Pino 2009.

Study characteristics
Patient sampling	Primary objective: to investigate the association between MMP‐1 and MMP‐9 activities and ICAM1 cleavage mediated by TNF in eutopic endometrial stromal cells from women with and without endometriosis during culture Participants: women without endometriosis who underwent laparoscopic tubal sterilisation or hysterectomy for a benign non‐endometrial gynaecologic condition (control group), and women undergoing laparoscopy for diagnosis of endometriosis, which was later surgically confirmed Selection criteria: exclusion criteria: hormonal treatment or contraceptives during the previous 6 or 3 months, respectively; neoplastic, endocrine or infectious diseases Study design: observational two‐gate, prospective sample collection
Patient characteristics and setting	Clinical presentation: endometriosis group ‐ not specified, controls ‐ eumenorrhoeic reproductively normal women without endometriosis who underwent laparoscopic tubal sterilisation Age: mean age 35.2 ± 0.7, endometriosis group; 38.4 ± 0.5 years, controls Number enrolled: 20 women Number available for analysis: 20 women (7 in proliferative and 13 in secretory phase of the cycle) Setting: Institute of Maternal and Child Research, San Borja Arriarán Clinical Hospital, University of Chile Place of study: Santiago, Chile Period of study: not reported Language: English
Index tests	Index test: MMP‐1, MMP‐9, ICAM1, sICAM1 mRNA and protein Description of positive case definition by index test as reported: MMP‐1, MMP‐9, ICAM1 and sICAM1 mRNA expression by RT‐PCR (internal control 18S rRNA, semi quantification by using agarose gel electrophoresis); ICAM1 and MMP1 protein levels by Western blot; activities of MMP1 and MMP9 by using casein zymography in cell culture (results are expressed as arbitrary units regarding the total µg of proteins loaded (MMP1) or as values normalised to the internal standard (MMP9)); concentration of active MMP‐1 by using enzymatic activity assay (Human Active MMP‐1 Fluorescent Assay kit (R&D System Inc., Minneapolis, USA) per manufacturer’s instructions; detection limit 0.052 ng/ml); sICAM concentration by using ELISA (Inmunoassay Human sICAM1 kit (R&D System) per manufacturer’s instructions, minimal detectable concentration of 0.096 ng/mL); sample processing and laboratory technique described in details; thresholds not provided Examiners: none stated; unclear if blinded to the results of reference standard Interobserver variability: intra‐ and interassay reproducibility for sICAM: 3.3%‐4.8% and 6.0%‐10.1%, for MMP‐1: 9.6%‐10.0% and 8.7%‐17.7%
Target condition and reference standard(s)	Target condition: endometriosis Prevalence of target condition in the sample: n/N = 10/20 (50%): stage I‐II 6, stage III‐IV 4; controls 10 Reference standard: laparoscopy/laparotomy Description of positive case definition by reference test as reported: staging according to the rASRM classification Examiners: none stated
Flow and timing	Time interval between index test and reference standard: samples were collected at surgery Withdrawals: none reported
Comparative
Notes	Conclusion: The deregulation of MMP‐9, and the TNF participation in the MMP‐1 and proMMP‐9 secretions, in the MMP‐9 expression and in the expression and cleavage of ICAM1 may contribute to the pathophysiology of this disease Comment: The reported estimates for the assessed biomarkers are for basal conditions only, prior to incubation with TNF For ICAM mRNA, ICAM protein, sICAM protein, MMP‐9 mRNA, ‐ protein and MMP‐1 protein there was no statistically significant difference between the groups ‐ no data available for meta‐analysis (Appendix 7) For MMP‐1 mRNA there was statistically significant difference between the groups, but there were insufficient data to construct 2 × 2 tables; not included in this review
Methodological quality
Item	Authors' judgement	Risk of bias	Applicability concerns
DOMAIN 1: Patient Selection
Was a consecutive or random sample of patients enrolled?	No
Did the study avoid inappropriate exclusions?	Yes
Was a 'two‐gate' design avoided?	No
		High	High
DOMAIN 2: Index Test All tests
Were the index test results interpreted without knowledge of the results of the reference standard?	Unclear
If a threshold was used, was it pre‐specified?	No
Was a menstrual cycle phase considered in interpreting the index test	Yes
		High	Low
DOMAIN 3: Reference Standard
Is the reference standards likely to correctly classify the target condition?	Unclear
Were the reference standard results interpreted without knowledge of the results of the index tests?	Yes
		Unclear	Low
DOMAIN 4: Flow and Timing
Was there an appropriate interval between index test and reference standard?	Yes
Did all patients receive the same reference standard?	Yes
Were all patients included in the analysis?	Yes
		Low