Evaluation of M1 and M2 Macrophages in Endometriotic Tissue of Women Affected by Endometriosis at Different Stages.
Condition: Endometriosis
MeSH terms: Endometriosis
Study type: Observational

Summary: 
      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.
    

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.
    

Intervention name(s): Evaluation of M1 and M2 macrophages by flow cytometry.
Intervention type(s): Diagnostic Test
Intervention description(s): 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.
Observational model: Case-Control
Time perspective: Prospective
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.
      
Biospecimen description: 
      -  Endometriotic tissue (histologically confirmed) from patients affected by endometriosis
           at different stages (cases).

        -  Tissue samples from ovarian functional cysts (controls).
    
Keywords: Macrophages; Macrophages, Peritoneal; Endometriosis