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STUDY QUESTION Are the primary cell cultures and cell lines used in endometriosis research of sufficient quality? SUMMARY ANSWER Primary cells used in endometriosis research lack purity and phenotypic characterisation, and cell lines are not genotypically authenticated. WHAT IS KNOWN ALREADY The poor reproducibility of in vitro research and the lack of authenticity of the cell lines used represent reasons of concern in the field of reproductive biology and endometriosis research. STUDY DESIGN, SIZE, DURATION In the present study, past in vitro research in the field of endometriosis was systematically reviewed to determine whether the appropriate quality controls were considered. In addition, we explored the performance of Paired Box 2 (Pax2) as an endometrium specific marker in endometrial and endometriotic primary cell cultures; we also characterised the most diffused endometriosis cell lines with respect to important markers including the short tandem repeat (STR) profile. PARTICIPANTS/MATERIALS, SETTING, METHODS Literature review part: almost 300 published protocols describing the isolation and creation of primary cell cultures from endometriosis were reviewed. Wet-lab part: primary cells isolated from 13 endometriosis patients were analysed by immunohistochemistry, immunofluorescence and FACS for the expression of Pax2. Cell lines Z11 and Z12, the most diffused endometriosis cell lines, were characterised with respect to the expression of Pax2, steroid hormone receptors and STR profile. MAIN RESULTS AND THE ROLE OF CHANCE From the literature review work, we underscored the lack of sufficient cell purity and phenotypic characterisation of primary cell cultures, which present high risk of contaminations from surrounding non-endometriotic tissues. Past work based on the use of cell lines was reviewed as well, and it emerged that cell line authentication was never performed. In an effort to address these weaknesses for future research, we present data on the performance of Pax2, a suitable marker to exclude ovarian (and other non-endometrial) cell contaminations from primary cell cultures; STR profiles of cell lines Z11 and Z12 were analysed and indicated that the cells were authentic. These profiles are now available for authentication purposes to researchers wishing to perform experiments with these cells. A quality control pipeline to assure sufficient quality of in vitro research in the field of reproductive biology and endometriosis is proposed. We encourage scientists, research institutes, journal reviewers, editors and funding bodies to raise awareness of the problem and adopt appropriate policies to solve it in the future. LARGE-SCALE DATA STR profiles of cell lines Z11 and Z12 are deposited at the Cellosaurus database—web.expasy.org. LIMITATIONS, REASONS FOR CAUTION There may be additional markers suitable to assess cell quality. WIDER IMPLICATIONS OF THE FINDINGS Future in vitro research in endometriosis and the reliability of outcomes can be improved by using the recommendations presented in this study. STUDY FUNDING/COMPETING INTEREST(S) The study was partly financed by the ‘Stichting Fertility Foundation’ (The Netherlands). The authors declare no existing conflict of interest. TRIAL REGISTRATION NUMBER Non-applicable.

Endometriotic cell culture contamination and authenticity: a source of bias in in vitro research?