Association between FOXP3+ regulatory T-cells and occurrence of peritoneal lesions in women with ovarian endometrioma and dermoid cysts

doi:10.1016/j.rbmo.2019.01.011

Reproductive BioMedicine Online

Volume 38, Issue 6, June 2019, Pages 857-869

https://doi.org/10.1016/j.rbmo.2019.01.011 Get rights and content

Abstract

Research question

Is there any relationship between numbers of FOXP3⁺ regulatory T-cells (Treg) and occurrence of peritoneal lesions in women with ovarian endometrioma and dermoid cysts?

Design

Retrospective and prospective case-controlled cohort study. Peritoneal lesions were collected from 27 women with ovarian endometrioma and 25 women with dermoid cysts. Peritoneal fluid was collected from 36 women with ovarian endometrioma and 42 women with dermoid cysts. Tissue expression of Forkhead box P3 (FOXP3), one of the transcription factors of Treg cells, and transforming growth factor-beta (TGF-β) were examined by immunohistochemistry. Interleukin-6 (IL-6) and TGF-β levels in the peritoneal fluid were measured by enzyme-linked immunosorbent assay.

Results

Ovarian endometrioma cases with coexisting peritoneal lesions were significantly more frequent than dermoid cyst cases with coexistent peritoneal lesions (269/350 [76.9%] versus 74/414 [17.9%]; P < 0.001). Numbers of FOXP3⁺ Treg cells were significantly higher in peritoneal lesions of women coexistent with ovarian endometrioma (F = 21.52, P < 0.001) and dermoid cysts (F = 22.01, P < 0.001) compared with women without peritoneal lesions. Higher FOXP3⁺ Treg cell numbers in pathological lesions corresponded with significantly higher TGF-β (P < 0.001) and lower IL-6 (P = 0.020) levels in peritoneal fluid of women with peritoneal lesions compared with women without lesions.

Conclusions

This study confirms current speculation that endometriosis is related to alteration in Treg cells, causing survival and implantation of ectopic endometrial lesions in women with endometrioma and dermoid cysts. The findings may clarify why only 10% of women in the general population develop endometriosis despite cyclic menstruation with retrograde flow occurring in >90% of women.

Introduction

Endometriosis is a multifactorial chronic inflammatory condition affecting 6–10% of women of childbearing age (Giudice and Kao, 2004) and is clinically manifested by dysmenorrhoea, chronic pelvic pain and infertility. Many theories have been proposed to explain the development of endometriosis and one of the most accepted is the retrograde menstruation theory (Sampson, 1927). It is still unclear why endometriosis occurs in only 10% of women while retrograde menstruation occurs in more than 90% of women. It has been proposed that a complementary theory may be involved by which defective immune response could determine survival and implantation of ectopic endometrial cells (Berbic and Fraser, 2011, Helme et al., 1984, Kralickova and Vetvicka, 2015, Olovsson, 2011). According to this theory, inflammatory reactions send signals to immune systems to scavenge endometrial cells at the ectopic sites, while in women with endometriosis an impairment of this process promotes reduced attack to the ectopic endometrial cells with consequent survival and implantation in the pelvis.

Changes in the type of cells that participate in the innate immune response (e.g. macrophages, neutrophils, natural killer cells) have been described in the endometrium, peritoneal fluid and peripheral blood of women with endometriosis when compared with controls without endometriosis (Ahn et al., 2015, Bacci et al., 2009, Ho et al., 1995, Podgaec et al., 2012). An emerging area of interest in endometriosis pathogenesis is the role of specialized anti-inflammatory populations of T lymphocytes, called regulatory T (Treg) cells. Upon their first molecular characterization as CD4⁺CD25⁺Treg cells in 2001, Treg cells were detected in a range of inflammatory pathologies such as allergies, autoimmune diseases and cancer (Sakaguchi et al., 2010). Treg cells are potent suppressors of inflammatory immune responses and are essential in preventing destructive immunity in all tissues. Currently, Treg cells are characterized by the expression of CD25⁺CD127^–FOXP3⁺ cells, because CD25 and Forkhead box P3 (FOXP3) are constitutive markers to isolate Treg cells (Josefowicz et al., 2012, Li et al., 2016, Liu et al., 2006). Among three subsets of T-cells (Tr1/Th3/Treg) involved in the suppression of immune response, FOXP3⁺Treg cells have been recognized as the main subset in the female reproductive tract (Guerin et al., 2009).

A number of recent elegant studies have demonstrated that Treg cells are in higher concentration in tissues and body fluids (eutopic endometrium, peritoneal fluid and peripheral blood) of women with endometriosis when compared with women without endometriosis (Chen et al., 2012, Olkowska-Truchanowicz et al., 2013, Podgaec et al., 2012). The authors speculated that an increased amount of Treg cells could promote an anti-inflammatory environment by suppressing a possible immune response against lesion development and permitting ectopic endometrial implantation and propagation. However, none of these studies could describe the pattern of Treg cells in different peritoneal lesions coexistent with ovarian endometrioma and dermoid cysts and their association with the revised American Society for Reproductive Medicine (ASRM) staging of endometriosis (American Society for Reproductive Medicine, 1997). We previously demonstrated a significantly higher association of pelvic pain in women with ovarian endometrioma who had coexisting peritoneal lesions compared with women with ovarian endometrioma without coexisting peritoneal lesions (Khan et al., 2013). In clinical practice, there is a lower occurrence of peritoneal lesions in women with dermoid cysts, serous cyst adenoma (SCA) and mucinous cyst adenoma (MCA), even though these women experience similar cyclic menstruation with retrograde menstrual flow. A thorough survey of the coexistence of peritoneal lesions in women with dermoid cysts/SCA/MCA and how this varies from that of ovarian endometrioma is still unknown.

This study consisted of two parts: a retrospective part, reviewing recorded files of laparoscopic surgery including women with ovarian endometrioma, dermoid cysts, SCA and MCA. The second part was a prospective study analysing biopsy samples and peritoneal fluid samples, including only women with dermoid cysts and endometriomas. In the retrospective part of this study, the occurrence of peritoneal lesions in the pelvis of women with coexistent ovarian endometrioma and dermoid cysts/SCA/MCA was analysed. In the prospective part of the study, the expression pattern of FOXP3⁺Treg cells was investigated in biopsy samples collected from women with ovarian endometrioma and dermoid cysts during laparoscopic surgery with or without coexistent peritoneal lesions. Secondly, the number of FOXP3⁺Treg cells based on the phases of the menstrual cycle and revised ASRM staging of endometriosis (American Society for Reproductive Medicine, 1997) were analysed. Finally, levels of transforming growth factor-beta (TGF-β), one of the differentiation factors of Treg cells, was measured, together with levels of interleukin-6 (IL-6), one of the potential cytokines released by the effector immune cells in the peritoneal fluid collected from these two groups of women.

Section snippets

Materials and methods

In the retrospective part of this study, cases of ovarian endometrioma and dermoid cysts/SCA/MCA were searched for from the recorded files of laparoscopic surgery during the period between September 1982 and April 2008, where there was surgery for the indication of ectopic pregnancy, tubal infertility and other benign gynaecological diseases. During the same time period, laparoscopic surgery was performed for cases with dermoid cysts/SCA/MCA, uterine myoma and adenomyosis in addition to cases

Results

During the period between September 1982 and April 2008, searches revealed 2988 cases had laparoscopic surgery for a variable indication (see Materials and methods). Among the 2988 cases who had surgery, 350 cases (11.7%) were found to have ovarian endometrioma, 414 cases (13.9%) had mature cystic teratoma (dermoid cysts), and 101 cases (3.4%) had combined SCA and MCA. Detailed clinical characteristics could only be found for women with ovarian endometrioma and dermoid cysts from the recorded

Discussion

This retrospective cohort study using collective data from the past 25 years demonstrated for the first time that women with dermoid cysts/SCA/MCA had significantly less coexistence of peritoneal lesions in their pelvis compared with women with ovarian endometrioma, even though women with dermoid cysts/SCA/MCA experience similar retrograde flow during menstruation. In an attempt to investigate the possible mechanistic basis, a significantly lower accumulation of FOXP3⁺Treg cells was found in

Acknowledgements

We thank Miss Kyoko Ishida of Nagasaki University Graduate School of Biomedical Sciences for her excellent technical assistance. The authors also thank all doctors of Saiseikai Nagasaki Hospital and Kyoto Prefectural University of Medicine for their kind assistance in collecting biopsy samples and sample preparation for this study. This work was supported in part by Grants-in-Aid for Scientific Research (grant nos. 24592474 and 15K10675 to KNK) from the Japan Society for the Promotion of

Khaleque N Khan is an Associate Professor and Project Coordinator of the Kyoto Prefectural University of Medicine in Kyoto, Japan. His main research interest is investigating the pathogenesis and physiopathology of endometriosis, adenomyosis and uterine myoma. He has published over 80 articles in peer-reviewed journals.

Key message

Coexistence of peritoneal lesions was significantly lower in women with dermoid cysts compared with women with ovarian endometrioma. An alteration of

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Key message

Coexistence of peritoneal lesions was significantly lower in women with dermoid cysts compared with women with ovarian endometrioma. An alteration of FOXP3⁺ Treg cells in the peritoneal lesions of these two groups of women may be involved in the survival and implantation of ectopic endometrial lesions.

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ArticleAssociation between FOXP3+ regulatory T-cells and occurrence of peritoneal lesions in women with ovarian endometrioma and dermoid cysts

Abstract

Research question

Design

Results

Conclusions

Introduction

Section snippets

Materials and methods

Results

Discussion

Acknowledgements

Am J Pathol

J Reprod Immunol

Lancet

J Biol Chem

Fertil Steril

Fertil Steril

Fertil Steril

Pathophysiology and immune dysfunction in endometriosis

Biomed. Res. Int.

Revised American Society for Reproductive Medicine classification of endometriosis: 1996

Fertil Steril

Expansion of CD4+CD25+ and FOXP3+ regulatory Treg cells during the follicular phase of the menstrual cycle: implications for human reproduction

J Immunol

The biological role of Treg cells in ectopic endometrium homeostasis

Histol. Histopathol.

Reciprocal development pathways for the generation of pathogenic effector Th17 and regulatory T cells

Nature

The role of FoxP3+ regulatory T-cells in endometriosis: a potential controlling mechanism for a complex, chronic immunological condition

Hum Reprod

Expression of T regulatory cell transcription factor FoxP3 in peri-implantation phase endometrium in infertile women with endometriosis

Reprod. Biol. Endocrinol.

Regulatory t-cells and immune tolerance in pregnancy: A new target for infertility treatment?

Hum Reprod Update

Retrograde menstruation in healthy women and in patients with endometriosis

Obstet Gynecol

Peritoneal natural killer cytotoxicity and CD25+CD3+ lymphocyte subpopulation are decreased in women with stage III-IV endometriosis

Hum Reprod

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Association between FOXP3⁺ regulatory T-cells and occurrence of peritoneal lesions in women with ovarian endometrioma and dermoid cysts