Evaluation of M1 and M2 Macrophages in Endometriotic Tissue of Women Affected by Endometriosis at Different Stages.

March 23, 2020 updated by: Antonio Simone Laganà, University of Messina

Evaluation of Evaluate M1 and M2 Macrophages in Endometriotic Tissue of Women Affected by Endometriosis at Different Stages.

Accumulating evidence suggests that the peritoneal microenvironment of women affected by endometriosis undergoes a number of local inflammatory-reparative phenomena, with the involvement of resident macrophages, and the attraction and recruitment of peripheral mononuclear cells (monocytes and lymphocytes) from the blood into the peritoneal cavity: during endometriosis a breakdown occurs in endometrial and peritoneal homeostasis caused by cytokine-addressed cell proliferation and dysregulation of apoptosis.

The surrounding microenvironment may address the macrophage plasticity towards a transient and reversible polarization. These polarized phenotypes reflects the proinflammatory or anti-inflammatory status and may change over the time. They could be functionally classified in two main populations: "classically activated" macrophages (M1) and "alternatively activated" macrophages (M2).

Considering that published data so far are still not robust enough to drawn firm conclusion, the aim of this research project will be to evaluate M1 and M2 macrophages in endometriotic tissue from women affected by endometriosis at different stages.

Study Overview

Status

Completed

Conditions

Intervention / Treatment

Detailed Description

Background and rationale: Endometriosis is an estrogen-dependent disease defined by the ectopic presence and growth of functional endometrial tissue, glands and stroma, outside the uterine cavity (Kavoussi et al, 2016). The aetiopathogenesis of endometriosis still remains controversial: immune, hormonal, genetic, and epigenetic factors may be all involved, and several theories have been proposed to explain it.

The disease affects 2-10 % of women of reproductive age and 50 % of those infertile (Dunselman et al, 2014) and may cause pelvic pain (Triolo et al, 2013; Butticè et al, 2013), abnormal bleeding, infertility/sterility and, consequently, important psychological problems (Laganà et al, 2015). Endometriosis is classified depending on the number, size, and superficial and/or deep location of endometrial implants, plaques, endometriomas and/or adhesions. The most used classification of endometriosis was developed by the American Society for Reproductive Medicine (Rock JA, 1995), although other classification can be used for deep infiltrating endometriosis (Haas et al, 2011) or to correlate endometriosis and infertility (Adamson et al, 2010).

As widely evidenced (Donnez et al, 2016), it is generally accepted that moderate/severe endometriosis-related sterility is due to mechanical factors, namely to the distortion/subversion of the regular pelvic anatomy. On the contrary, the factors behind infertility/subfertility related to minimal/mild endometriosis are less clear. Nevertheless, to date none of the hypothesized mechanisms exhaustively explained the infertility related to endometriosis, while it is possible that such disease is caused by multiple factors altogether, including genetic and epigenetic modifications (Sofo et al, 2015; Maniglio et al, 2016).

Accumulating evidence (Laganà et al, 2013; Laganà et al, 2016) suggests that the peritoneal microenvironment of women affected by endometriosis undergoes a number of local inflammatory-reparative phenomena, with the involvement of resident macrophages, and the attraction and recruitment of peripheral mononuclear cells (monocytes and lymphocytes) from the blood into the peritoneal cavity: during endometriosis a breakdown occurs in endometrial and peritoneal homeostasis caused by cytokine-addressed cell proliferation (Pizzo et al, 2002) and dysregulation of apoptosis (Sturlese et al, 2011; Salmeri et al, 2015).

Monocytes play a key role during adaptive immunity, since they migrates in sites of infection or inflammation, differentiate in macrophages and act as Antigene Presenting Cells (APCs).

Since their first identification of phagocytosis by Elie Metchnikoff in Messina (JAMA, 1968), after experimenting on the larvae of starfish, many steps forward allowed us to have a wide overview of macrophages apart from their role as phagocytic cells.

The surrounding microenvironment may address the macrophage plasticity towards a transient and reversible polarization. These polarized phenotypes reflects the proinflammatory or anti-inflammatory status and may change over the time. They could be functionally classified in two main populations: "classically activated" macrophages (M1) and "alternatively activated" macrophages (M2) (Sica & Mantovani, 2012). Although it is widely accepted that M1 and M2 represent two terminals of the full spectrum of macrophage activation (Liu et al, 2014), recent data (Locati et al, 2013; Mantovani et al, 2013; Capobianco & Rovere-Querini, 2013) allowed to characterize these two populations as follow:

  • M1 Macrophages: CD14+ CD68+ CCR7+ CD80+
  • M2 Macrophages: CD14+ CD68+ CD163+ CD206+

Study Population: The investigators will select patients affected by histologically confirmed endometriosis (cases) and by ovarian functional cysts (controls), who will undergo laparoscopic surgery. In accordance with the revised American Society for Reproductive Medicine classification for endometriosis (Rock JA, 1995), endometriotic patients will be divided into 4 groups: minimal, mild, moderate and severe stages of endometriosis.

All the laparoscopic procedure will be performed during the proliferative phase of the menstrual cycle. The investigators will exclude from the study women affected by other pelvic disorders, chronic circulatory, autoimmune or neoplastic disease and who took any anti-inflammatory or hormonal or immunomodulatory medication in the preceding 6 months.

Tissue sample preparation and macrophage characterization: Tissue sections (endometriotic tissues for cases, ovarian functional cysts for controls) will be minced and incubated in an enzyme cocktail containing final concentrations of 3.4 mg/ml pancreatin, 0.1 mg/ml hyaluronidase and 1.6 mg/ml collagenase I in Hank's Buffered Saline Solution (HBSS) containing 2 mg/ml D-glucose at 37°C for 2 hours. Following digestion, cells will be dispersed by straining through a 250 μm mesh screen and washed with HBSS. Tissue cells will be stained and fixed for flow cytometric analysis. Prior to macrophage isolation, dead cells will be removed from the culture using the dead cell removal kit. Cell viability will be assessed by trypan blue exclusion.

To assess surface expression of macrophage markers, tissue samples will be stained for flow cytometry with fluorochrome-conjugated monoclonal antibodies against CD14, CD68, CCR7 and CD80 to identify M1, whereas fluorochrome-conjugated monoclonal antibodies against CD14, CD68, CD163 and CD206 to identify M2.

Statistical analysis: The assumption of normal distribution for continuous variables will be tested by Kolmogorov-Smirnov test for goodness of fit. All inferential analyses will be performed using nonparametric statistical tests. Non-normally distributed variables between the groups will be compared using the Kruskal-Wallis test. The range of statistical significance will be P < .05. In addition, The investigators will use post hoc multiple comparison tests either versus controls or versus each stage to analyze data.

Study Type

Observational

Enrollment (Actual)

45

Contacts and Locations

This section provides the contact details for those conducting the study, and information on where this study is being conducted.

Study Locations

  • Italy
      • Messina, Italy, 98122
        • University of Messina

Participation Criteria

Researchers look for people who fit a certain description, called eligibility criteria. Some examples of these criteria are a person's general health condition or prior treatments.

Eligibility Criteria

Ages Eligible for Study

16 years to 33 years (Adult)

Accepts Healthy Volunteers

No

Genders Eligible for Study

Female

Sampling Method

Non-Probability Sample

Study Population

Reproductive-aged patients affected by histologically confirmed endometriosis (cases) and by ovarian functional cysts (controls), who will undergo laparoscopic surgery.

Description

Inclusion Criteria:

  • Patients affected by histologically confirmed endometriosis (cases)
  • Patients affected by ovarian function cysts (controls)

Exclusion Criteria:

  • Other pelvic disorders (apart ovarian functional cysts)
  • Chronic circulatory disorders
  • Autoimmune disorders
  • Neoplastic disorders
  • Patients who were treated with any anti-inflammatory or hormonal or immunomodulatory medication in the preceding 6 months.

Study Plan

This section provides details of the study plan, including how the study is designed and what the study is measuring.

How is the study designed?

Design Details

  • Observational Models: Case-Control
  • Time Perspectives: Prospective

Cohorts and Interventions

Group / Cohort
Intervention / Treatment
Endometriosis
Patients affected by endometriosis (histologically confirmed) at different stages, who will undergo laparoscopic surgery.
Diagnostic test: Evaluation of M1 and M2 macrophages by flow cytometry.
To assess surface expression of macrophage markers, tissue samples will be stained for flow cytometry with fluorochrome-conjugated monoclonal antibodies against CD14, CD68, CCR7 and CD80 to identify M1, whereas fluorochrome-conjugated monoclonal antibodies against CD14, CD68, CD163 and CD206 to identify M2.
Ovarian functional cysts
Patients affected by ovarian functional cysts, who will undergo laparoscopic surgery.
Diagnostic test: Evaluation of M1 and M2 macrophages by flow cytometry.
To assess surface expression of macrophage markers, tissue samples will be stained for flow cytometry with fluorochrome-conjugated monoclonal antibodies against CD14, CD68, CCR7 and CD80 to identify M1, whereas fluorochrome-conjugated monoclonal antibodies against CD14, CD68, CD163 and CD206 to identify M2.

What is the study measuring?

Primary Outcome Measures

Outcome Measure
Measure Description
Time Frame
Quantification of M1 and M2 macrophages in endometriotic tissue for each stage of endometriosis.
Time Frame: Day 1
To assess surface expression of macrophage markers, endometrial tissue cells will be stained for flow cytometry with fluorochrome-conjugated monoclonal antibodies against CD14, CD68, CCR7 and CD80 to identify M1, whereas fluorochrome-conjugated monoclonal antibodies against CD14, CD68, CD163 and CD206 to identify M2.
Day 1

Secondary Outcome Measures

Outcome Measure
Measure Description
Time Frame
Correlation of M1 and M2 macrophages quantity in endometriotic tissue to the presence of endometriosis-associated infertility.
Time Frame: Day 1
To assess surface expression of macrophage markers, endometrial tissue cells will be stained for flow cytometry with fluorochrome-conjugated monoclonal antibodies against CD14, CD68, CCR7 and CD80 to identify M1, whereas fluorochrome-conjugated monoclonal antibodies against CD14, CD68, CD163 and CD206 to identify M2. Female infertility will be defined by the failure to achieve a clinical pregnancy after 12 months or more of regular unprotected sexual intercourse.
Day 1
Correlation of M1 and M2 macrophages quantity in endometriotic tissue to the presence of endometriosis-associated chronic pelvic pain.
Time Frame: Day 1
To assess surface expression of macrophage markers, endometrial tissue cells will be stained for flow cytometry with fluorochrome-conjugated monoclonal antibodies against CD14, CD68, CCR7 and CD80 to identify M1, whereas fluorochrome-conjugated monoclonal antibodies against CD14, CD68, CD163 and CD206 to identify M2. Chronic pelvic pain will be defined as pain that occurs below the umbilicus (belly button) that lasts for at least six months.
Day 1

Collaborators and Investigators

This is where you will find people and organizations involved with this study.

Sponsor

University of Messina

Collaborators

University of Padova
University of Ljubljana

Investigators

  • Principal Investigator: Antonio Simone Laganà, M.D., University of Messina

Publications and helpful links

The person responsible for entering information about the study voluntarily provides these publications. These may be about anything related to the study.

General Publications

Study record dates

These dates track the progress of study record and summary results submissions to ClinicalTrials.gov. Study records and reported results are reviewed by the National Library of Medicine (NLM) to make sure they meet specific quality control standards before being posted on the public website.

Study Major Dates

Study Start (Actual)

December 1, 2016

Primary Completion (Actual)

December 1, 2018

Study Completion (Actual)

December 1, 2019

Study Registration Dates

First Submitted

April 27, 2017

First Submitted That Met QC Criteria

April 27, 2017

First Posted (Actual)

May 2, 2017

Study Record Updates

Last Update Posted (Actual)

March 24, 2020

Last Update Submitted That Met QC Criteria

March 23, 2020

Last Verified

March 1, 2020

More Information

Terms related to this study

Keywords

Additional Relevant MeSH Terms

Other Study ID Numbers

  • ENDOMAC

Plan for Individual participant data (IPD)

Plan to Share Individual Participant Data (IPD)?

Yes

Drug and device information, study documents

Studies a U.S. FDA-regulated drug product

No

Studies a U.S. FDA-regulated device product

No

This information was retrieved directly from the website clinicaltrials.gov without any changes. If you have any requests to change, remove or update your study details, please contact register@clinicaltrials.gov. As soon as a change is implemented on clinicaltrials.gov, this will be updated automatically on our website as well.

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