead to changes in the behaviour of menstruated cells, which form the Anaerococcus menstrual effluent and undergo retrograde menstruation. We were therefore intrigued by a Aquabacterium 50 Achromobacter recent study that described the endometrial microbiome in women undergoing assisted Atopobium reproductive therapy (ART). Sequencing of the V1-2 region of the 16S ribosomal RNA gene in Propionibacterium Sneathia endometrial biopsies revealed the presence of belonging to the Bacteroidetes and Staphylococcus Fusobacterium Proteobacteria phyla which are normally associated with the gastrointestinal tract, as well as 25 Relative Abundance (%) Abundance Relative Veillonella Firmicutes and Actinobacteria that are also typically found in the vaginal microbiome (2). Streptococcus Specific bacterial genera that have been identified in the endometrium include Acinetobacter, Ruminococcaceae** Porphyromonas Blautia, Corynebacterium, Lactobacillus, and Staphylococcus. Since the gut microbiome has Finegoldia Others been associated with diseases of the gastrointestinal tract and behavioural function, we 0 Adenomyosis 5 Adenomyosis 11Control 10 Control Control 13 Control 6 Control 7 Control 8 Control 9 Control Endometriosis 1 Endometriosis & Adenomyosis postulated that differences in the endometrial microbiome may predispose some women to Table 1: Participant Characteristics. Demographic and reproductive characteristics were 12 Control

develop endometriosis. Furthermore, of the endometrial microbiome has recently compared between with endometriosis, endometriosis and adenomyosis vs. a control group.

been associated with dysfunctional menstrual bleeding (3) and endometrial cancer (4). Therefore, we sought to compare the endometrial of women with endometriosis, Figure 4. Cluster dendrogram illustrating dissimilarities in the micriobiome of women endometriosis and adenomysosis, and controls. with adenomyosis (2/5) and controls (8/8). Calculated using the Bray-Curtis dissimilarity distance. Note that one woman with adenomyosis also had endometriosis. r Hypothesis ) 1 0 0 0 2 0 0 0 6 o s x t e U a d T m n i

8 0 0 I O

t ( 1 5 0 0

y The endometrial microbiome of women with endometriosis and endometriosis and s t s i E 4

e s i s adenomyosis will be less diverse and differ in composition compared to women undergoing 6 0 0 r c s e e e 1 0 0 0 v i benign gynecological surgeries. p n D S h

4 0 0 c n d i 2 e o R v

5 0 0 n r 1 2 0 0 n e o a s a h b h Methods S O

0 C 0 0 0 5 0 0 0 1 0 0 0 0 0 5 0 0 0 1 0 0 0 0 0 5 0 0 0 1 0 0 0 0 § Twenty-one women (N=21) undergoing gynecologic laparoscopy for pelvic pain at S e q u e n c e s p e r S a m p le S e q u e n c e s p e r S a m p le S e q u e n c e s p e r S a m p le McMaster University Medical Center participated in this pilot study. Written informed Figure 5. Bacterial alpha diversity was assessed in all 8 controls and 2 women with consent was obtained from each study participant prior surgery according to a protocol adenomyosis, using (a) observed species (estimated bacterial richness), (b) Chao1 approved by the Hamilton integrated Research Ethics Board (REB# 12-083T). (estimated bacterial richness), and (c) Shannon Diversity Index (estimated evenness § The presence or absence of endometriosis was determined at the time of laparoscopy and and richness). confirmed using pathology reports. Women with adenomyosis were identified by imaging and confirmed through review of surgical and pathology reports. Study participants were Figure 1: Bacterial community structure in eutopic endometrium samples of controls and free of other co-morbidities and were grouped as exclusively endometriosis (N=8) and patients with adenomyosis. Twenty most representative OTUs are shown. OTUs were Conclusions endometriosis/adenomyosis (N=5) with the exception of one study participant with identified using NCBI BLAST. *= genus level identification, **= family level identification. exclusively adenomyosis. § To our knowledge, this is the first study that explores the endometrial microbiome in the context of endometriosis and adenomyosis. § Women with tubal cysts or fibroids were assigned to the control group. § Data on the bacterial species present in the eutopic endometrium of all symptomatic § All study participants received prophylactic intravenous antibiotics. During the surgery, a sample of eutopic endometrium was collected by pipelle biopsy. controls and 2/5 adenomyosis study participants was consistent with existing reports on the endometrial microbiome. However, the 16S rRNA gene could not be detected § Eutopic endometrium samples were transferred to RNAlater (Sigma-Aldrich, Mississauga, ON), refrigerated at 4°C overnight, and subsequently stored in -80°C until required for in the endometrium of all women with endometriosis and most adenomyosis samples analysis. (3/5). Inflammation or increased immune surveillance are potential explanations for § PCR was used to amplify the hypervariable V3 region of the bacterial 16S rRNA gene, and failure to detect bacterial species in women with endometriosis. attach unique forward and reverse barcoded primers for sequencing using the Illumina MiSeq platform at the McMaster Genome Facility References § Resulting sequence reads were processed through an in-house bioinformatics pipeline 1. Burney RO, Giudice LC. Pathogenesis and pathophysiology of endometriosis. Fertil Steril 2012; 98:511-519. which included trimming reads that exceeded the V3 region, aligning paired-end 2. Verstraelen H, Vilchez-Vargas R, Desimpel F, Jauregui R, Vankeirsbilck N, Weyers S, Verhelst R, De Sutter P, Pieper DH, Van sequences with PANDAseq, culling sequences with mismatches/ambiguous bases, and De Wiele T. Characterisation of the human uterine microbiome in non-pregnant women through deep sequencing of the V1-2 clustering sequences into operational taxonomic units (OTUs) region of the 16S rRNA gene. PeerJ 2016; 4:e1602. 3. Pelzer ES, Willner D, Buttini M, Huygens F. A role for the endometrial microbiome in dysfunctional menstrual bleeding. Antonie § Taxonomic assignment was performed using the Ribosomal Database Project classifier Van Leeuwenhoek 2018; doi: 10.1007/s10482-017-0992-6. against the Greengenes 2011 reference database 4. Walther-Antonio MR, Chen J, Multinu F, Hokenstad A, Distad TJ, Cheek EH, Keeney GL, Creedon DJ, Nelson H, Mariani A, Chia N. Potential contribution of the uterine microbiome in the development of endometrial cancer. Genome Med 2016; 8:122 § Graphical analysis was conducted using the Phyloseq R package Acknowledgements

Figure 2: Bray-Curtis dissimilarity Figure 3. Similarity between bacterial communities in The authors gratefully acknowledge the assistance of Annette Bullen with study participant recruitment, sample collection and data extraction. Sample collection by the Endo@Mac clinical team is greatly appreciated. Funding support for this project was distance principle coordinate endometrial samples calculated using the Bray-Curtis provided by the Canadian Institutes of Health Research (CIHR; grant MOP142230; to WGF), and the Vanier Canada Graduate analysis. dissimilarity distance. Scholarship-CIHR (to JMW). Laura Rossi from the Michael G. Surette laboratory for her expertise in DNA sequencing. The funding bodies had no role in study design, collection, analysis, or interpretation of data.