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

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.