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Foxp3 expression in deep rectosigmoid endometriosis lesions and its association with chronic pelvic pain Sergio Podgaec a,b,∗ , Denise Frediani Barbeiro c , Bárbara Yasmin Gueuvoghlanian-Silva b , Patrick Bellelis a , Maurício Simões Abrão a , Edmund Chada Baracat a a Department of Obstetrics and Gynecology, Faculty of Medicine, University of São Paulo, Av. Dr. Enéas de Carvalho Aguiar, 255, Cerqueira César CEP 05403-000, Sao Paulo, Brazil b Jewish Teaching and Research Institute, Albert Einstein Hospital, Avenida Albert Einstein 627, Morumbi CEP 05652-900, Sao Paulo, Brazil c Medical Investigation Laboratory #51, Faculty of Medicine, University of São Paulo, Av. Dr. Arnaldo, 455, Cerqueira César CEP 01246903, Sao Paulo, Brazil

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Article history: Received 24 February 2014 Received in revised form 11 May 2014 Accepted 22 May 2014 Keywords: Endometriosis Endometrium Forkhead transcription factor – Foxp3 T regulatory cells

a b s t r a c t Endometriosis is a benign gynecological disease that is related to immune response alterations. T regulatory cells modulate immune response, and Foxp3 seems to be the best marker of these cells. This study evaluated Foxp3 mRNA expression in eutopic endometrium from women with endometriosis and healthy controls, and its expression in deep rectosigmoid endometriosis lesions, one of the more aggressive types of the disease. Foxp3 expression was higher in lesions than in eutopic endometrium in the two groups. Moreover, eutopic endometrium Foxp3 expression of women with endometriosis was associated with chronic pelvic pain and cyclic urinary pain. © 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Endometriosis is a gynecological disease characterized by endometrial stroma and/or glandular epithelium implantation outside the uterus (Giudice, 2010). It has been shown that immunological response changes may be involved in the development of the disease. It is hypothesized that in women with endometriosis that the immune system does not react against the implants from

∗ Corresponding author at: Avenida Albert Einstein 627, Bloco A1, sala 113, Morumbi CEP 05652-900, Brazil. Tel.: +55 11 30 81 70 03; fax: +55 11 30 81 70 03. E-mail addresses: [email protected] (S. Podgaec), [email protected] (D.F. Barbeiro), [email protected] (B.Y. Gueuvoghlanian-Silva), [email protected] (P. Bellelis), [email protected] (M.S. Abrão), [email protected] (E.C. Baracat).

retrograded menstrual flux and thus, endometrial cells survive, new vessels are developed, and the cells proliferate (Burney and Giudice, 2012). One mechanism that could be involved in this process includes alteration in the presence of some T cell subtypes. Regulatory T cells (Tregs) have immunomodulatory propriety, regulating Th1/Th2 response and produce mainly transforming growth factor beta (TGF-B) and interleukin10 (IL-10) (Sakaguchi et al., 2007; Corthay, 2009). Moreover, forkhead transcription factor (Foxp3) is the main marker of these cells and is essential for differentiation and suppressor activity of Tregs (Josefowicz et al., 2012). There is already some evidence regarding the relationship between Treg cells and endometriosis. The presence of these cells may inhibit the recruitment of immune cells, increasing the chances of survival of the endometriotic implants (Berbic and Fraser, 2011). A higher frequency of

http://dx.doi.org/10.1016/j.jri.2014.05.002 0165-0378/© 2014 Elsevier Ireland Ltd. All rights reserved.

Please cite this article in press as: Podgaec, S., et al., Foxp3 expression in deep rectosigmoid endometriosis lesions and its association with chronic pelvic pain. J. Reprod. Immunol. (2014), http://dx.doi.org/10.1016/j.jri.2014.05.002

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TCD4+ CD25high cells expressing Foxp3 has been observed in the peritoneal fluid of women with endometriosis (Podgaec et al., 2012; Olkowska-Truchanowicz et al., 2013). Although Berbic et al. (2010) observed higher expression of Foxp3 in eutopic endometrium of women with the disease, Basta et al. (2010) did not. Deep infiltrating lesions affecting the bowel are one of the most aggressive types of this disease, and may differ from more benign lesions. To date, there have been no studies evaluating Treg cells in deep endometriosis. Thus, the objective of the present study was to evaluate Foxp3 mRNA expression in deep rectosigmoid endometriosis lesions. 2. Materials and methods This case–control exploratory study included 22 patients with endometriosis and 30 control women. The study has IRB approval, and all participants signed the written informed consent. Patients were 18–40 years of age with eumenorrheic cycles of 26–34 days. All patients were clinically evaluated and had undergone transvaginal ultrasound with bowel preparation. Surgery in the endometriosis group was indicated for patients in whom deep rectosigmoid lesions had been observed on ultrasound and who had tried 12 months of hormonal treatment without acceptable improvement in their pain symptoms. Controls were women undergoing elective tubal ligation and without endometriosis observed during surgery. Exclusion criteria included autoimmune, inflammatory or malignant disease, and use of hormonal therapy for 3 months prior to the study. After induction of adequate anesthesia, a sample of eutopic endometrium was collected using a Pipelle curette (both groups). During surgery, after removal of the disease, tissue samples were obtained from endometriotic lesions (endometriosis group). Histological analysis confirmed endometriosis in all lesions and the menstrual cycle phase (follicular or secretory) was determined by eutopic endometrium histological analysis. Gene expression was determined by reversetranscriptase polymerase chain reaction (RT-PCR) analysis using beta-2-microglobulin (B2M) gene as the housekeeping gene. Total RNA was isolated from eutopic endometrium and from endometriotic lesion in biopsy samples according to a standard Trizol RNA isolation protocol (Invitrogen). Samples of total RNA were quantified by measuring the optical density at 260 nm (Nano Vue Plus Spectrophotometer; GE). All samples had a degree of purity between 1.8 and 2.0. The integrity was analyzed by electrophoresis using 1% agarose gel stained with ethidium bromide. The reactions were made with 100 ng/mL mRNA concentration. All RT-PCR reaction mixtures were prepared using Superscript Platinum One-Step kits with incorporated SYBR Green (11736-051; Invitrogen) on a Step One thermocycler (Applied Biosystems) with the following primers: B2M forward 5 -GATGAGTATGCCTGCCGTGTG-3 ; B2M reverse 5 -CCATCCAAATGCGGCATCCT-3 ;

Foxp3 forward 5 -GACCAAGAAGTGAGGTTTCCAC-3 ; Foxp3 reverse 5 -TTCCAGCCCTGAAGTAATCTGT-3 . Reverse transcription was performed at 50 ◦ C for 10 min. After that, PCR was performed under the following conditions: 95 ◦ C for 30 s, 60 ◦ C for 30 s, and 72 ◦ C for 1 min for 35 cycles. The fold change was represented as 2−CT . Categorical variables were analyzed using Fisher’s exact test for data with two rows and two columns, or Chisquared test for large values. Kolmogorov–Smirnov or Shapiro–Wilk tests and skewness and kurtosis values were used to assess the distribution of all quantitative variables. For analysis of variance between groups was used oneway ANOVA with Bonferroni’s multiple comparison test. Student’s t or Mann–Whitney tests were used to analyze continuous variables, for normal and non-normal variable distribution respectively. Welch’s correction was applied when the continuous variables had normal distribution, but the samples had unequal variances. Pearson or Spearman correlation was applied to calculate the correlation coefficients, for normal and non-normal variable distribution respectively. All tests were considered significant at p < 0.05. Statistical analyses were performed using standard software (GraphPad Prism 5 for Windows). 3. Results and discussion There were no differences between the groups regarding age, body mass index, or menstrual cycle phase. The clinical symptoms, dysmenorrhea, chronic pelvic pain, deep dyspareunia, infertility, cyclic dyschezia, and cyclic urinary pain were present in the endometriosis group, while some women in the control group had dysmenorrhea. On this point, there was a significant difference. While 85.7% of women with endometriosis presented this symptom, 36.7% of healthy women had dysmenorrhea (p = 0.0006, Fisher’s exact test). As shown in Fig. 1a, we observed higher Foxp3 expression in deep rectosigmoid lesions than in eutopic endometrium from both healthy women and those with endometriosis (mean ± standard error: 5.97 ± 0.50 × 1.22 ± 0.47 × 1 ± 0.59 respectively; p < 0.0001, one-way ANOVA with Bonferroni’s multiple comparison test). Despite there being some other markers usually used to identify Treg cells, such as CD4+ , CD25high , CD127low by flow cytometry, in routinely fixed paraffin-embedded tissue Foxp3 seems to be the best marker (Giatromanolaki et al., 2008). Our results are in keeping with recent work of our group that observed an increase in Treg cells (TCD4+ CD25high ) in the peritoneal fluid of women with endometriosis compared with controls and an increase in Foxp3 mRNA expression in Treg cells from women with endometriosis (Podgaec et al., 2012). Our results are also in agreement with those of Olkowska-Truchanowicz et al. (2013) who observed a higher percentage of CD25high Foxp3+ cells in the peritoneal fluid of women with the disease. Basta et al. (2010) evaluated the expression of Foxp3 in CD4+ and CD4+CD25+ lymphocytes in ectopic endometrium (ovarian endometrioma) from women with

Please cite this article in press as: Podgaec, S., et al., Foxp3 expression in deep rectosigmoid endometriosis lesions and its association with chronic pelvic pain. J. Reprod. Immunol. (2014), http://dx.doi.org/10.1016/j.jri.2014.05.002

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Fig. 1. (a) Foxp3 mRNA relative expression in eutopic endometrium from healthy women (controls) and women with endometriosis and endometriotic lesions; (b) Foxp3 mRNA relative expression in eutopic endometrium from women with endometriosis according to chronic pelvic pain; (c) Foxp3 mRNA relative expression in eutopic endometrium from women with endometriosis according to cyclic urinary pain.

endometriosis and eutopic endometrium from control women and did not observe differences between the groups. However, Berbic et al. (2010) showed that eutopic endometrium of women with endometriosis has higher Foxp3 expression than women without the disease, regardless of the menstrual cycle. Analyzing each menstrual phase separately, they observed high expression of Foxp3 only in the secretory phase. Moreover, they observed that peritoneal endometriotic lesions expressed higher levels of Foxp3 than the eutopic endometrium. However, they were unable to detect Foxp3 in all ectopic samples and could not correlate Foxp3 expression to endometriosis symptoms. We found no association between menstrual cycle phase and Foxp3 expression (data not shown). In women with endometriosis, Foxp3 expression in eutopic endometrium was lower in patients who had chronic pelvic pain (mean ± standard deviation, 0.70 ± 0.60 vs 4.40 ± 4.46, p = 0.01 by unpaired t test with Welch’s correction; Fig. 1b) and lower in patients who presented cyclic urinary pain (mean ± standard deviation, 0.79 ± 0.63 vs 3.69 ± 4.28, p = 0.02 by unpaired t test with Welch’s correction; Fig. 1c). No other associations were found (data not shown). It is important to highlight that the majority of women with cyclic urinary pain also had endometriosis in the urinary tract, which is the possible explanation for the observed association. Also, lower expression of Foxp3 in eutopic endometrium could be a sign of higher expression in endometriotic lesions. Moreover, there is an immunological balance between Treg and Th17 cells (Noack and Miossec, 2014). Perhaps, the lower frequency of Treg cells in eutopic endometrium of women with endometriosis is associated with a higher frequency of Th17 cells, providing an inflammatory environment and causing chronic pelvic

pain. Given that Th17 cells are involved with the development of pain (Murphy et al., 2014), our findings may prove to have diagnostic value in women presenting with chronic pelvic pain. 4. Conclusion To our knowledge, this is the first study to show high Foxp3 expression in deep rectosigmoid lesions. This may be related to the suppression of the local immune response that allows the endometriosis to develop. Our finding of lower Foxp3 expression in eutopic endometrium of women with endometriosis and chronic pelvic pain should be investigated as an auxiliary tool in the diagnosis of endometriosis. Conflict of interest The authors have no conflicts of interest to declare. References Basta, P., Majka, M., Jozwicki, W., Lukaszewska, E., Knafel, A., Grabiec, M., Stasienko, E., Wicherek, L., 2010. The frequency of CD25+CD4+ and FOXP3+ regulatory T cells in ectopic endometrium and ectopic decidua. Reprod. Biol. Endocrinol. 8, 116. Berbic, M., Fraser, I.S., 2011. Regulatory T cells and other leukocytes in the pathogenesis of endometriosis. J. Reprod. Immunol. 88, 149–155. Berbic, M., Hey-Cunningham, A.J., Ng, C., Tokushige, N., Ganewatta, S., Markham, R., Russell, P., Fraser, I.S., 2010. The role of Foxp3+ regulatory T-cells in endometriosis: a potential controlling mechanism for a complex, chronic immunological condition. Hum. Reprod. 25, 900–907. Burney, R.O., Giudice, L.C., 2012. Pathogenesis and pathophysiology of endometriosis. Fertil. Steril. 98, 511–519.

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Corthay, A., 2009. How do regulatory T cells work? Scand. J. Immunol. 70, 326–336. Giatromanolaki, A., Bates, G.J., Koukourakis, M.I., Sivridis, E., Gatter, K.C., Harris, A.L., Banham, A.H., 2008. The presence of tumor-infiltrating FOXP3+ lymphocytes correlates with intratumoral angiogenesis in endometrial cancer. Gynecol. Oncol. 110, 216–221. Giudice, L.C., 2010. Clinical practice. Endometriosis. N. Engl. J. Med. 362, 2389–2398. Josefowicz, S.Z., Lu, L.F., Rudensky, A.Y., 2012. Regulatory T cells: mechanisms of differentiation and function. Annu. Rev. Immunol. 30, 531–564. Murphy, S.F., Schaeffer, A.J., Thumbikat, P., 2014. Immune mediators of chronic pelvic pain syndrome. Nat. Rev. Urol. 11, 259–269.

Noack, M., Miossec, P., 2014. Th17 and regulatory T cell balance in autoimmune and inflammatory diseases. Autoimmun. Rev. 13, 668–677. Olkowska-Truchanowicz, J., Bocian, K., Maksym, R.B., Bialoszewska, A., Wlodarczyk, D., Baranowski, W., Zabek, J., Korczak-Kowalska, G., Malejczyk, J., 2013. CD4(+) CD25(+) FOXP3(+) regulatory T cells in peripheral blood and peritoneal fluid of patients with endometriosis. Hum. Reprod. 28, 119–124. Podgaec, S., Rizzo, L.V., Fernandes, L.F., Baracat, E.C., Abrao, M.S., 2012. CD4(+) CD25(high) Foxp3(+) cells increased in the peritoneal fluid of patients with endometriosis. Am. J. Reprod. Immunol. 68, 301–308. Sakaguchi, S., Wing, K., Miyara, M., 2007. Regulatory T cells – a brief history and perspective. Eur. J. Immunol. 37 (Suppl. 1), S116–S123.

Please cite this article in press as: Podgaec, S., et al., Foxp3 expression in deep rectosigmoid endometriosis lesions and its association with chronic pelvic pain. J. Reprod. Immunol. (2014), http://dx.doi.org/10.1016/j.jri.2014.05.002

Foxp3 expression in deep rectosigmoid endometriosis lesions and its association with chronic pelvic pain.

Endometriosis is a benign gynecological disease that is related to immune response alterations. T regulatory cells modulate immune response, and Foxp3...
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