Hallamaa 2012.

Study characteristics
Patient sampling	Primary objective: to investigate whether serum HE4 concentration varies within the normal menstrual cycle and whether common gynaecological hormonal treatments have an effect on HE4 values Participants: patients undergoing laparoscopy for suspected endometriosis or tubal ligation Selection criteria: exclusion criteria: suspicion of malignancy, pregnancy or infection Study design: cross‐sectional, two‐gate design, prospective collection of samples
Patient characteristics and setting	Clinical presentation: endometriosis ‐ not specified; controls ‐ women requesting tubal ligation; hormonal medication was used by 78 (43.3%) women Age: mean age 34 years, range 18‐48 years Number of participants enrolled: 180 women Number of participants available for analysis: 175 (7 in menstrual, 32 in proliferative and 60 in secretory cycle phase; 61 had inactive/atrophic endometrium) Setting: 2 central hospitals and 2 university central hospitals Place of study: Turku, Finland Period of study: October 2005 ‐ October 2007 Language: English
Index tests	Index test: HE4, CA‐125 Details of the index test procedure as stated: serum HE4 and CA‐125 concentrations were analysed by ELISA analysis (Fujirebio Diagnostics inc, Malvern, PA, USA) according to the manufacturer's instructions Threshold for positive result: For HE4 not provided, for CA‐125 > 35 U/l, not pre‐specified Examiners: no information provided; unclear if were blinded to the result of reference standard Interobserver variability: not reported
Target condition and reference standard(s)	Target condition: endometriosis Prevalence of target condition in the sample: n = 123/175 (70%): stages I‐IV, the number of participants per each stage not reported; controls n = 52 Reference standard: laparoscopy N = 175 (100%) + histology Description of positive case definition by reference standard test as reported: visual inspection confirmed by histopathology; staging according to the rASRM classification Examiners: no information provided
Flow and timing	Time interval between index test and reference standard: blood samples were collected 24 h before surgery Withdrawals: 5 women were excluded when endometrial biopsy was non‐conclusive regarding cycle phase
Comparative
Key conclusions by the authors	HE4 measurement in healthy pre‐menopausal women as well as in women with endometriosis can be carried out at any phase of the menstrual cycle, and irrespective of hormonal medication, extending the benefits of HE4 use in clinical practice
Conflict of interest	One of the authors received lecture honoraria from several pharmaceutical companies; other authors declared no conflict of interest; the study was supported by the Finnish Funding Agency for Technology and Innovation (projects 40343/05 and 599/05); Hormos Medical Ltd, Finland (subsidiary of QuatRx Pharmaceutical, USA); Biotop Oy, Finland; Genolyze Oy, Finland
Notes	For HE4 there was no difference between the groups ‐ no data available for meta‐analysis
Methodological quality
Item	Authors' judgement	Risk of bias	Applicability concerns
DOMAIN 1: Patient Selection
Was a consecutive or random sample of patients enrolled?	Unclear
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 cycle phase considered in interpretation of the result of index test?	Yes
		High	Low
DOMAIN 3: Reference Standard
Is the reference standards likely to correctly classify the target condition?	Yes
Were the reference standard results interpreted without knowledge of the results of the index tests?	Yes
		Low	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