1 Microbiome in EndometriosisNiket Yadav Burns Lab

2 Endometriosis: a Challenging Disease5.5 million women $22 billion annually Significant morbidity and loss of productivity There are no diagnostic biomarkers for endometriosis There is no cure for endometriosis Ovary Endometrium Cervix Uterus Vagina Endometriosis lesions Fallopian Tube Chronic pelvic pain Infertility Chronic pain leads to emotional health problems Functioning endometrium outside uterine cavity >90% of women have retrograde menstruation Affects 6-10 % of females 35-50 % in infertile women (Giudice and Kao, Lancet 2004; Bulun et al, J Steroid Biochem Mol Biol 2005 ) Posterior cul-de-sac Ovaries Ovarian Fossa Anterior cul-de-sac Bowel/appendix Ligaments Treatments alter hormone regulation Etiology and pathogenesis remain unknown Functioning endometrium outside the uterine cavity Affects 6-10 % of female population Found % in infertile women Believed to be an E2-dependent disease Gynecology Specialists of Utah (Giudice and Kao, Lancet 2004; Bulun et al, J Steroid Biochem Mol Biol 2005 )

3 Female Reproductive SystemOvary Endometrium Cervix Uterus Vagina Endometriosis lesions Fallopian Tube Mouse uterus PR is important here The regulation of estrogen receptor (ER) and progesterone receptor (PR) genes is critical to estrogen and progesterone responsiveness of the uterus during the estrous cycle. The female reproductive system is designed to carry out several functions. produces the egg necessary for reproduction Conception occurs in the fallopian tubes. fertilized egg implants into the walls of the uterus, beginning the initial stages of pregnancy. If fertilization and/or implantation does not take place, the system is designed to menstruate (the monthly shedding of the uterine lining). female reproductive system produces female sex hormones that maintain the reproductive cycle. What Happens During the Menstrual Cycle? Females of reproductive age experience cycles of hormonal activity that repeat at about one-month intervals. With every cycle, a woman's body prepares for a potential pregnancy, whether or not that is the woman's intention. The term menstruation refers to the periodic shedding of the uterine lining. (Menstru means "monthly.'') occurs in phases: the follicular phase, the ovulatory phase (ovulation), and the luteal phase. four major hormones in the menstrual cycle: follicle-stimulating hormone, luteinizing hormone, estrogen, and progesterone. Follicular Phase of the Menstrual Cycle This phase starts on the first day of your period. follicle stimulating hormone (FSH) and luteinizing hormone (LH), are released from the brain stimulate the growth of about 15 to 20 eggs in the ovaries called a follicle. FSH and LH also trigger an increase in the production of estrogen. estrogen levels rise turns off the production of follicle-stimulating hormone. one follicle in one ovary becomes dominant and continues to mature. This dominant follicle continues to produce estrogen. Ovulatory Phase of the Menstrual Cycle The ovulatory phase is the midpoint of the menstrual cycle, with the next menstrual period starting about two weeks later. The rise in estrogen triggers a surge in the amount of luteinizing hormone that is produced by the brain. This causes the dominant follicle to release its egg from the ovary. As the egg is released it is captured by finger-like projections on the end of the fallopian tubes Also during this phase, there is an increase in the amount and thickness of mucus produced by the cervix. If a woman were to have intercourse during this time, the thick mucus captures the man's sperm, nourishes it, and helps it to move towards the egg for fertilization. Luteal Phase of the Menstrual Cycle The luteal phase of the menstrual cycle begins right after ovulation and involves the following processes: Once it releases its egg, the empty follicle develops into a new structure called the corpus luteum. The corpus luteum secretes the hormone progesterone. Progesterone prepares the uterus for a fertilized egg to implant. If intercourse has taken place and a man's sperm has fertilized the egg (a process called conception), the fertilized egg (embryo) will travel through the fallopian tube to implant in the uterus. The woman is now considered pregnant. If the egg is not fertilized, it passes through the uterus. Not needed to support a pregnancy, the lining of the uterus breaks down and sheds, and the next menstrual period begins. (Adapted from Staley & Scharfman, Nature Neuroscience, 2005)

4 Mouse Model of Endometriosis1 cm Donor Host (ovariectomized) 40x vehicle or Estradiol (E2) Host (ovariectomized) Immunocompetent model! Vehicle E2 7.5x Uterine lesion Intestinal lesion Stroma Epithelial lined Gland (Burns et al, Endo 2012) 3 week histology 3 week histology Epithelial lined Gland Stroma

5 Role of Microbiome ? 1 cm Donor Host (ovariectomized) E2or EDC treatment vehicle or Estradiol (E2) Uterus microbiome ? Host (ovariectomized) Immunocompetent model! Hypothesis: Microbiome promotes endometriosis in a mouse model of endometriosis Vehicle E2 7.5x (Burns et al, Endo 2012)

6 What is Microbiome ? Microbiome refers to the collective genomes of the microorganisms within an ecosystem (e.g Human body). Vs. Microbiota / microflora refers to all microorganisms of a particular ecosystem (e.g. human body).

7 Human body microbiome ?

8 More Facts on human microbiome………………….Microbial cells in human body outnumber human cells 10:1! More than 10,000 microbial species have been identified as living in human body thus far. Microbes account for 1-3% of human body weight [up to 4-6 lbs of our body mass]. The microbiome contributes 8 million protein-coding genes.

9 Analyzing Microbiome?

10 Human Microbiome includes species from each major biological domain“Extremophile” Archaebacteria Bacteria Fungi

11 Organ-specific MicrobiomePCoA2 (4.4%) PCoA1 (13%) In healthy American adults, the microbial community composition in each part of the body is unique. Human Microbiome Project Consortium (2012)

12 The Sterile Womb ParadigmThe sterile womb paradigm, coined by French paediatrician Henry Tissier at the turn of the 20th century [that human infants develop within a sterile environment ], has been subsequently challenged!

13 Urogenital Microbiome (Female)Microbiome has been identified from the following parts: Uterus (endometrium) microbiome (Verstraelen et al 2016) Vaginal microbiome (Mitchell CM et al 2015) Placental microbiome (Aagaard K el 2014) Urinary bladder microbiome (Brubaker L, Wolfe AJ. 2015) Verstraelen H, Vilchez-Vargas R, Desimpel F, Jauregui R, Vankeirsbilck N, Weyers S, Verhelst R, De Sutter P, Pieper DH, Van De Wiele T. (2016) Characterisation of the human uterine microbiome in non-pregnant women through deep sequencing of the V1-2 region of the 16S rRNA gene. PeerJ 4:e1602 https://doi.org/ /peerj.1602 Mitchell CM, Haick A, Nkwopara E, et al. Colonization of the upper genital tract by vaginal bacterial species in nonpregnant wome n. Am J Obstet Gynecol 2015;212:611.e1-9. Brubaker L and Wolfe AJ The new world of the urinary microbiota in women. Am. J Obstetrics Gynacol Volume 213, Issue 5, Pages 644–649. Aagaard K, Ma J, Antony KM, Ganu R, Petrosino J, Versalovic J The placenta harbors a unique microbiome. Science Translational Medicine 6 237ra65

14 Microbiome of human uterus (endometrium)The endometrial bacterial community structure of the 19 human subjects (non-pregnant women with reproductive failure): Total 183 phylotypes 60 phylotypes with ≥ 1% abundance Verstraelen et al 2016, Peer J

15 Distribution of upper genital tract bacterial colonization (Human)A). Proportion of participants with detection of bacteria by species-specific qPCR in the vagina alone, both vagina and upper genital tract (UGT), and UGT alone. B). Comparison of the mean quantity of bacteria in the vagina and UGT of women with either vaginal or UGT detection of that species. C). Proportion of women with vaginal detection of a given species by qPCR who also had UGT detection of that species. A, Proportion of participants with detection of bacteria by species-specific qPCR in the vagina alone, both vagina and UGT, and UGT alone. B, Comparison of the mean quantity of bacteria in the vagina and UGT of women with either vaginal or upper genital tract detection of that species. AsteriskIndicatescomparisonsthat are significantly different (P<.05) by Student t test. C, Proportion of women with vaginal detection of a given species by qPCR who also had UGT detection of that species. qPCR, quantitative polymerase chain reaction; UGT, upper genital tract. Mitchell et al Intrauterine bacterial colonization. Am J Obstet Gynecol

16 Role of Microbiome in Endometriosis (Mouse model studies)Overall Working hypothesis Carry over of Microbiome from uterus (endometrium) tissue into the peritoneal cavity may have a role in : Establishment of uterine tissue fragments as lesions of endometriosis Exacerbation of endometriosis lesions by modulating the immune mediators

17 The Study design

18 Microbiome DNA from peritoneal fluid (40 samples)Peritoneal fluid sample in a microfuge tube Isolate Cell pellet (>10k rpm/10 min) Lyse cells by enzymatic and bead beating Process sample using Qiagen’s DNeasy Blood and Tissue kit / Purify DNA Elute microbiome DNA in 200 µl volume of elution buffer

19 16S rDNA PCR on Microbiome DNA for NextGen Sequencing (40 samples)The first round of amplification did not yield amplicons, possibility because of low abundance of microorganisms in the mouse peritoneal fluid

20 Next steps/Future DirectionFor Peritoneal fluid samples - Concentrate the DNA for amplification - Use Multi-round amplification approach - Use genus-specific PCR approach (for bacterial genera known to colonize uterus) Include Uterus tissue samples for comparison (to understand the link between uterus microbiome and peritonial microbiome)

21 Thanks to…… Dr. Katie Burns Dr. Sukanta BhattacharyaMs. Stephanie Lang