“What do we know about regulatory T cells and endometriosis? A systematic review”

Accepted Manuscript Title: “What do we know about regulatory T cells and endometriosis? A systematicreview” Author: Isabela Bottura Leite de Barros Helena Malvezzi Barbara Yasmin Gueuvoghlanian-Silva Carla de Azevedo Piccinato Luiz Vicente Rizzo Sergio Podgaec PII: DOI: Reference:

S0165-0378(16)30513-7 http://dx.doi.org/doi:10.1016/j.jri.2017.04.003 JRI 2468

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Please cite this article as: Barros, I.B.L., Piccinato, C.A.,“What do we know about regulatory T cells and endometriosis? A systematicreview”, Journal of Reproductive Immunology (2017), http://dx.doi.org/10.1016/j.jri.2017.04.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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"What do we know about regulatory T cells and endometriosis? A

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systematicreview"

3 4 Isabela Bottura Leite de Barrosa, Helena Malvezzia, Bárbara Yasmin

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Gueuvoghlanian-Silvaa,#, Carla de Azevedo Piccinatoa, Luiz Vicente Rizzoa, Sergio

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Podgaeca

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Hospital Israelita Albert Einstein, São Paulo, SP, Brazil, 05652-900. Av. Albert

Einstein 627, Morumbi, CEP: 05652-900, Sao Paulo, Brazil

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([email protected];

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[email protected];

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[email protected];

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[email protected];

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[email protected];

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[email protected])

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#

an M d

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Corresponding author

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Abstract

2 Endometriosis is a benign, chronic inflammatory disease that presents

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alterations in immune response that can be detected in eutopic endometrium,

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peritoneal fluid and peripheral blood of affected women. Regulatory T (TReg)

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cells are a subpopulation of T lymphocytes specialized in immune regulation

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that seem to participate in the development of endometriosis, by suppressing

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the immune response and favoring the establishment of lesions. Our aim was

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to review the scientific literature that evaluates TReg cell phenotypes in the

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context of endometriosis. PRISMA statement for systematic reviews was

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applied, using “regulatory T cells” and “endometriosis” as keywords in the

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following databases: PubMed, Cochrane, EMBASE and Lilacs. The initial

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search and abstract review yielded 41 papers relating to the subject. At the

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end, 12 studies, published between 2009 and 2016, were included. Most

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studies that analyzed TReg cells did not characterize these cells with current

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Bona Fide markers. In peritoneal fluid and endometriotic lesions, there was a

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higher concentration of TReg cell phenotype and/orTReg cell expression

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markers in patients with endometriosis when compared with controls.

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However, there is still not a consensus about TReg cells concentration in

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eutopic endometrium and peripheral blood between the revised studies.

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Taken together, this data collection suggests that endometriosis is related to

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TReg cells alterations, although further studies are necessary to reach more

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precise conclusions, especially regarding the percentage of these cells in

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eutopic endometrium and peripheral blood. This systematic review attempted

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to provide instructive and up-to-date collection of data that may help better

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design future studies.

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Keywords: Endometriosis, Immune response, Regulatory T cell

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Highlights

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There are few studies that evaluated Bona Fide TReg cells in endometriosis

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endometriosis

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There is not a consensus about the amount of TReg cells in eutopic endometrium

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Sub-optimal TReg cells seem to be in high concentration in endometriotic lesions

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Bona Fide Treg cell concentration is high in peritoneal fluid from

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Results of TReg cell quantity in peripheral blood is contradictory in

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endometriosis

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11 1. Introduction

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Endometriosis is a benign, chronic inflammatory disease defined by the presence and development of endometrial tissue outside the uterine cavity

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(Vercellini et al., 2014). Although it is a prevalent disease that affects about

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10% of reproductive age women, itsexactetiopathogenesis remain unknown

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(Ahn et al., 2015). Many theories have been proposed to explain the

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development of endometriosis and one of the most accepted is the retrograde

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menstruation theory (Sampson, 1927). However, as this phenomenonoccurs

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in 90% of women and only 10% develop endometriosis, it has been proposed

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a complementary theory by which defective immune response could

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determine survival and implantation of ectopic endometrial cells (Halme et al.,

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1984, Berbic and Fraser, 2011, Olovsson, 2011, Kralickova and Vetvicka,

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2015). This theory hypothesizes that endometrial cells trigger inflammation

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and fibrosis upon ectopic implantation. In healthy women, inflammatory

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reactions send signals to immune systems to scavenge these cells at the

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ectopic sites (Pradeu and Cooper, 2012). However, still according to this

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theory, women with endometriosis would have impairment in this process,

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promoting a reduced attack to the ectopic endometrial cell (Burney and

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Giudice, 2012).

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The first indications of the involvement of the immune response in endometriosis pathogenesis come from the 80s. Dmowski et al. (1981)

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4 observed that cellular immune response to autologous endometrial antigens

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was altered in monkeys with spontaneous endometriosis. Based on that,

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Gleicher et al. (1984) studied the association between different T cell subsets

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(helper T cells, suppressor T cells) and monocytes in peripheral blood from

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women with endometriosis, but failed to find any significance. Subsequently,

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new studies have attempted to clarify the mechanisms underlying the

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association between altered immune response and endometriosis.

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Changes in the type of cells that participate in the innate

[macrophages, neutrophils, dendritic cells (DC) and natural killer (NK) cells]

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and adaptive (T and B lymphocytes) immune responses have been described

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in the endometrium, peritoneal fluid and peripheral blood of women diagnosed

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with endometriosis when compared to non-endometriosis controls (Podgaec

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et al., 2012, Olkowska-Truchanowicz et al., 2013, Ahn et al., 2015).

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Regarding innate immune response, high expression of alternative markers of

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activation was observed in macrophages of peritoneal fluid and endometriotic

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lesions of women with endometriosis (Bacci et al., 2009). Moreover, there are

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differences in the presence of immature and mature dendritic cells in eutopic

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and ectopic endometrium when women with endometriosis are compared to

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healthy control (Schulke et al., 2009). Additionally, although there seems to be

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no differences in number of NK cells between patients with and without

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endometriosis, NK cells from peritoneal fluid of women with endometriosis

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presented lower cytotoxicity (Ho et al., 1995). Also, stromal cells from

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endometriotic lesions seem to have an inhibitory effect on NK cell cytotoxicity

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(Somigliana et al., 1996).

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Adaptive immune cells seem to have a role in endometriosis

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pathogenesis as well, despite there are still some controversies (Ahn et al.,

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2015). It was observed high expression of B Lymphocyte Stimulator (BlyS) in

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endometriotic lesions and serum from women with endometriosis. This protein

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is essential to lymphocyte B development (Hever et al., 2007). Regarding T

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cell subpopulations, Takamura et al. (2015) observed that the proportion of

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Th1 lymphocytes was significantly lower in endometriotic lesions in

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comparison to eutopic endometrium and Th17 lymphocyte fraction was

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significantly higher in the lesions when compared to eutopic endometrium.

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Besides, in peripheral blood, Th1 lymphocyte fraction was significantly higher

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in patients with endometriosis when compared to women without the disease.

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One emerging focus on endometriosis pathogenesis is the role of specialized anti-inflammatory populations of T lymphocytes termed regulatory

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T (TReg) cells. Upon its first molecular characterization as CD4+CD25+TRegcells

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in 2001, TReg cells were detected in a diversity of inflammatory pathologies

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such as allergies, autoimmune diseases and cancer (revised by Sakaguchi et

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al. (2010). Regulatory T cells are potent suppressors of inflammatory immune

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responses, and are essential in preventing destructive immunity in all tissues.

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They are mainly produced in the thymus from where they migrate to the

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circulation (natural TRegs or nTRegs) and, in smaller proportion, some

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differentiate from naïve conventional T cells in the periphery (induced TRegs or

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iTRegs) (Ohkura et al., 2013).These cells are indispensable for maintaining

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antigen-specific T-cell tolerance and immune homeostasis (Ohkura et al.,

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2013, Geginat et al., 2014). Therefore, nTRegs, seem to prevent auto-immunity

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and transplant rejection, whereas iTRegs regulate maternal immune tolerance

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(Corthay, 2009, Sakaguchi et al., 2010, Berbic and Fraser, 2011, Ohkura et

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al., 2013, Clark, 2016). Currently, TReg cells are characterized by

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CD25highCD127-Foxp3+cells, since CD25 and Foxp3 are constitutive markers

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used to isolated these cells, and CD127 expression is inversely correlated

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with Foxp3 expression and with suppressive function of CD4+TReg cells (Liu et

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al., 2006, Josefowicz et al., 2012, Li et al., 2016). However, depending on the

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year of publication, many studies did not use all these markers to evaluate

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TReg cells. Besides the year of publication, the technique used to evaluate TReg

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cells also limits its exact identification.

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It has been suggested that TReg cells are in higher concentration in

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tissues (eutopic endometrium, peripheral blood and peritoneal fluid) of women

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with endometriosis when compared to non-endometriosis control women

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(Chen et al., 2012, Podgaec et al., 2012, Olkowska-Truchanowicz et al.,

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2013). Such increased amount of TReg cells could promote an anti-

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inflammatory environment, by suppressing a possible immune response

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against lesion establishment and permitting ectopic endometrial implantation

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and propagation, as occurs in allogeneic organ grafts and pregnancy (Basta

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et al., 2010). Conversely, TReg cells could be moving towards the ectopic

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6 endometrial implant in an attempt to restrain an exacerbated inflammatory

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response (Basta et al., 2014). For implantation and progression of lesions, it is

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necessary the development of neovascularization to promote endometriotic

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lesion growth. For this, there is a complex involvement of angiogenic factors

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and estrogen produced locally, that will in turn facilitate lesion growth and

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survival. Overall, it is known that these mechanisms influence and are

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influenced by immune cells, although there is still need to clarify the exact

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functions (Ahn et al., 2015). Based on this, the aim of this review was to

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examine articles that evaluated the association between endometriosis and

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changes in TReg cells. To our knowledge, this is the first systematic review on

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this topic.

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This study was designed according to PRISMA statement for

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systematic reviews at Hospital Israelita Albert Einstein in São Paulo, Brazil.

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Studies were selected by searching electronic database and revising

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reference list of articles. The keywords “regulatory T cells” and

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“endometriosis” were submitted to PubMed, Cochrane, EMBASE and Lilacs

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databases (the last search was performed in September 4th 2016).

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All studies that associated TReg cells with the etiopathogenesis, clinical

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symptoms or other aspects of endometriosis were included. No data limits

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were imposed. Two reviewers (I.B.L.B and S.P.) performed eligibility

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assessment independently in an unblinded standardized manner.

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Disagreement on study inclusion was solved by consensus. A systematic

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review was performed analyzing year of publication, number of patients

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involved in the study, type of study, TReg cell characterization criteria, results

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and conclusions of each study.

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2.1 Selection and Exclusion criteria

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A total of 78 articles were retrieved through database searching: 38

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from PubMed and 40 from Lilacs – Medline. No articles were found in the

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other databases. Abstracts of all studies identified by the search strategy were

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7 examined and, if relevant, the papers were read in full. The reference lists of

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these papers were also searched. One more study was found from the

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reference list of one of the articles. After removing duplicates, there were 41

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studies. We also excluded 5 literature reviews (Berbic et al., 2010, Khoufache

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et al., 2012, Basta et al., 2014, Fazleabas et al., 2015, Parkin and Fazleabas,

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2016) and 24 studies with discordant themes from this review, e.g. non TReg

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lymphocytes responsiveness; spontaneous abortion; regulatory

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mechanisms of the fallopian tube epithelium; genetic polymorphisms in Foxp3

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gene and others (Gleicher et al., 1984, Cunningham et al., 1992, Vigano et

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al., 1994, Nava-Loya et al., 1996, Lebovic et al., 2001, Akoum et al., 2002,

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Szyllo et al., 2003, Antsiferova et al., 2005, Ginsburg et al., 2005,

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Lambropoulou et al., 2006, Hirata et al., 2008, Patel and Lessey, 2011, Prieto,

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2011, Saito et al., 2011, Bianco et al., 2012, Coleman et al., 2012, Uchiyama

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et al., 2012a, Uchiyama et al., 2012b, Bellelis et al., 2013, Chaouat, 2013,

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Illanes et al., 2013, Kisielewski et al., 2013, Oda et al., 2013, Wang et al.,

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2014).

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To be included in the review, the study should contain evaluation of one of the following TReg cells markers and endometriosis. As the acceptable

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markers of TReg cells changed in recent years, we considered all TReg cell

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phenotypes. A variety of studies used immunohistochemistry to search for

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Foxp3+ or CD4+Foxp3+ cells and RT-PCR to detect Foxp3 gene expression.

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When flow cytometric analyses were performed, a wider range of cell surface

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markers for TReg cells characterization were considered: CD3+CD25high,

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CD4+CD25high, CD4+Foxp3+, CD3+CD4+Foxp3+, CD4+CD25+Foxp3+ and

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CD4+CD25highFoxp3+. Finally, as shown in Figure 1, 12 studies fulfilled the

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selection criteria and were included, in the review (Budiu et al., 2009, Basta et

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al., 2010, Berbic et al., 2010, Braundmeier et al., 2012, Chen et al., 2012,

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Podgaec et al., 2012, Olkowska-Truchanowicz et al., 2013, Slabe et al., 2013,

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Gogacz et al., 2014, Li et al., 2014, Podgaec et al., 2014, Takamura et al.,

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2015). Among them, relevant animal models used to evaluate the relationship

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between endometriosis and TReg cells were included: Budiu et al. (2009)

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evaluated mouse experimental model and humans and Braundmeier et al.

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(2012) evaluated only primate experimental model.

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3. Results

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3.1 Relevant animal model data

4 Budiu et al. (2009) conducted an experiment crossing loxP-stop-loxP-

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KrasG12D/+ mice (Kras mice) with MUC1 transgenic mice, thereby creating

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MUC1Kras transgenic animals. The MUC1Kras mice enable the study of

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induction of endometriosis at the same time that human MUC1 is expressed.

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Hence, induction of ovarian endometriosis lesions was performed by

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intraovarian injection of Cre-encoding adenovirus (AdCre) to study changes in

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the expression of MUC1 during disease progression.

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The results showed that the expression of MUC1 in the affected ovaries increased significantly after the injection of AdCre and it remained

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throughout the disease, thus stimulating the production of anti-MUC1-specific

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antibodies. Secondary outcomes showed that MUC1Kras and Kras mice with

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lesions similar to endometriosis had higher percentages of

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CD4+Foxp3+TRegcells in paraaortic lymph nodes when compared with controls

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without AdCre injection and to MUC1 mice injected with AdCre. Such increase

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in cell number was not observed in the early stages, even without injury. An

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increase in the percentage of TReg cells in the spleen and lymph nodes of

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injured mice compared with control mice was also observed, suggesting that

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the immune environment was leading to immune suppression.

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In order to study the TReg cells in endometriosis, Braundmeier et al.

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(2012) carried out a cohort study with baboons. The control and experimental

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endometriosis groups consisted of 7 and 20 animals, respectively. On the

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second day of the menstrual cycle, the animals received warm saline

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injections (control group) or inoculation of menstrual endometrium, to induce

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endometriosis (endometriosis group). Using flow cytometry,

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immunohistochemistry and RT-PCR, the authors were able to analyze the

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CD4+CD25+Foxp3+and CD4+CD25+Foxp3- T cellsubpopulations.

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The CD4+CD25+Foxp3+TReg cells decreased in peripheral blood in the

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secretory phase when compared to proliferative and menstrual phases

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(p<0.05). Among the T cell populations the CD4+CD25+Foxp3- T cells, known

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as Tr1, were found most prevalent during all cycle phases. Moreover, the

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9 induction of the disease significantly reduced the population of

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CD4+CD25+Foxp3+ T cells in peripheral blood in both proliferative and mid-

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secretory phases (p<0.05). The CD4+CD25+Foxp3+ and CD4+CD25-Foxp3- T

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cells phenotypes presented lower concentration in the first month after

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inoculation, maintaining low levels throughout the study. Conversely, the

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CD4+CD25-Foxp3- T cell phenotype showed a non-linear reduction during

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proliferative phase and a later reduction during mid-secretory phase.

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Similarly, the quantitative analysis of Foxp3+cells in the eutopic endometrium showed significant reduction after the induction of

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endometriosis. Even in the group that underwent surgical excision, this

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pattern was maintained. The number of Foxp3+ cells in the eutopic

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endometrium of animals subjected to induction of endometriosis was

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significantly lower than the control group.Moreover, evaluations after 1 month

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showed that the expression and the number of Foxp3+ cells in the lesions of

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animals subjected to surgical excision were significantly lower when

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compared with animals treated with non-surgical therapy and control animals.

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Both groups, with the disease treated with non-surgical therapy and the group

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treated with therapy surgery after 6 months, showed an increase ofFoxp3

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expression in the lesions. The group of animals treated with non-therapeutic

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surgery with 6 months of disease showed the highest number of Foxp3+ cells.

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In the highly invasive lesions, Foxp3+cells were abundantly found in

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mesenchymal/endometrial border and this pattern was detected especially in

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invasive lesions of the peritoneum, abdominal organs, and fatty tissue of the

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abdominal wall. The regression of all lesions was significantly higher in the

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groups with surgical excision treatment of highly invasive lesions, after 6

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months than at 1 month. Thus, according to the authors, the surgical removal

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of lesions can alter the rate of regression of all other lesions throughout

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disease, and that TReg are important for maintenance of the disease.

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3.2 Bona Fide TReg cells (CD4+CD25+/hiFoxp3+TReg cells phenotypes)

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In a prospective case-control study conducted by Podgaec et al.

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(2012), a total of 98 women (70 diagnosed with endometriosis and 28 control

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patients without endometriosis after investigative laparoscopy) were enrolled

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10 to study the presence of TReg cells in peritoneal fluid. These samples were

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analyzed for CD4+CD25high cells using flow cytometry, and after isolation of

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the cells by sorting, RT-PCR analysis was performed to identify the Foxp3

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expression. Furthermore, the concentrations of interleukin (IL)-6, IL-10, IL-17

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and transforming growth factor-β (TGF-β) were evaluated and increased

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levels of IL-6 and TGF-β in the group with endometriosis (p<0.001) were

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observed. Among the differences with statistical significance (p <0.001), both

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the percentage of CD4+CD25high cells as the expression of Foxp3 were higher

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in samples of peritoneal fluid of patients with endometriosis when compared

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to the control group. Considering menstrual cycle, disease stage or location of

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the lesions, no differences were found.

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In a case-control study, Olkowska-Truchanowicz et al. (2013) compared a group of 17 women diagnosed with ovarian

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endometriomasstages III/IV, confirmed by laparoscopy and histology, with a

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control group of 15 women with no visible foci of endometriosis, pelvic

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inflammatory disease or other associated diseases, which were undergoing

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laparoscopy due to dermoid ovarian cyst or diagnostic laparoscopy. Patients

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with chronic inflammatory or autoimmune diseases were excluded. Samples

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of peripheral blood and peritoneal fluid of both groups were collected between

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days 5 and 10 of the menstrual cycle and subjected to analysis of antinuclear

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antibodies by immunofluorescence and identification of TReg cells using flow

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cytometry, evaluating CD4, CD25 and Foxp3 expression. The mean

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percentage of CD4+CD25+Foxp3+ T cells in the peripheral blood of women

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with ovarian endometriomas (4.4%) was not significantly different from the

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control group (5.2%). However, when analyzing the peritoneal fluid, a

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significantly higher mean percentage of CD4+CD25+Foxp3+TReg cells

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(p<0.003) was observed in the group with the disease (6.1%) when compared

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with the control group (3.4%). An additional observation described by the

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researchers showed a higher proportion (70-90%) of cells that expressed

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CD25highFoxp3+. This TReg cell subpopulation was in significantly lower

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proportion in the peripheral blood and in increased proportion in the peritoneal

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fluid of ovarian endometriomas group when compared with non-endometriosis

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controls.

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In a prospective study (Basta et al., 2010), the authors performed comparisons between 15 eutopic endometrial samples from control patients,

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with ectopic samples from ovarian endometriomas of 16 women with

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endometriosis as well as 16 ectopic samples from decidua obtained during

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laparoscopy performed for treatment of tubal ectopic pregnancies. The

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patients were classified into subgroups according to the phase of the

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menstrual cycle determined by the histopathological analysis of tissue

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samples. The percentage of CD4+CD25+Foxp3+TReg cells was analyzed by

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flow cytometry after tissue disintegration. TReg cells were found in all samples

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of ovarian endometriomas, 72% of eutopic endometrial samples of the control

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group, and only 29% of decidua samples of patients with ectopic pregnancies.

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There was no difference in the percentage of TReg cells between menstrual

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cycle phases in samples of ovarian endometriomas. Moreover, it was found a

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significantly higher percentage of these cells in samples of ovarian

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endometriomas and control group, when compared to ectopic pregnancy

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group. There was no significant difference between ectopic ovarian

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endometriosis and eutopic control group.

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Another prospective case-control study recently performed by

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Takamura et al. (2015) analyzed ovarian endometrioma samples of 10

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patients and eutopic endometrium from 10 women without endometriosis who

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underwent hysterectomy for fibroids. Additionally, peripheral blood samples

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were collected from 10 endometriosis patients and 10 non-endometriosis

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control patients who underwent laparoscopy due to other benign

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gynecological condition. Flow cytometry was performed to detect

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CD4+CD25+Foxp3+TReg cell phenotype and other immune cells. In this study,

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the endometriomas samples did not show any significant difference in

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CD4+CD25+Foxp3+ TReg cell percentage. There was no significant difference

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in percentage of CD4+CD25+Foxp3+ TReg cells in the peripheral blood of

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women with endometriosis when compared to the control group. Similar

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results were obtained for CD4+CD25+Foxp3+ TReg cells percentage, both on

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tissue and peripheral blood samples, in the proliferative and secretory phase

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of the menstrual cycle.

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The case-control study from Gogacz et al. (2014) enrolled 42 patients who underwent laparoscopy. Flow cytometry was used to determine the

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12 percentage of CD4+CD5+Foxp3+TReg cells in peritoneal fluid and peripheral

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blood. This study revealed no significant differences in the percentage of TReg

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cells in peripheral blood and peritoneal fluid of patients with endometriosis

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when compared with non-endometriosis controls. In addition, the percentages

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of TReg cells did not change according to the stage of endometriosis. However,

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the percentage of TReg cells was found to be higher in the peritoneal fluid

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compared with the peripheral blood of patients in the endometriosis and

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control groups. Considering that, significance was found only in those in the

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control group, lack of significant difference between the peritoneal fluid and

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peripheral blood TReg cell percentage in women with endometriosis, point

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towards deficient local host-defense mechanisms and immune responses in

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those patients.

Finally, although not evaluating CD25 expression, an interesting study

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evaluated CD4+Foxp3+ cells that showed activation signs specific of TReg cells

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by secreting the following cytokines: IL-10, TGF-β and thymus-expressed

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chemokine (TECK). Comparing eutopic endometrium and endometriotic

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lesions in the proliferative phase of the menstrual cycle of 49 patients

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presenting endometriosis with eutopic endometrium from 11 women without

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the disease (control group), Li et al. (2014), carried out a case-control study to

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analyze the correlation of endometriosis with the number of CD4+Foxp3+ T

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cells. The concentration of IL-10, TGF-β and TECK as well as the number of

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CD4+Foxp3+ cells in the peritoneal fluid were positively correlated with the

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progression of endometriosis.

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3.3 Sub-optimal TReg cell determination (CD4+CD25highTReg cells phenotypes) The percentages of lymphocyte subsets (CD19+, CD3+, CD4+, CD8+,

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CD56+ CD16+ and CD3+ CD25high) in the peripheral blood of women with

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endometriosis (n=65) and controls (n=61) in different menstrual cycle phases

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were evaluated by Slabe et al. (2013) using flow cytometry. In non-

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endometriosis control women, the concentration of TReg cells (CD3+CD25high

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phenotype) remained at stable levels in peripheral blood at different stages of

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the menstrual cycle. In contrast, in the endometriosis group, there was a

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significant increase in the concentration of TReg cells in the secretory phase

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when compared with the proliferative phase and to the group without the

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disease in the secretory phase.

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3.4 Foxp3 expression analysis

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In order to verify the gene expression of Foxp3 in patients with deep rectosigmoid endometriosis, Podgaec et al. (2014) conducted a case-control

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study with a group of 22 endometriosis patients and 30 non-endometriosis

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control women. The control group consisted of patients without endometriosis

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identified during laparoscopy that underwent elective surgery for sterilization.

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Eutopic endometrium was obtained from patients of both groups and

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endometriotic lesions were collected from the group with endometriosis. The

13

phases of the menstrual cycle were identified in all women by histological

14

analysis of the endometrial tissue. The Foxp3 gene expression of all samples

15

was evaluated using RT-PCR technique and increased expression of Foxp3

16

was observed in the rectosigmoid lesions when compared to the eutopic

17

endometrium of both groups (p<0.0001). No difference was observed

18

between eutopic endometrium of endometriosis women and the control group.

19

There was also no association between the phase of the menstrual cycle and

20

Foxp3 expression.

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The same study also evaluated the association of clinical symptoms

22

characteristic of endometriosis with Foxp3 expression. It was observed lower

23

expression of Foxp3 on the eutopic endometrium of patients with

24

endometriosis and chronic pelvic pain or urinary cyclic pain.

25

In a prospective study conducted by Chen et al. (2012) Foxp3

26

expression was analyzed in the endometrium of 27 women with primary

27

infertility and endometriosis (7 in the minimal and mild stages and 20 in the

28

moderate and severe stages according to the American Society for

29

Reproductive Medicine, ASRM, 1996) (American Society for Reproductive

30

Medicine, 1997) and 20 fertile women without endometriosis and no history of

31

miscarriage, ectopic pregnancy or premature birth (control group). The

32

samples were collected with the use of curettage technique between days 19

33

and 23 of the menstrual cycle. Exclusion criteria for both groups were:

34

autoimmune disease history, pelvic inflammatory disease, intrauterine

Page 13 of 28

14 contraception for less than 6 months, endometrial hyperplasia, endometrial

2

polyps, adenomyosis or uterine fibroids. In RT-PCR analysis, Foxp3

3

expression was significantly higher in the group of infertile patients with

4

endometriosis when compared with the control group (p<0.05). Furthermore,

5

Foxp3 expression was also significantly higher in patients with advanced

6

endometriosis when compared with both mild endometriosis group and

7

controls. The immunohistochemical analysis of the endometrium stromal cells

8

of infertile women with endometriosis III/IV, showed no significant difference in

9

the number of Foxp3+ cells compared to the endometriosis I/II group and the

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control group.

A retrospective case-control study on peritoneal and ovarian

12

endometriosis presented immunohistochemical data of Foxp3+ cells in 127

13

eutopic endometrial samples of women with and without endometriosis

14

(Berbic et al., 2010). The authors also analyzed 59 samples of peritoneal

15

endometriotic lesions and, as controls, 15 peritoneal biopsies from women

16

undergoing laparotomy for conditions unrelated to endometriosis. The phase

17

of the menstrual cycle was determined by histological examination of

18

endometrial samples. A significant increase in the number of Foxp3+ cells was

19

found in the endometrium of women with endometriosis compared to the

20

control group throughout all subgroups of the secretory phase. Nevertheless,

21

a significant increase of Foxp3+ cell was also observed in the early

22

proliferative phase when comparing women with and without endometriosis.

23

The analysis of biopsies from peritoneal endometriosis lesions showed

24

positive results for Foxp3+ cells in 18 of 59 samples, expression restricted to

25

the lesion and not found in the adjacent tissue, and unrelated to the phase of

26

menstrual cycle. None of the 15 peritoneal biopsies in the control group

27

showed Foxp3 expression.

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28 29

4. Discussion

30 31

In this systematic review, we divided the results into groups according

32

to markers chosen to phenotypically characterize TReg cells (Tables 1 and 2).

33

The latest research data that came out in the literature help to elucidate many

34

questions about the relationship between TReg cells and endometriosis.

Page 14 of 28

15 1

However, there are still important points that need clarification, especially

2

those concerning the discrepancies between results, which in part can be

3

explained by the different selection / exclusion criteria, distinct applied

4

methodologies and molecular markers employed.

5

Indeed, the molecular marker strategy used to define the different TReg cell phenotypes is the first issue to be clarified. Depending on the phenotype

7

concerned and the methodology applied, different cell types may be the target

8

of the study, making the results incomparable between different studies. This

9

type of uncertainty contributes to the current confusion regarding the

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Another important issue to be considered is the variation of the amount of TReg cells in the different phases of the menstrual cycle. In order to avoid

13

the menstrual cycle bias, some studies proposed to collect samples in only

14

one phase of the menstrual cycle, as for instance during the menses (Chen et

15

al., 2012) or during the proliferative phase (Olkowska-Truchanowicz et al.,

16

2013, Li et al., 2014). Although this strategy avoids cycle bias, it may also

17

promote partial analysis of the disease, as it only considers one menstrual

18

cycle phase, which could probably mask the spectral feature of endometriosis

19

as a complex disease with many facets.

20

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presence, activity and function of TReg cells in endometriosis.

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The results described by Basta et al. (2010), Podgaec et al. (2012) and

21

Podgaec et al. (2014) showed no interference of menstrual cycle phase in the

22

percentage of Foxp3 expression in eutopic endometrium or endometriotic

23

lesion. Berbic et al. (2010) also observed that in peritoneal lesions, there was

24

no change in the number of Foxp3+ cells during the different phases of the

25

menstrual cycle. Paradoxically, they showed differences in the amount of

26

Foxp3+ cells in the eutopic endometrium of women with endometriosis versus

27

non-endometriosis controls in some stages of the menstrual cycle phases,

28

especially in the secretory phase. Considering that there are fluctuations of

29

Foxp3+ cell levels in eutopic endometrium of non-endometriosis women

30

throughout the menstrual cycle, these data suggest that the observed lack of

31

variation in Foxp3+ cell levels in the eutopic endometrium of endometriosis

32

women could indicate a defective local immunity.

33

The results described by Podgaec et al. (2012) and Olkowska-

34

Truchanowicz et al. (2013) showed a higher number of CD25highTReg cells in

Page 15 of 28

16 peritoneal fluid of patients with endometriosis when compared with the control

2

group. These results were supported by the outcomes of Berbic et al. (2010)

3

and Li et al (2014) that found a higher number of Foxp3+ cells in peritoneal

4

fluid of patients with endometriosis, suggesting that a local suppression of

5

immune cells by TReg cells is taking place in the peritoneal cavity, hampering

6

the elimination of ectopic endometrial cells. The only contradictory data

7

concerning peritoneal fluid TReg phenotypes came from Gogacz et al. (2014)

8

which indicate that there was no difference in the concentration of

9

CD4+CD25+Foxp3+TReg cells in the peritoneal fluid between endometriosis

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and non-endometriosis women.

Instead, when peripheral blood was analyzed, no clear conclusions

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1

could be obtained about the involvement of TReg cells in endometriosis.

13

Olkowska-Truchanowicz et al. (2013) detected a decrease in

14

CD4+CD25highFoxp3+TReg cells in the blood of patients with endometriosis

15

when compared with the control group. Conversely, Slabe et al. (2013) found

16

an increase in the percentage of CD3+CD25highTRegcells in peripheral blood of

17

patients with endometriosis during the secretory phase, with no oscillation in

18

the levels of such cells in non-endometriosis control patients. When

19

evaluating CD4+CD25+Foxp3+ TReg cells, Takamura et al. (2015), found no

20

difference in the percentage of these cells in the peripheral blood. In addition,

21

when Olkowska-Truchanowicz et al. (2013) also evaluated

22

CD4+CD25+Foxp3+ or CD4+CD25+ T cells, no difference was found in the

23

peripheral blood of patients with endometriosis and controls, which is

24

consistent with the results of Takamura et al. (2015).

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an

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Similar results observed in human were reported in animal models.

26

There is an increase of TReg cells in lymph nodes and spleen of mice with

27

lesions similar to endometriosis when compared to the mice with no apparent

28

lesions and to the controls. In peripheral blood of non-human primates, the

29

induction of disease significantly reduced all peripheral TReg populations

30

during both cycle phases, besides observing a greater presence of Foxp3+

31

cells and higher expression of Foxp3 in endometriotic lesions along time.

32

So far, little is known about the association of TReg cells and clinical

33

symptoms of endometriosis. The results reported by Chen et al. (2012) were

34

different from those already reported previously by other authors (Jasper et

Page 16 of 28

17 al., 2006), which demonstrated an association between unexplained infertility

2

and reduced Foxp3 gene expression in the eutopic endometrium. Therefore,

3

the study performed by Chen et al. (2012) concluded that the presence of

4

greater number of Foxp3+ cells in the eutopic endometrium, during the

5

secretory phase, could be associated with the pathogenesis of advanced

6

endometriosis, with no correlation with infertility per se. This is consistent with

7

the data presented by Berbic et al. (2010) that showed an increase in Foxp3

8

expression in eutopic endometrium of women with endometriosis during the

9

secretory phase.

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The association between Foxp3 expression and clinical symptoms of endometriosis were also analyzed by Podgaec et al. (2014). Despite no

12

correlation with infertility was detected, there was a lower expression of Foxp3

13

in eutopic endometrium of women with endometriosis with chronic pelvic and

14

cyclic urinary pain. Although the sole expression of Foxp3 does not clearly

15

identify TReg cells, this suggests that investigation of Foxp3 expression in the

16

eutopic endometrium may be a possible auxiliary tool in the diagnosis of

17

endometriosis, especially in patients with these symptoms.

an

M

Analyzing the articles in this systematic review, we could observe a

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11

similarity in the data collected from peritoneal fluid and endometriotic lesions.

20

Additionally, the results are conflicting when analyzing different percentages

21

of TReg cell phenotypes in the eutopic endometrium, with the majority of the

22

studies showing an increase of TReg cells in the endometriosis group when

23

compared with controls. The results of peripheral blood evaluations also

24

exhibit inconsistencies, since some studies show an increase of TReg cell

25

phenotypes in endometriosis, while others show a decrease or similarity in the

26

proportion of TReg cells in relation to the control groups. This inconsistency is

27

seen among studies that used different cell surface markers to characterize

28

TReg cells. Together, these data showed that the relationship between the

29

presence or number of TReg cells in endometriosis women is still a question

30

that should be better investigated. Nevertheless, it is important to note that all

31

studies so far have taken a static picture of cell populations that are

32

continuously maturing and changing plasticity, be it in the peritoneal fluid,

33

blood or ectopic endometrium. The studies reported here, as well as others

34

took into account cell markers and genes expressed at the moment samples

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Page 17 of 28

18 1

were collected. We now know that TReg cells, as well as other cells in the

2

immune system, may change surface markers as they mature throughout an

3

immune response.

4 5

5. Conclusion

6 Despite the already known data about the relationship of TReg cells with

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endometriosis, it is still necessary to clarify some points. Studies with similar

9

laboratory methods, tissue samples, TReg cell phenotypes, besides inclusion

10

and exclusion criteria are essential to reach more accurate conclusions. Due

11

to the complexity of data about the involvement of TReg cells in the

12

pathophysiology of endometriosis, this systematic review attempted to provide

13

instructive and an up-to-date data collection that may help better design future

14

studies. These studies should help to understand more clearly the

15

pathogenesis of endometriosis and possibly create new diagnostic and

16

therapeutic strategies for this high prevalent disease, with great

17

socioeconomic impact.

20 21

Funding

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SP and BYG-S are supportedby Fundação de Amparo à Pesquisa do

22

Estado de São Paulo (FAPESP - #2013/27092-2 and #2014/08227-7,

23

respectively). CAP is a visitingscientistsupportedbyaninstitutionalgrantfrom

24

Sociedade Beneficiente Israelita Brasileira Albert Einstein, LVR has a

25

personalgrant for scientificachievementfrom Conselho Nacional de

26

Desenvolvimento Científico de Tecnológico (CNPq).

27 28

Acknowledgement

29

We would like to thank Sheyla Inés Castillo-Mendéz for critically reading the

30

manuscript.

31 32

Conflict of interest

33 34

The authors have no conflict of interest to declare.

Page 18 of 28

19 1

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Somigliana, E., Vigano, P., Gaffuri, B., Candiani, M., Busacca, M., Di Blasio, A.M., et al., 1996.

26

Modulation of NK cell lytic function by endometrial secretory factors: Potential role in

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endometriosis. Am. J. Reprod. Immunol. 36, 295-300

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Szyllo, K., Tchorzewski, H., Banasik, M., Glowacka, E., Lewkowicz, P., Kamer-Bartosinska, A.,

29

2003. The involvement of T lymphocytes in the pathogenesis of endometriotic tissues

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overgrowth in women with endometriosis. Mediators Inflamm.12, 131-138

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Takamura, M., Koga, K., Izumi, G., Hirata, T., Harada, M., Hirota, Y., et al., 2015.

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Simultaneous detection and evaluation of four subsets of CD4+ T lymphocyte in lesions

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and peripheral blood in endometriosis. Am. J. Reprod. Immunol.74, 480-486

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Uchiyama, M., Jin, X., Zhang, Q., Amano, A., Watanabe, T., Niimi, M., 2012a. Induction of

2

regulatory CD4+ cells and prolongation of survival of fully allogeneic murine cardiac

3

grafts by danazol. Transplant. Proc.44, 1067-1069

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Uchiyama, M., Jin, X., Zhang, Q., Hirai, T., Bashuda, H., Watanabe, T., et al., 2012b. Danazol

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6

generation of regulatory CD4+ cells in mice. Transpl. Int.25, 357-365

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ip t

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danazol on lymphocyte-mediated cytotoxicity toward human endometrial stromal cells.

11

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us

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Wang, W.J., Liu, F.J., Xin, L., Hao, C.F., Bao, H.C., Qu, Q.L., et al., 2014. Adoptive transfer of

13

pregnancy-induced CD4+CD25+ regulatory T cells reverses the increase in abortion rate

14

caused by interleukin 17 in the CBA/JxBALB/c mouse model. Hum. Reprod.29, 946-952

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cr us

25

Table 1. Table showing the main findings of included studies. Noteworthy is the lead author and year, methodological

2

design, method of analysis of samples and results found, showing increase ( ), decrease ( ) or no difference (=) in TReg

3

cells concentrations in eutopic endometrium, endometriotic lesions, peritoneal fluid and peripheral blood of research

4

subjects with endometriosis and the non-endometriosis controls.

5

Basta, 2010

a

Case Control a

Endometriosis

Control

Proliferative Secretory Proliferative Secretory

Cytometry

-

Peritoneal Fluid Peripheral Blood Endometriotic Lesions Endometriosis Control Endometriosis

=

=

-

-

-

-

OlkowskaCytometry + Case Control Truchanowicz, 2013a immunofluorescence

-

-

-

-

-

Gogacz, 2014a

-

-

-

-

-

-

-

-

-

pt

-

Li, 2014a

Case Control

a

Ac Berbic, 2010

Cytometry

Case Control Cytometry + ELISA

Takamura, 2015 b

Chen, 2012b

7

Endometrium

Method of sample analysis

Case Control Cytometry + RT PCR

ce

Podgaec, 2012

6

Experimental Design

ed

Main Author and Year

M

an

1

Case Control

Cytometry

Immunohistochemistr y Immunohistochemistr Case Control y + RT PCR + ELISA

Case Control

Slabe, 2013b

Case Control

Cytometry

Podgaec, 2014b

Case Control

RT PCR

a

-

+

=

-

-

+

=

=

=

-

=

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

FoxP3 Treg cells phenotypes). Studies with Sub-optimal Treg cells

Treg phenotypes or FoxP3 expression)

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=

-

-

b

-

-

high

(CD4 CD25

-

-

=

+/hi

Studies with Bona Fide Treg cells (CD4 CD25 +

=

-

-

cr us

26

Table 2. Table showing the main findings of included relevant animal model studies. Noteworthy is the lead author and

2

year, methodological design, method of analysis of samples and results found, showing increase ( ), decrease ( ) or no

3

difference (=) in TReg cells concentrations in eutopic endometrium, endometriotic lesions, peritoneal fluid, peripheral blood,

4

lymph nodes and spleen of research subjects with endometriosis and the non-endometriosis controls.

M

an

1

5

Method of sample analysis

Cohort

Cytometry + Immunohistochemistry + RT PCR

pt

Braundmeier, 2012**

Experimental Design

ed

Main Author and Year

Cohort

Endometriosis

Control

Endometriotic Lesions

Peritoneal Fluid

Peripheral Blood

Endometriosis

Control

-

-

Para aortic lymph nodes and Spleen Transgenic mice with endometriotic lesions Transgenic mice without lesions =

* Study in baboons; ** Study in mice

Ac

6

ce

Budiu, 2009*

Cytometry + Immunohistochemistry + RT PCR

Endometrium

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Endometriosis

Control

Control =

27 1

Figure legends

2 3

Figure 1. Flow diagram of the studies included in the systematic review, after

4

searching for the keywords “endometriosis” and “regulatory T cells” and

5

excluding duplications, reviews and discordant themes.

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an

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6

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